308a Tuesday, February 14, 2017

Symposium: Transporters Symposium: Destroying the Cytoskeleton

1512-Symp 1515-Symp Principles of Energy Transduction and Alternating Access of ABC The Relationship between Microtubule Birth and Death Exporters Gary Brouhard. Hassane S. Mchaourab. McGill University, Canada, Montreal, QC, Canada. Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Microtubules are born and reborn continuously, even during quiescence. TN, USA. These polymers are nucleated from templates, namely gamma-tubulin ring ATP binding cassette (ABC) transporters to drive the energetically uphill complexes and severed microtubule ends. Interestingly, the rate of micro- translocation of substrates including the export of amphiphilic molecules tubule nucleation increases as cells enter mitosis, and all cells nucleate out of the cell by harnessing the energy of ATP hydrolysis. Dedicated motor microtubules in distinct regions of their cytoplasms. How are these spatial domains, the nucleotide binding domains (NBDs), bind and turn ATP over to and temporal profiles for microtubule nucleation established? I will discuss power conformational rearrangements in the transmembrane domain (TMD) my lab‘s recent attempts to answer this question using a mix of single- which binds and transports substrates. Although numerous investigations molecule biophysics, cell biology, and structural biology. I will describe have defined the architecture and described the ATP-coupled conformational how the process of microtubule ‘‘death’’ (catastrophe) relates to the process rearrangements for a number of ABC transporters, a general model of the of microtubule ‘‘birth’’ (nucleation). I will argue that understanding how the transduction of ATP energy to the work of substrate remains elusive. We uti- microtubule cytoskeleton is destroyed is critical to understanding how it is lized double electron-electron resonance (DEER) and molecular dynamics first built. (MD) simulations to describe the ATP- and substrate-coupled conforma- tional cycle of the mammalian ABC efflux transporter P-glycoprotein 1516-Symp (Pgp), or ABCB1, which has been extensively studied owing to its role in Architecture Dependence of Actin Filament Network Disassembly the clearance of xenobiotics and clinical implications in cancer resistance Laurent Blanchoin. to chemotherapy. Pairs of spin labels were introduced at residues selected CytoMorphoLab, Biosciences & Biotechnology Institute, Grenoble, France. to track the putative inward-facing (IF) to outward-facing (OF) transition. Turnover of actin networks in cells requires the fast disassembly of aging actin Our findings illuminate how ATP energy is harnessed in the NBDs in a structures. Two key protein factors are involved during actin disassembly, two-stroke cycle and elucidate the consequent conformational motion that ADF/cofilin and myosin, both acting on actin filament mechanics. We used a reconfigures the TMD, two critical aspects of Pgp transport mechanism. classic micropatterning method or a high-resolution surface structuration assay Along with a novel, fully atomistic model of the OF conformation in mem- to spatially control actin network assembly. These actin templates were used to brane, the insight into Pgp conformational dynamics harmonizes mechanistic evaluate the response of oriented actin structures to myosin or ADF/cofilin- and structural data into a novel perspective on ATP-coupled transport. induced disassembly. We found that the actin architecture (branched, parallel When considered in the context of previous studies of ABC exporters, these or antiparallel) and its density govern network disassembly. We also found findings reveal mechanistic divergence within the efflux class of ABC that they modulate the accessibility of proteins within the actin network. We transporters. established therefore some general principles on how the cell can modulate locally the turnover of a given structure by varying both density and architec- 1513-Symp ture of its actin network. CLC Chloride/Proton Transporters Merritt Maduke. Platform: Membrane Dynamics Stanford University, Stanford, CA, USA. The conventional alternating-access mechanism for transporter function re- 1517-Plat quires outward-facing, inward-facing, and occluded conformational states. The Interplay of Structure and Dynamics in Mixed Lipid Bilayers For the CLC Cl-/Hþ antiporters, only the occluded state has been accessible Elizabeth Kelley1, Rana Ashkar2, Robert Bradbury3,4, Paul Butler4, to crystallography. We are combining a variety of spectroscopic and computa- Michihiro Nagao4,5. tional approaches to gain insight into the elusive outward- and inward-facing 1Center for Neutron Research, NIST, Gaithersburg, MD, USA, 2ORNL, Oak states. Ridge, TN, USA, 3U. of Indiana, Bloomington, IN, USA, 4NIST, Gaithersburg, MD, USA, 5University of Indiana, Bloomington, IN, USA. 1514-Symp The implication of lipids in membrane mediated processes has motivated signif- Rigorous Representation of Transporters as Allosteric Machines Enables a icant theoretical and experimental research interest to understand the functional Quantitative Understanding of their Functional Mechanisms significance of lipid chemical structure variations on the biomembrane structure Harel Weinstein, George Khelashvili, Michael V. LeVine, and dynamics. In particular, hydrophobic mismatch between lipids with different Michel A. Cuendet. acyl chain lengths is expected to play an important role in determining the local Physiology and Biophysics, Weill Cornell Med Col, Cornell Univ, New membrane structure and collective membrane dynamics. Herein we investigated York, NY, USA. these phenomena in large unilamellar vesicles composed of binary mixtures The sodium-coupled transporters are prototypical molecular machines of the of lipids with a 4 carbon mismatch in tail length, dimyristoylphosphatidylcholine cell that maintain communication with the environment by moving substances (DMPC, 14:0 C) and distearoylphosphatidycholine (DSPC, 18:0 C), using across the membrane. How they use energy to perform the transfer of substrates neutron scattering techniques. Structural studies using small angle neutron scat- against their chemical gradient, i.e., by sodium symport along its gradient, is of tering (SANS) revealed that the mixed lipid bilayers were thinner than expected special biophysical interest. New structural information, and powerful compu- for a simple composition-weighted average of the pure component bilayers. The tational methods applied to the analysis of extensive molecular dynamics tra- structural differences were accompanied by dramatic differences in the mixed jectories, make it possible to incorporate mechanistic elements of ion and lipid membrane dynamics. Neutron spin echo spectroscopy (NSE) experiments substrate transport in structure-informed allosteric models that can be interro- showed that the mixed lipid bilayers were more dynamic than their single gated for specific and quantitative inferences. This will be illustrated for the component analogs, with the mixed bilayers having a lower bending modulus dopamine transporter, DAT, a member of the neurotransmitter sodium:sym- and increased thickness fluctuation amplitude than the pure DMPC or DSPC bi- porter (NSS) family of proteins that is of great interest not only because of layers. Both of these enhanced dynamics were consistent with a decrease in the its essential physiological role, but also because of increasing evidence for area expansion modulus of the mixed systems. Together our results demonstrate its involvement in various diseases, from neuropsychological impairments to the influence of lipid composition on the bilayer biophysical properties and high- cancer. We show how rigorous physics-based analyses of extensive computa- light the complex interplay between structure and dynamics in lipid membranes. tional simulations of DAT in the context of structural and functional informa- tion can reveal specifics of the molecular mechanisms of substrate and ion 1518-Plat movements, and of the functional association of the transporters with regulators Microfluidic Device to Study Phosphoinositide Gradients in Model and membrane components that are essential for function, and also uncover Membranes some surprisingly specific effects of disease-causing mutations in humans. Brittany M. Neumann1, Devin Kenney1,QiWen2, Arne Gericke1. Computational resources: Oak Ridge Leadership Computing Facility (ALCC- 1Chemistry Biochemistry, Worcester Polytechnic, Worcester, MA, USA, BIP109) at ORNL, and NERSC (m1710), supported by Office of Science of 2Physics, Worcester Polytechnic, Worcester, MA, USA. U.S-DoE under Contracts No. DE-AC05-00OR22725; and No. DE-AC02- The availability of phosphoinositide-(3,4,5)-trisphosphate (PI(3,4,5)P3) in the 05CH11231, respectively. plasma membrane (PM) dictates downstream activation of cellular division,

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growth and motility via the PI3K-Akt pathway. PI(3,4,5)P3 composition in the and Young’s modulus. The high-tension, non-linear regime was also studied PM is regulated by the competition of PI3K kinase versus the phosphatase for the cell wall, leading to the observation of stress stiffening. PTEN, whose role is to dephosphorylate PI(3,4,5)P3 to produce PI(4,5)P2. 1521-Plat These opposing actions in motile cells result in a gradient of PI(3,4,5)P3 at Geometric Diversity of Living Organisms and Viruses the leading edge giving way to PI(4,5)P2 at the trailing. Despite this important feature in cells like Dictyostelium and neutrophils, there are no current model David V. Svintradze. systems available to study gradients of phosphoinositides. The work presented Department of Physics, Tbilisi State University, Tbilisi, Georgia. Recently, it has been celebrated a hundred year anniversary of Einstein’s here shows a novel methodology using microfluidics to generate PI(4,5)P2 gra- dients in supported lipid bilayers (SLB). theory of general relativity. Since, underlying geometry of space and time We have successfully developed a tapered microfluidic device where a gradient became main source of inspiration of new theories. Though, much attention was paid to the geometric construction of the universe, geometric diversity of PI(4,5)P2 can be generated with both temporal and spatial control. The tapered design allows for a choice of channel width to observe the gradient of living organisms got largely omitted. Of course such reluctant approach ranging from 500 mmto50mm. Using total internal reflectance fluorescence has fundamental explanation. General relativity is formulated to explain the (TIRF) microscopy we can quantify single molecule dynamics in the channel. gravity, while the role of gravitation on the molecular level in the formation In addition to this, we demonstrate Ca2þ gradient dependent domain formation of living organisms is negligibly small. However, mathematical approach used in general relativity can be carried out for two dimensional surfaces. in SLBs composed of PI(4,5)P2, PI(4,5)P2 and cholesterol, and well as POPS containing membranes. Therefore, scientists started to develop analogical differential geometric This novel method will allow for the investigation of gradients in membranes approach to predict static shapes of living organisms. As a result associated that where not attainable before. The advantage of having a choice to work with Euler-Lagrange equations, so called ‘‘shape equations’’ have been formu- a model system in addition to the cellular systems is the ability to test hypoth- lated. But, shape equations turned to be fourth order partial nonlinear differ- eses outside the complex cellular milieu to see if a trend is preserved. With this ential equations, and finding a general analytical solution is a difficult device we hope to start piecing together the most basic components necessary problem, even though it has been numerically solved for some specific to maintain or degrade cellular gradients whose results could continue to cases. The numerical solutions generated a large body of beautiful explana- answer the ultimate question of how lipid organization affects the information tion of experimental results especially observed in diversity of lipid transduction within a cell. membranes shapes. Because of shape equations as well as numerical calcu- lations are not exact equations of motion and analytical solutions, we have 1519-Plat decided to employ different approach to the problem. Namely, we have used Advances in the Theoretical and Computational Modeling of Lipid Bilayer tensor calculus of moving surfaces to deduce exact equations of surface mo- Membranes tion in electromagnetic field and then to analytically solve it for some Roger A. Sauer1, Kranthi K. Mandadapu2, Thang X. Duong1, simplified cases. Simplifications have produced such outstanding equations Amaresh Sahu2, Yannick Omar1. as Maxwell equations for electrodynamics and Navier-Stokes equations for 1Mechanical Engineering, RWTH Aachen University, Aachen, Germany, dynamic fluid are (D. V. Svintradze, (2016), arXiv:1609.07765 [cond- 2Chemical and Biomolecular Engineering, University of California at mat.soft]). Also, analytic solutions to simplified equations for homogeneous Berkeley, Berkeley, CA, USA. surfaces, in equilibrium with environment, (D. V. Svintradze, (2016), ar- This contribution presents an overview of theoretical and computational Xiv:1608.01491 [physics.bio-ph]) have explained why constant mean curva- models for lipid bilayer membranes focusing on recent advances based on ture (CMC) surfaces are such abundant shapes among living organisms and non-linear shell theory. Lipid bilayers are generally characterized by complex viruses. mechanical behavior that has both fluid-like and solid-like properties, leading to complex three-dimensional deformation states. This requires solving par- 1522-Plat tial differential equations on evolving surfaces. To analyze those equations, Simulations Help Unravel Flavivirus Envelope Structure and Function 1,2 1 1 a new computational model is proposed based on so-called isogeometric finite Jan K. Marzinek , Daniel Holdbrook , Roland G. Huber , Chandra Verma1,2, Peter J. Bond1,2. element methods. These methods are capable of representing surface topog- 1 2 raphy, curvature and flow to high accuracy. The new model is suitable for Bioinformatics Institute (BII) A*STAR, Singapore, Singapore, Department analyzing and predicting the shapes changes observed during tethering, of Biological Sciences, National University of Singapore (NUS), Singapore, budding and endocytosis. The computational ingredients required for Singapore. capturing the mechanics of lipid bilayers, like the bending kinematics, area- Over 40 flaviviruses worldwide are human pathogens. These include dengue incompressibility, viscous surface flow and numerical stabilization tech- virus (DENV), responsible for more than 400 million infections per year, and niques, are discussed. Several numerical examples are presented to illustrate emerging infectious diseases such as Zika virus (ZIKV). The flavivirus nucle- the current simulation capabilities. The considered surface representation is ocapsid core is encapsulated by a phospholipid bilayer, within which are also highly suitable for formulating surface interactions, like contact and embedded envelope and membrane proteins. Low pH conditions trigger adhesion, which are also discussed. This contribution also presents new conformational changes in the envelope protein and a dimer-to-trimer transi- work on the coupling of deformation to mass transport caused by diffusion tion, leading to exposure of the hydrophobic fusion peptide (FP). Subsequent or phase transitions and proposes computational formulations to handle these interaction of FPs with the host endosomal membrane initiates fusion, result- complex cases. ing in release of the RNA genome into the cell. The pre-fusogenic flaviviral membrane exhibits significant curvature and deformation, indicated by 1520-Plat cryo-electron microscopy (cryo-EM) maps. Here, multiscale simulations of Engineering the Cell Envelope of Gram-Negative Bacteria the complete mature flavivirus envelope particle have been used to refine Sunny Hwang, James C. Gumbart. the envelope structure in unprecedented detail. The final structures of Georgia Tech, Atlanta, GA, USA. DENV were morphologically consistent with cryo-EM maps and revealed The cell envelope in Gram-negative bacteria is made of two distinct mem- the structural basis for membrane curvature, whilst interesting differences branes and a cell wall between them. Although there is growing interest in are highlighted in simulations of the more robust ZIKV envelope. Extra- the mechanical adaptation of this cell envelope to turgor pressure, how the membranous protein components are shown to serve as scaffolds which posi- cell wall, the inner membrane (IM), and the outer membrane (OM) couple tion transmembrane domains and help to determine lipid bilayer architecture, together to provide this adaption remains unexplored. In this study, we have dependent upon specific electrostatic interactions with anionic lipids. The used molecular dynamics (MD) simulations to resolve how lipid membranes observed lipid-protein coupling in the mature virus has potential functional and cell wall respond to changes in lateral tension, such as might result from significance for pH-dependent envelope protein conformational changes, sub- an osmotic gradient. Simulations of individual membranes as well as mem- sequently investigated by constructing a viable structure of the fusogenic fla- branes coupled to the cell wall were also carried out. Our inner membrane vivirus particle, guided by biophysical data, and simulating its interaction model consists of saturated, unsaturated, and cycle-containing lipids while with models of late endosomal membranes. Clustering of trimeric envelope our outer membrane model contains an outer leaflet of lipopolysaccharides; protein complexes was observed to be dependent upon the viral membrane both membranes accurately reflect the diverse population of lipids found in lipid composition, whilst contact between the envelope protein FP tips and en- each experimentally. We have measured the mechanical properties of each dosomal lipid bilayer induced ‘‘micro-budding’’ in the target membrane. membrane at protein concentrations ranging from 0 to 50%. In order to describe Remarkably, many FPs could simultaneously interact with and embed them- the membranes’ elasticity, we focused on the low-tension regime (elastic selves within the endosomal membrane, challenging traditional mechanistic regime), which can be exploited to measure the area compressibility modulus views of viral fusion and resultant infective states.

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1523-Plat the model organism Candida albicans, are presently thought to lack motility Modulation of Membrane Rigidity by the ESCRT-III Complex appendages and to be non-motile both in planktonic and adherent forms. In Nicola De Franceschi1, Maryam Alqabandi1, Nolwenn Miguet2, this study, we investigated the early-stage adhesion of C. albicans to fiber- Christophe Caillat2, Winfried Weissenhorn2, Patricia Bassereau1. textured surfaces (diameter: 0.5, 1, and 2 mm) at high spatiotemporal resolu- 1UMR168, Institut Curie, Paris, France, 2Institut de Biologie Structurale, tion to gain insight into the mechanisms involved in recognition and response Grenoble, France. to surface topography in absence of motility. We report that upon adhering to The ESCRT-III complexes is an evolutionary conserved membrane scission textured surfaces, C. albicans continually adjust their location until an equi- machinery and it is essential in many cellular processes such as cytokinesis, librium attachment location is achieved. This behavior was observed to multivesicular bodies formation, HIV release and nuclear membrane repair. occur more vigorously on substrates textured with larger diameter fibers Membrane scission by ESCRT-III is accomplished by constricting the nega- (1.0-2.0 mm). We hypothesized this to occur due to the spatial gradient in total tively curved membrane present inside the neck connecting two membrane- energy experienced on textured surfaces. To quantitate the total energy of delimited compartments. In Homo Sapiens there are at least 12 ESCRT-III adherent cells as a function of fiber diameter, we developed a biophysical proteins, called CHMPs. However, only a subset of them, namely CHMP4B, model. The model verified that the experimentally obtained equilibrium CHMP2A/B and CHMP3 appear to be strictly required in all these processes, resting positions correspond to locations of energy minima, especially on sur- indicating that they might constitute the minimal scission machinery. faces where significant energy gradient is experienced. Notably, we find this Several CHMP2B mutations have been reported to cause neurological dis- behavior to be biologically-driven, as adjustment of initial adhesion location functions; however the function of this protein is still unknown. We inves- was not observed in UV-treated cell. We further demonstrate that the single tigated the mechanical properties of CHMP2B polymer at the mesoscale cell total energy quantitation enables prediction of the population-level cell and found that it can modulate membrane rigidity, providing a possible mo- attachment density, which can be utilized towards ab initio design of surfaces lecular mechanism explaining the pathogenic property of the mutated with controlled biofilm growth. CHMP2B. Our results indicate that modulation of membrane rigidity is an important aspect of ESCRT-III function and assign a novel function for 1526-Plat CHMP2B. Exploring the pH Sensitivity of E. Coli MscS Jordyn Veres, Hannah R. Malcolm. 1524-Plat Chemistry, University of North Florida, Jacksonville, FL, USA. Design Principles for Robust Vesiculation in Clathrin-Mediated Endo- The mechanosensitive channel of small conductance (MscS) from Escheri- cytosis chia coli (E. coli) gates in response to mechanical tension that is generated Julian Hassinger1, George Oster2, David Drubin2, Padmini Rangamani3. from hypo-osmotic shock. The gating mechanism of E. coli MscS, in 1Biophysics, University of California, Berkeley, Berkeley, CA, USA, response to mechanical tension, is similar to that of a Jack-in-the-Box, the 2Molecular and Cell Biology, University of California, Berkeley, Berkeley, channel springs into the open state when the applied extrinsic tension over- CA, USA, 3Mechanical and Aerospace Engineering, University of California, comes the intrinsic lateral tension of the membrane. E. coli MscS is the San Diego, San Diego, CA, USA. founding member of the MscS superfamily, a superfamily of 15 subfamilies Budding of membranes by protein coats is a universal phenomenon that is that all contain significant homology to the pore lining helix of E. coli MscS. critically important in cellular trafficking pathways. Recent experiments In the early identification of MscS a rapid desensitization of MscS when have demonstrated that elevated membrane tension inhibits the ability of pro- pressure is applied at low pH is observed, however when patched at physi- tein coats to deform membranes into buds. However, the robustness of ological pH E. coli MscS does not desensitize. To identify the region of the clathrin-mediated endocytosis (CME) across a diverse range of organisms channel that evokes pH sensitivity we will use asymmetric and symmetric and mechanical environments suggests that the protein machinery in this pH patch buffer. A recent study explored how significant alterations to the process has evolved to take advantage of some set of design principles to most conserved residues impacted E. coli MscS function, one residue ensure robust vesiculation against opposing forces like membrane tension. Us- F127 showed significant alteration to the gating phenotype when mutated ing a modified Helfrich model for membrane mechanics and membrane pro- to different residues. The results of the previous study show that the rapid tein interaction, we have investigated the influence of membrane rigidity, desensitization classically observed in low pH are observed in a F127 muta- curvature induced by the protein coat, area covered by the protein coat, mem- tions. This suggests that F127 is involved in a long-range interaction, poten- brane tension and force from actin polymerization on robust bud formation. tially a cation-p with K60. Based on the location of this interaction in Under low tension, the membrane smoothly evolves from a flat to budded multiple crystal structures, the cation-p is potentially involved in the desen- morphology as the coat area or spontaneous curvature increases, whereas sitized state of the channel. Through patch clamp electrophysiology we will the membrane remains essentially flat at high tensions. At intermediate, phys- understand the role of a pH sensitive region in the gating cycle of E. coli iologically relevant, tensions, the membrane undergoes a snapthrough insta- MscS. bility in which small changes in the coat area, spontaneous curvature or membrane tension cause the membrane to snap‘‘ from an open, U-shape to 1527-Plat 2D a closed bud. Through systematic analyses of the different parameters contrib- In Situ Measurements of Protein Forces and Intracellular Ca under uting to membrane budding, we have identified potential ways of overcoming Fluid Shear Stimuli the energy barrier associated with the instability. For example, increasing the Susan Z. Hua1, Mohammad Mehdi Maneshi2, Frederick Sachs1. 1 bending rigidity of the coat smooths out this instability, allowing for success- Physiology and Biophysics, SUNY-Buffalo, Buffalo, NY, USA, 2 ful budding at higher membrane tensions. Additionally, applied force from Mechanical and Aerospace Eng., SUNY-Buffalo, Buffalo, NY, USA. actin polymerization can induce the transition from an open to a closed During traumatic brain injury, brain cells experience transient shear bud, bypassing the instability. Finally, a combination of increased coat rigidity stresses that alter the forces in structural proteins. An increase in intracel- 2þ and force from actin polymerization ensures robust vesiculation, even at high lular Ca is seen universally in cells subjected to shear forces, but how 2þ membrane tensions. are mechanical forces coupled to the Ca influx is not well understood. We used a microfluidic chamber driven with a high-speed pressure servo Platform: Mechanosensation to generate controlled fluid shear stress to cultured astrocytes, and simulta- neously measured the intracellular responses using FRET-based force sen- 1525-Plat sors actinin-cpstFRET and Ca2þ probes jRCaMP1h. We found that fluid Candida Albicans Yeast Seek to Adhere in Energetically Optimal Locations shear generated non-uniform stresses in actinin. The time dependent force Zhou Ye1, Amrinder Nain2, Bahareh Behkam1. distribution is highly sensitive to the feature of the stimulus such as ampli- 1Mechanical Engineering Dept, Virginia Tech, Blacksburg, VA, USA, tude, duration rise time, and the frequencies of stimulation. In cells with 2Mechanical Engineering Dept, Virginia Tech, Christiansburg, VA, USA. weaker stress fibers, a rapid shear pulse (23 dyn/cm2, 2 ms rise time, 400 Formation of surface associated multicellular communities of microorgan- ms duration) produced an immediate and long-lasting increase in actinin isms known as biofilms is of concern in environmental, medical, and indus- stress at the upstream end of the cell and minimal changes at the downstream trial settings. Biofilm formation starts with adhesion of microorganisms on end. In contrast, a slow ramp to the same amplitude caused a minimal, and a substrates, as such understanding the microbe-substrate interaction at more uniform increase in actinin stresses. Simultaneous Ca2þ imaging single-cell resolution is crucial to advancing the current understanding of showed that the initial Ca2þ rise began at the upstream end of the cell where the biofilm formation. Previous works have demonstrated that bacteria there was high strain, and it propagated to the entire cell within ~4 s. More- motility appendages are critical to recognition of substrate nanostructures over, the Ca2þ peaked much faster (~4 s) at the front end and slower in the and modulation of adhesion behavior. In contrast, fungal pathogens such as center of cell body (~10 s). This behavior occurred in ~40% cells. In cells

BPJ 7813_7824 Tuesday, February 14, 2017 311a with abundant strong actin bundles, there was minimal response to shear the production of vasoregulatory and anti-atherogenic substances for the treat- stress. The actinin stress increased transiently and uniformly over the entire ment of vascular disease. cell during the period of stimulation, and the Ca2þ rise appeared starting from the cell body. These results suggest that the force distribution directly 1530-Plat alter the initial Ca2þ dynamics in different cellular domains. This work was Mechanically-Induced Gating in PKD2L1 (Trpp2): Calcium-Induced funded by NINDS. Activation Masquerading as Force Sensitivity? Charles D. Cox, Chai Ann Ng, Navid Bavi, Boris Martinac. 1528-Plat Victor Chang Cardiac Research Institute, Sydney, Australia. Mechanosensetive ATP Release Involves a Non-Conductive Pathway: The polycystic kidney disease 2-like protein (PKD2L1) is a member of the TRP Evidence from Large Field of View Real-Time Imaging channel superfamily and is highly expressed in the developing fetus, especially 1,2 2 1 in the cardiovascular system. In complex with PKD1L1 it forms a complex Olga Ponomarchuk , Francis Boudreault , Sergei N. Orlov , 2þ Ryszard Grygorczyk2. involved in Ca -signaling in primary cilia. Given the ubiquitous expression 1Department of Biophysics, Lomonosov Moscow State University, Moscow, of PKD2L1 and its localization within primary cilia, long thought of as mecha- Russian Federation, 2De´partement de me´decine, Universite´ de Montre´al, nosensing organelles, we sought to robustly address its mechanosensitivity Centre de recherche du CHUM, Montre´al, QC, Canada. alone and in the presence of putative binding partners including PKD1L1, Cell swelling is a potent ATP secretion stimulus. While the sources of ATP are TRPC1/6, TRPV4, Piezo1 and PKD1/2. In order to do so we created a stable known, it’s mechanisms of secretion in epithelial cells remain unclear. Two PKD2L1 expressing doxycycline inducible Flp-In HEK293 cell line. In the secretion pathways have been proposed: exocytosis of nucleotide-containing cell-attached configuration we could elicit large robust mechanically- vesicles or conductive release through pore-forming channels, like pannexins activated PKD2L1 currents over pressure ranges largely overlapping with or connexins. In this project, we have tested whether ATP is released or Piezo1. However, in comparison to the mechanically-gated channel Piezo1 not through a conductive pathway in A549 lung epithelial cells. Real-time whose response was instantaneous to applied force the PKD2L1 channels ATP secretion from glass-adherent A549 cells was imaged with a custom- had a much longer latency period. Once this period was overcome the channel designed lens system, combining a large field of view and a high light- responded to subsequent mechanical stimuli like a bona fide mechanosensitive gathering power, mounted directly on a PhotometricsÒ EvolveTM 512 EMCCD channel. The activity in cell-attached patches was highly dependent on the pres- ence of divalent cations. PKD2L1 is known to activate in response to rises in camera. ATP was detected with a light-emitting luciferin-luciferase reaction. 2þ Secretion was stimulated by acute cell swelling or exposure to ionomycin intracellular Ca and then subsequently desensitize. We found that multiple responses to mechanical stimuli was only possible in the absence of Ca2þ (10 mM). Plasma membrane permeability to large molecules was assessed in 2þ 2þ parallel by propidium iodide (PI) uptake with epifluorescence Zeiss Axio (n=45) in the pipette. When 1 mM Ca or Ba were present in the pipette cur- Observer microscope. Cells were grown in 10% (control cells) or 0.1% FBS rent rapidly and irreversibly desensitized. Activity could still be elicited in the presence of BAPTA or EGTA in the extracellular solution indicating that the (fragile cells). Our novel imaging system revealed a strong homogeneous 2þ ATP secretion lasting several minutes from control A549 cells stimulated response was not mediated by external Ca influx. The activity was boosted with 50% hypotonic shock. Yet, no PI-stained cells were detected under these by the addition of cytochalasin D (10 mM)(n=11) or GsMTx-4 (5 mM)(n=7) conditions. In contrast, cells rendered fragile, and thus prone to cell lysis, and inhibited by colchicine (10 mM)(n=9). This data suggests that PKD2L1 showed an appreciable amount of PI-stained cells post-hypotonic shock and channel activity can be modulated by mechanical force but the exact mecha- a punctuate pattern of ATP secretion co-localizing with PI-positive cells. Sud- nism still remains to be elucidated. den intracellular calcium elevation with ionomycin provoked a rapid, albeit 1531-Plat small, ATP secretion. Based on the fact that hypotonically-induced ATP Microgravity Modulates Drug-Induced Enhancement of Cancer Cell release in normal conditions was not accompanied with detectable permeation Migration of PI, we conclude that ATP in A549 cells is released by a non-conductive Devika Prasanth1, Sindhuja Suresh2, Michael Mimlitz3, Noah Zetocha4, pathway. Andrew E. Ekpenyong3. 1Biology, Creighton University, Omaha, NE, USA, 2Computer Science, 1529-Plat Creighton University, Omaha, NE, USA, 3Physics, Creighton University, The Short Term Response of PECAM-1 to Mechanical Loading: Omaha, NE, USA, 4Chemistry, Creighton University, Omaha, NE, USA. Cyclooxygenase-2 and Nitric Oxide Production with AFM Pulling and Microgravity experienced during spaceflights, causes bone, muscular and im- Shear Stress mune dysfunctions in astronauts. These dysfunctions correlate with changes Anne Marie T. Weber, Rick Mathews, Zahin Haq, Limary M. Cancel, induced in individual bone cells, muscle cells and white blood cells, both by John M. Tarbell. simulated microgravity on earth as well as microgravity conditions in outer Biomedical Engineering, The City College of New York, New York, space (as in the international space station). Reported changes include struc- NY, USA. tural/morphological abnormalities, changes in gene expression, protein expres- Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a transmembrane sion, metabolic pathways and signaling pathways, suggesting that cells mount protein located at the intercellular junction of endothelial cells (EC). PECAM-1 some response or adjustment to microgravity. However, the implications of in adjacent EC bind to each other through homophilic interactions and regulate such adjustments on many cellular functions and responses are not clear largely endothelial function. Recent studies have identified the role of PECAM-1 in because the primary mechanism of gravity sensing in animal cells is unknown. mechanotransduction, sensing luminal shear stress and coordinating the pro- Here we used a rotary cell culture system developed by NASA, to subject duction of both prostacyclin/prostaglandin I2 (PGI2) and nitric oxide. While leukemic cancer cells to microgravity for 48 hours and then quantified their most of these studies use inhibition methods, such as PECAM-1 knockdown innate immune-response to common anti-cancer drugs. We found that leukemic cells or knockout animals, our group has implemented novel methods to cancer cells treated with daunorubicin show increased chemotactic migration selectively activate PECAM-1 on the EC surface for 10-30 minutes. Using (p < 0.01) following simulated microgravity (mg) compared to normal gravity AFM pulling with PECAM-1 antibody coated cantilevers on rat fat pad endo- on earth (g). However, cells treated with doxorubicin showed enhanced migra- thelial cell (RFPEC) monolayers, we have found significant activation of tion both in g and following mg. Our results show that microgravity modulates cyclooxygenase-2 (COX-2), the mediating enzyme of prostacyclin (PGI2) pro- cancer cell response to chemotherapy in a drug-dependent manner. This sug- duction. Conversely, AFM pulling with PECAM-1 coated probes did not gests using simulated microgravity as an immunomodulatory tool for the devel- induce any significant change in NO production. These data suggest that fluid opment of new immunotherapies for both space and terrestrial medicine. shear stress acts directly on PECAM-1 to induce PGI2 production but indirectly to induce NO production. We have shown that pulling with probes targeting the 1532-Plat heparan sulfate proteoglycan glypican-1 initiates NO production, suggesting TREK-2 Has an Asymmetrical Response to Force Changes in the Membrane that glypican-1 is upstream of PECAM-1 in the mechanotransduction cascade Michael Voldsgaard Clausen1, Viwan Jarerattanachat2, Jackie Ang2, that produces NO. We are currently investigating the potential interaction be- Liz Carpenter2, Stephen Tucker2. tween glypican-1 and PECAM-1 in shear-induced NO production. Heparin and 1Aarhus University, Aarhus C, Denmark, 2University of Oxford, Oxford, surfen were used to inhibit extracellular heparan sulfate-to-PECAM-1 binding United Kingdom. and shear-induced NO production was studied at both 10 and 30 minutes. The Perception of force is a key component in our sensation of touch, hearing and inhibition of PECAM-1 expression and intermediate intracellular signaling pain as well as in regulation of blood and osmotic pressures, but unlike the well- pathways may further illuminate the role of PECAM-1 in glycocalyx- described nature of taste and odorant receptors and the photoreceptors in the dependent shear-induced NO production. This work aims to identify critical eye, we have not yet a clear idea of how this sense operate at the molecular mechanosensing structures on the surface of endothelial cells that mediate level. Fundamental to the concept is that force must be translated into electrical

BPJ 7813_7824 312a Tuesday, February 14, 2017 conductance, that is perceived by the nervous system, and we believe this trans- 1535-Plat lation occur through the action of mechanosensitive ion channels. Recently it A Novel DNA Binding Mode of H-NS Drives Chromosome Compaction has been demonstrated that eukaryotic mechanosensitive channels, just like and Gene Silencing in Single Bacterial Cells their prokaryotic counterparts, directly sense force changes in the lipid bilayers, Linda J. Kenney. so now the important question is: how does membrane modulation couple to Mechanobiology Institute, National University of Singapore, Singapore, channel activity? To address this question we use purified mechanosensitive Singapore. TREK-2 channels, which we reconstitute into planar lipid patches at a concen- Nucleoid-associated proteins (NAPS) facilitate chromosome organization in tration that allow us to study channel activity at the single molecule level. We bacteria, but the precise mechanism remains elusive. H-NS is a NAP that find that the reconstituted channels have a random orientation, and that we can also plays a major role in silencing pathogen genes acquired by horizontal clearly distinguish between the two possible orientations by analyzing electro- gene transfer. We used genetics, single-particle tracking, super-resolution physiological characteristics as conductance and open dwell times. To investi- fluorescence microscopy, atomic force microscopy and molecular dynamics gate the sensitivity of the channels to mechanical modulation, we apply simulations to examine H-NS/DNA interactions in single bacterial cells. We different degrees of negative or positive pressure to the patches, and analyze discovered a role for the unstructured linker region connecting the N-terminal how such stimulations affect the activity of differently oriented channels. oligomerization domain and the C-terminal DNA binding domain. Amino With this approach we can demonstrate that mechanical activation of TREK- acids in the linker stabilize initial H-NS/DNA binding, facilitating polymeri- 2 depends on the direction of the pressure pulse, i.e. one orientation is activated zation of H-NS along DNA. In the absence of linker contacts, single particle only by negative pressure while the other orientation is activated only by pos- tracking experiments indicated that H-NS binding was significantly reduced itive pressure. We carefully suggest that these results demonstrate that TREK-2 and in PALM images, the chromosome was de-condensed. In contrast to pre- channels are sensitive to the direction of membrane curvature. Furthermore we vious reports, H-NS was not localized to two distinct foci, rather it was scat- are in the process of deducing a kinetic model that describes TREK-2 activation tered all around the nucleoid. Amino acids in the linker make DNA contacts by pressure. that are required for both gene silencing and chromosome compaction, linking these two important functions. Supported by NIH-R21123640, VAIBX- Platform: Optical Microscopy and Super- 000372 and an RCE in Mechanobiology, NUS from the Ministry of Educa- tion, Singapore. Resolution Imaging: Applications to Cellular 1536-Plat Molecules Investigating RNAP Search Dynamics in Live E. Coli Cells using Single Molecule and Statistical Methods 1533-Plat Kelsey Bettridge1, Chris Bohrer1,2, Jie Xiao1. Illuminating Bacterial Electrophysiology 1Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Giancarlo N. Bruni, Benjamin Dodd, Joel Kralj. Baltimore, MD, USA, 2Jenkins Department of Biophysics, Johns Hopkins Biofrontiers Institute, MCDB, University of Colorado Boulder, Boulder, CO, University, Baltimore, MD, USA. USA. Transcription is an essential component of gene regulation and one of the The revelation that bacteria undergo rapid voltage transients sparked a po- most highly regulated processes in the cell. Because bacteria lack a nuclear tential union between the fields of microbiology and electrophysiology. envelope, genes transcribed by RNA polymerase (RNAP), the key enzyme Voltage transients in biology are canonically studied in neuronal and car- of transcription, are essentially immediately translated into protein, leaving diac contexts, where a voltage depolarization induces calcium fluctuations. the kinetics of transcription significantly influencing the resulting protein Using genetically encoded voltage and calcium indicators, we sought to un- expression timing and levels. However, while the kinetics of transcription is cover a physiological role for voltage depolarizations in E. coli. We found greatly studied in vitro, the in vivo dynamics of RNAP, including the typical bacteria are similar to electrically excitable eukaryotic cells; voltage depo- search time and transcription time, are difficult to obtain. Our lab takes advan- larization induces calcium influx, hinting at a potential signaling mecha- tage of the sensitivity and fast time scale of single-molecule tracking to deter- nism. Surprisingly, we found that changing the mechanical environment mine the kinetics of RNAP binding and transcription in live E. coli cells. We can trigger calcium influx and also alters gene expression. We hypothesize used a strain of E. coli in which the b’ subunit (rpoC) of RNAP is tagged with that, similar to sensory neurons, E. coli mechanosensation is mediated by a a photoactivatable fluorescent protein, PAmCherry, replacing the endogenous voltage-gated calcium channel. Further, we hypothesize that the calcium chromosomal copy. We discovered there are three states of RNAP with dynamics we observe mediate the correlated gene expression changes, distinct diffusion coefficients. Using genetic manipulations and drug treat- akin to eukaryotic cells. Understanding how bacteria utilize voltage fluctu- ments, we were able to assign each diffusion state to a particular function ations to enact physiological change could lead to the development of novel of RNAP, i.e. RNAP that is bound to the DNA, RNAP that is undergoing antibiotics, and potentially give clues as to the origins of voltage as a signal rapid association and dissociation from the nucleoid, and freely diffusing in biology. RNAP. Further, using a Markov chain Monte Carlo algorithm, we were able to form a hidden Markov model of our data to parse out the dynamics 1534-Plat of RNAP switching states, which enabled us to parse out a complete dynam- Understanding the Asymmetric MipZ Gradient in Caulobacter Crescentus ical model of RNAP in living cells. We obtainined a wide variety of important Matthew D. Stilwell, Nikolai P. Radzinski, James C. Weisshaar, parameters, including the non-specific dwell time, the typical promoter search Douglas B. Weibel. time, and the typical transcription time for an average gene. These parameters University of Wisconsin-Madison, Madison, WI, USA. will be useful for future gene regulations studies of all kinds, as well as to help Caulobacter crescentus is a freshwater a-proteobacterium that divides into understand general protein-nucleic acid interactions and dynamics within the two asymmetric daughter cells. These curved, rod-shaped cells differ in their crowded cytoplasm of a live cell. chromosome replication fates and in their cell length at division. C. crescentus divides into a replication-competent ‘stalked’ cell and a smaller, replication- 1537-Plat quiescent ‘swarmer’ cell. The site of cell division is dictated by the polymeri- Collective Live-Cell Superresolved Traces Reveal Nonaxonomal Dynamics zation of the eukaryotic tubulin homologue, FtsZ. MipZ is an essential ATPase of Intraflagellar Transport Particles at the Ciliary Base and inhibitor of FtsZ polymerization. MipZ forms an intracellular bipolar Tony Yang, Nguyet Thi, Minh Tran, Weng Man Chong, Jung-Chi Liao. gradient that directs FtsZ to the lowest concentration of MipZ, which is found Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, slightly towards the new pole as opposed to the exact mid-cell. This localiza- Taiwan. tion bias leads to two differently sized daughter cells. To better understand The primary cilium is an essential organelle responsible for multiple sensory how MipZ directs FtsZ to this off-center location, we employed single- and signaling activities. Ciliogenesis is achieved by delivery of precursors particle tracking photoactivated localization microscopy (sptPALM) to charac- such as tubulins along the axoneme through intraflagellar transport (IFT), terize the diffusive states and transition state kinetics of MipZ. We then used which is mediated by molecular motors and various IFT particles. Distal ap- deterministic and stochastic simulations with these biophysical parameters to pendages (DAPs) are known to serve as the recruiting site of IFT proteins. recreate the asymmetric MipZ gradient in silico. These simulations produce During ciliogenesis, IFT proteins must go through several different zones a distribution of cell lengths after cell division similar to that observed in cilia. One of missing links of the IFT dynamics is how IFT particles in vivo. These studies provide more insight into the asymmetric division of move between the DAPs and the ciliary axoneme. The major obstacle comes C. crescentus, but further questions remain regarding the nature of the asym- from the tiny volume surrounding the DAPs and TZ and the high density of metric division. IFT particles in this region, which is far smaller than the diffraction-limited

BPJ 7813_7824 Tuesday, February 14, 2017 313a spot. Here we performed live-cell sptPALM-based superresolution tracking nanoscale spatial arrangement of these processes was then assessed by of short trajectories to demonstrate IFT particle dynamics at the ciliary dual colour Stimulated Emission Depletion (STED) microscopy. More spe- base with the optimization of particle density and trajectory duration suitable cifically, we used a novel approach recently introduced by our group (Sep- for IFT motion speed. Our results revealed the DAPs and TZ accommodate aration of Photons by Lifetime Tuning, SPLIT)1 to achieve the nanoscale not only axonemal but also transverse IFT88 movement. IFT particles move resolution required to image subnuclear structures. In this approach, the slower at the base than in the ciliary compartment. Moreover, diffusion anal- use of the phasor plot to represent the emission dynamics of both fluoro- ysis revealed that IFT particle movement was confined at the distal TZ while phores allows for the proper spatial tuning of each signal dynamics as a superdiffusive at the proximal TZ. This heterogeneous diffusion characteris- function of their respective positions within the detection volume. In this tics was likely attributed to a complex organization at the DAPs, spatially way, we are able to increase the spatial resolution of two fluorophore signals partitioned into some obstructed regions and some unhindered areas. simultaneously beyond the diffraction limit, ultimately leading to the proper Together, our live-cell superresolution studies revealed that IFT proteins spatial mapping of different structures within the cell nucleus at the adopt location-dependent stochastic paths in different regions of the ciliary nanoscale. base, with newly reported dynamic characteristics of IFT particles to shed [1] Lanzano`L.et al. (2015) Nat. Commun. 6: 6701-9 light on the mechanisms of IFT particle traffic and gating facilitating [Work partly funded by AIRC-Cariplo TRIDEO #17215] ciliogenesis. 1540-Plat 1538-Plat Time-Resolved Single Cell, Sub-Cellular Compartmentalized Proteomics, Real-Time Subcellular Localization Reveals Hidden Intraflagellar Trans- Combining Precise Microfluidics, Deconvolution and Ultrasensitive Single- port Mechanisms Molecule Microscopy Anthony Kovacs1, Jonathan Kessler1, Je-Luen Li2, HuaWen Lin3, Adam J.M. Wollman1, Sviatlana Shashkova2, Niek Welkenhuysen3, Susan Dutcher1, Yan Mei Wang4,5. Erik G. Hedlund1, Stefan Hohmann3, Mark C. Leake1. 1Physics, Washington University in St. Louis, St. Louis, MO, USA, 2D. E. 1Physics and Biology, University of York, York, United Kingdom, Shaw Research, New York, NY, USA, 3Genetics, Washington University in 2Department of Chemistry & Molecular Biology, University of Gothenburg, St. Louis, St. Louis, MO, USA, 4Physics, Princeton University, Princeton, NJ, Gothenburg, Sweden, 3Department of Biology and Biological Engineering, USA, 5Physics, Washington University In St. Louis, St. Louis, NJ, USA. Chalmers University of Technology, Gothenburg, Sweden. Subcellular localization, a process that determines the location of a protein at a Optical microscopy is emerging as a powerful tool in quantified in vivo pro- specific time during its trajectory in cell, is essential for understanding protein teomics, allowing protein copy number determination in live cells and even function and mechanisms. Current localization techniques such as CLEM sub-cellular compartments, but to date without dynamic information. Here, (correlative light-electron microscopy) and colocalization measurements, how- we have combined a novel microfluidics technology, Fluicell(1) with cell ever, compromise on accuracy and/or precision due to their limited temporal tracking and deconvolution software(2), to create a new high-throughput dy- and/or spatial resolutions. CLEM does not localize proteins at the time of im- namic proteomics platform, capable of quantifying copy number changes in aging; rather a delayed time of at least seconds due to cell fixation. During this sub-cellular compartments in live cells in response to extracellular micro- time, the proteins could have relocated. Colocalization measurements have a environmental changes. We used a canonical signal transduction pathway in spatial resolution of at best 10 nm, rendering adjacent organelles/structures eukaryotic budding yeast cells as a test system to observe the localisation not differentiable. changes of the Mig1 transcription factor between nucleus and cytoplasm in Here we report a real time subcellular localization method (Reticello) by us- response to extra-cellular glucose concentration changes. Combined with ing single-particle tracking measurements that can differentiate interfacing or- single-molecule Slimfield measurements of the dynamics of individual ganelles/structures in live cells with millisecond resolution. The underlying Mig1 molecular clusters, we find that clusters of Mig1 molecules translocate principle of this method is as follows: on or in different organelles/structures across the nuclear envelope and bind target genes, enabling a tuneable in live cells, a protein is confined to different geometrical spaces or surfaces response to the glucose concentration signal. By combining microfluidics con- and therefore exhibit different movement patterns that can be predicted and trol of the input signal, with molecular quantification of the output on a single tested. We demonstrate the Reticello method by revealing transient and un- cell level, this new platform generates novel insight into the precise mecha- known BBSome and kinesin-2 motor localizations in the 250 nm-wide nisms of cellular processes. C. reinhardtii flagella, differentiating among four interfacing structures. At 1. A. Ainla, G. D. Jeffries, R. Brune, O. Orwar, A. Jesorka, A multifunctional the flagellar tip, these proteins reorganize by free diffusion for a mean of pipette. Lab Chip. 12, 1255-1261 (2012). 2.2 sec on the membrane and 1.3 sec on the microtubules, respectively. After 2. A. J. M. Wollman, M. C. Leake, Millisecond single-molecule localization the reorganization, kinesin-2 motors perform retrograde travel by diffusion in microscopy combined with convolution analysis and automated image segmen- the flagellar matrix. tation to determine protein concentrations in complexly structured, functional cells, one cell at a time. Faraday Discuss.184, 401-24 (2015). 1539-Plat SpatialOrganizationof Nuclear Structuresby Dual Colour Super-Resolution Platform: Protein-Nucleic Acid Interactions II Microscopy Maria J. Sarmento1, Lorenzo Scipioni1,2, Melody Di Bona1,3, 1541-Plat Mario Faretta4, Laura Furia4, Gaetano I. Dellino4, Pier G. Pelicci4, Genomic RNA Binding Promotes Retroviral Gag Protein Interactions Paolo Bianchini1,5, Alberto Diaspro1,3, Luca Lanzano`1. in an Assembly-Competent Conformation Leading to Selective Genome 1Nanoscopy, Istituto Italiano di Tecnologia, Genoa, Italy, 2Department of Packaging Informatics, Bioengineering, Robotics and Systems Engineering, University Ioulia Rouzina1, Shuohui Liu1, Erik D. Olson1, Tiffiny Rye-McCurdy1, of Genoa, Genoa, Italy, 3Department of Physics, University of Genoa, Genoa, Christiana Binkley1, Joshua-Paolo Reyes1, Leslie J. Parent2, Italy, 4Department of Experimental Oncology, European Institute of Karin Musier-Forsyth1. Oncology, Milan, Italy, 5Nikon Imaging Center, Istituto Italiano di 1OSU, Columbus, OH, USA, 2Penn State College of Medicine, Hershey, Tecnologia, Genoa, Italy. PA, USA. The overall function of DNA in cell nuclei has long been related with In HIV-1 infected cells, full-length viral genomic RNA (gRNA) is selectively different levels of chromatin organization. In fact, besides its primary packaged by the HIV-1 Gag protein despite a vast excess of spliced viral and sequence, proper chromatin structure and dynamics are required for healthy host RNAs. The mechanism of this selective packaging is incompletely under- cell proliferation and maintenance. Errors in vital nuclear functions such as stood but a region of gRNA known as ‘‘Psi’’ mediates specific Gag interaction. DNA replication and transcription, and the consequent DNA damage, have HIV-1 Gag binds Psi and non-Psi RNA with similar affinity under physiolog- frequently been identified as the source of genomic instability responsible ical salt concentration (~150 mM NaCl), but the salt dependence of these two for numerous diseases, e.g., cancer development. Recently, new in vitro ap- binding events differs dramatically (Webb, JA, et al, RNA 2013). HIV-1 Gag proaches have been developed that allow the genome mapping of these nu- binds Psi RNA with a strong non-electrostatic binding component and a small clear processes and the identification of substantial differences between effective charge (Zeff ~ 5). In contrast, HIV-1 Gag binds non-Psi RNA with a health and disease. However, all genomic processes have also an underlying very weak non-electrostatic binding component and a larger effective charge spatio-temporal organization within the cell nucleus that goes well beyond (Zeff ~ 9). In this work, we use salt-titration binding assays to study the effect the pure linear sequence. of various Gag and Psi RNA mutations on the Psi and non-Psi binding interac- Here, our goal was to detect and visualize different nuclear structures tions of HIV-1 and Rous sarcoma virus Gag proteins. Our findings are consis- directly within the nuclei of intact cells. Upon immunostaining, the tent with a model in which Gag binding to cognate Psi RNA shifts the

BPJ 7813_7824 314a Tuesday, February 14, 2017 equilibrium from a non-assembling to an assembling Gag state, providing an binding kinetics were monitored in live U2OS cells by fast relaxation imaging advantage for nucleation of assembly only on gRNA. (FreI), which combines a temperature jump with fluorescence microscopy of the FRET (Fluorescence resonance energy transfer)-labeled RNA and protein. 1542-Plat U1A protein alone was found to diffuse across the cell, whereas SL2 RNA and Kinetics of dCas9 Target Search in Escherichia Coli the U1A-SL2 complex localized in the nucleus. This demonstrates that SL2 Daniel Jones, Cecilia Unoson, Prune Leroy, Vladimir Curic, Johan Elf. RNA mediates transport of the U1A protein to the nucleus. The binding affinity Cell and Molecular Biology, Uppsala University, Uppsala, Sweden. in live cells was reduced compared to in vitro. The dissociation rate was un- How fast can a cell locate a specific chromosomal DNA sequence specified by a changed in cells; however, the association rate was an order of magnitude single stranded oligonucleotide? To address this question we study the lower, resulting in a two order of magnitude decrease in the dissociation con- CRISPR-associated protein Cas9 which can be programmed by guide RNAs stant. Introduction of a macromolecular crowder, Ficoll 70, in vitro further sta- to bind essentially any DNA sequence. This targeting flexibility requires bilized the complex. This suggests that differences between binding affinities as Cas9 to unwind the DNA double helix to test for correct base pairing to the measured in vitro and in live cells cannot be explained by crowding alone. guide RNA. Here we study the search mechanisms of the catalytically inactive Instead, high binding affinities as measured in vitro may be necessary for selec- dCas9 in living Escherichia coli by combining single molecule fluorescence tivity in vivo, where competition exists between multiple binding partners. microscopy and bulk restriction protection assays. We find that it takes a single dCas9 ~100 h to find and bind a specific target, in stark contrast to transcription 1546-Plat factors such as LacI, which takes 5 minutes to locate its target. Thus, the price Visualizing Infection Initiation of Bacteriophage P22 by Cryo-Electron dCas9 pays for flexibility in targeting is time. We further identify a likely role Tomography for short-range (20-40) 1D sliding along DNA in dCas9 target search. The Chunyan Wang1, Jiagang Tu1,BoHu1, Ian Molineux2, Jun Liu1. physical limitations for Cas9 likely generalize to all systems that are pro- 1The University of Texas Medical School at Houston, Houston, TX, USA, grammed by single stranded oligonucleotides to locate sequences in dsDNA, 2The University of Texas at Austin, Austin, TX, USA. such as the homologous repair machinery. For successful infection, bacteriophages must overcome multiple barriers in the bacterial cell envelope to translocate viral DNA and proteins into the host cell. 1543-Plat However, the molecular mechanisms underlying the phage infection initiation Argonaute Target Search is Facilitated by Long Distance Diffusion remain poorly understood. Here, we use cryo-electron tomography of Salmo- Tao Ju Cui1, Stanley D Chandradoss1, Jorrit Hegge2, John van der Oost2, nella cells infected by phage P22 to capture intermediates during infection. Chirlmin Joo1. P22 particle initially attaches to the cell surface obliquely through interactions 1Delft University of Technology, Delft, Netherlands, 2Wageningen between phage tail spikes and bacterial lipopolysaccharides. Subsequently the University, Wageningen, Netherlands. phage needle penetrates the host membrane. Three ejection proteins (gp7, gp16, Argonaute proteins have a variety of functions ranging from post transcrip- gp20), which are originally stored in capsid, are ejected and assembled to create tional gene silencing in eukaryotes to host defence in prokaryotic systems. a 40 nm long trans-envelope channel between the phage tail and the cyto- Despite the well-established functional understanding of Argonautes, the bio- plasmic membrane. The novel channel serves as the conduit for genome trans- physical understanding of its targeting mechanism remains limited. How is it location. Using mutant particles lacking one or more of the ejection proteins, able to find a specific nucleic acid sequence among numerous decoys? Here our in situ structural analysis demonstrates that gp7 forms an extra-cellular we make use of the high spatiotemporal resolution of single-molecule FRET channel, extending from the phage tail to the outer membrane; gp20 forms a to elucidate the target search mechanism of a mesophilic bacterial Argonaute. mushroom-like structure spanning the periplasm, while gp16 is involved in We find that it makes use of lateral diffusion for rapid sampling of adjacent se- channel formation in the cytoplasmic membrane. Together with atomic resolu- quences while using three-dimensional diffusion in order to cover larger dis- tion information from mature particles and their proteins, our studies reveal a tances efficiently. Similar results have been obtained for thermophilic series of key intermediate infection structures at unprecedented detail. They bacterial Argonaute. The long-distance facilitated diffusion mechanism might also unveil a massive remodeling of both the infecting phage and the bacterial be a conserved feature across different Argonaute families and may also be a cell envelope. The structures we obtain provide the first direct evidence illumi- general feature for other nucleic-acid guide directed nucleoproteins such as nating the functions of the three ejection proteins and their distinct role in phage CRISPR systems. genome delivery. 1544-Plat How Conformational Dynamics Influences the Protein Search for Targets 1547-Plat on DNA Using Site Specific Fluorescent Probes to Examine Replication Fork Maria P. Kochugaeva, Alexey A. Shvets, Anatoly B. Kolomeisky. Destabilization by Regulatory Proteins of the Bacteriophage T4 DNA Chemistry, Rice University, Houston, TX, USA. Replication Complex Protein search and association to specific target sequences on DNA is essential Davis Jose1, Miya Mary Michael2, Wonbae Lee3, Thomas H. Steinberg2, Andrew H. Marcus3, Peter H. von Hippel1. for all fundamental biological processes. The detailed qualitative and quantita- 1 tive mechanism of fast target finding is still unknown in spite of numerous Institute of Molecular Biology and Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR, USA, 2Institute of experimental and theoretical efforts. Particularly, the role of protein conforma- 3 tional fluctuations in the search dynamics remains uncovered. We extend devel- Molecular Biology, University of Oregon, Eugene, OR, USA, Center for oped earlier theoretical method to analyze how the conformational dynamic Optical, Molecular and Quantum Science and Department of Chemistry and affects the process of finding the specific targets on DNA. Our approach is Biochemistry, University of Oregon, Eugene, OR, USA. based on discrete-state stochastic model that takes into account main The reconstituted T4 DNA replication complex serves as the simplest model physical-chemical processes. This allows us to evaluate explicitly the protein system to examine the ‘core’ replication mechanisms of higher organisms. In search for the targets on DNA at different conditions. Our calculations show this study we used site-specifically introduced fluorescent base analogues to that conformational fluctuations might strongly affect the protein search dy- track local conformational changes of the nucleic acid bases, as well as Cy3/ namics. We discuss the contribution of the shift in the conformational equilib- Cy5 dye-pairs inserted into the sugar-phosphate backbones to monitor back- rium in the target search kinetics. We utilized extensive Monte Carlo computer bone motions at defined positions within the DNA during interactions of the simulations to validate our theoretical predictions. fork with regulatory proteins. By combining low energy circular dichroism (CD) and fluorescent measurements of the site-specifically introduced fluores- 1545-Plat cent base analogues with single molecule (sm) FRET experiments with cyanine Spliceosomal U1A Protein-SL2 RNA Binding Affinity Decreases in Cells dyes, we have monitored the binding stoichiometries and interactions of the Caitlin Davis1, Irisbel Guzman2, David Gnutt3, Martin Gruebele1. regulatory proteins with model replication fork constructs. The assembly path- 1Physics, University of Illinois, Urbana, IL, USA, 2Biochemistry, University ways and binding stoichiometries of gp59 (the helicase loader protein), gp41 of Illinois, Urbana, IL, USA, 3University of Illinois, Urbana, IL, USA. (the hexameric helicase) and gp61 (the primase) were studied using functional While extensive biochemical and biophysical studies have been carried out to assays as well as single molecule imaging experiments. The results showed that elucidate protein-RNA binding mechanisms and dynamics in vitro, most of the stoichiometry of gp59/gp41/gp61 subunits within the functional complex is these studies do not take into consideration the effect of the cellular environ- 1:6:1. CD and fluorescence experiments with base analogue probes show that ment. Here we have experimentally tested the role of the cellular environment gp59 preferentially binds to and perturbs the bases at the junction of a forked on protein transport and binding affinity in one of the most widely studied RNA DNA construct, and that the addition of the gp41 helicase hexamer extends recognition motifs, the spliceosomal protein U1A, and its binding partner, stem this conformational perturbation deep into the DNA duplex. In contrast, the loop 2 (SL2) of the U1 small nuclear RNA. U1A-SL2 localization, stability and addition of gp61 perturbs the bases at the fork junction, but not base-pairs

BPJ 7813_7824 Tuesday, February 14, 2017 315a deep in the duplex sequence. Based on these results and our previous studies we 1550-Plat propose a possible model for DNA duplex unwinding by the helicase loader- Simulations and Experiments Provide a Convergent View of Protein primosome-single-strand binding protein complex within the T4 replication Unfolded States under Folding Conditions system. Alex S. Holehouse1, Ivan Perana2, Isaac S. Carrico2, Osman Bilsel3, Daniel P. Raleigh2, Rohit V. Pappu1. 1548-Plat 1Center for Biological Systems Engineering, Washington University in Saint Mechanism of Duplex DNA Destabilization by RNA-Guided Cas9 Nuclease Louis, St. Louis, MO, USA, 2Department of Chemistry, Stony Brook during DNA Interrogation University, Stony Brook, NY, USA, 3Department of Biochemistry and Vladimir Mekler1, Leonid Minakhin1,2, Konstantin Severinov1,3. 1 2 Molecular Pharmacology, University of Massachusetts Medical School, Waksman Institute, Rutgers University, Piscataway, NJ, USA, Peter the Worcester, MA, USA. Great State Polytechnical Institute, St. Petersburg, Russian Federation, 3 An accurate description of unfolded states under folding conditions is important Institutes of Molecular Genetics and Gene Biology, Russian Academy of for quantitative studies of protein stability, protein folding kinetics, and an un- Sciences, Moscow, Russian Federation. derstanding of how features of the unfolded state may have contributed to the The Cas9 endonuclease cleaves double-stranded DNA bearing sequences com- convergent evolution toward foldable sequences. Here, we present results from plementary to a 20-nucleotide segment in the guide RNA (gRNA), which are multi-pronged biophysical investigations that uncover a comprehensive flanked by a conserved PAM sequence. The Cas9-gRNA system has been suc- description of conformational ensembles accessed by unfolded states of pro- cessfully used for genome editing in various organisms. The Cas9-gRNA teins under folding conditions. We combined results from experiments based effector complex locates the target site by scanning and interrogating the on time-resolved Fo¨rster resonance energy transfer, using multiple non- genomic DNA. Unpairing of a short PAM-proximal segment of DNA is a crit- perturbing, amino-acid-sized dye pairs, and time-resolved small angle X-ray ical event in DNA interrogation that may determine the overall rate of target scattering, with novel molecular simulations and polymer physics theories. Pre- location. We sought to elucidate mechanism of initial DNA interrogation steps vious work showed that aqueous milieus are poor solvents for polypeptide that precede the pairing of target DNA with gRNA. Using fluorometric and backbones. This should drive chain collapse and globule formation. Our current biochemical assays, we studied Cas9-gRNA complexes with model DNA sub- results, obtained for the N-terminal domain of the ribosomal protein L9, show strates likely mimicking early intermediates on the pathway to the final Cas9- that under folding conditions, native and non-native intra-chain interactions gRNA-DNA R-loop complex. The results show that Cas9-gRNA binding to lead to transient globule formation, while large conformational fluctuations PAM favors separation of few nearest to PAM base pairs but causes no signif- brought about by favorable sidechain-solvent interactions lead to an unfolded icant effect on DNA duplex stability in the protospacer region distant from ensemble that behaves statistically like a flexible polymer would in a theta- PAM. The destabilization of DNA duplex is achieved primarily through inter- solvent. Therefore, the unfolded state under folding conditions may be actions of Cas9-gRNA with non-target DNA strand nucleotides and hindering described as an ensemble with fluctuating elements of native and non-native of duplex DNA entry into the Cas9-gRNA interior. The data also suggest that non-random elements of structure that are averaged over to yield random interaction of the target DNA strand with the ‘‘phosphate lock’’ loop of Cas9 is coil-like statistics of chains in theta solvents. This behavior derives from subsequent to the initial destabilization step and is coupled with the DNA- the sequence-encoded interplay between backbone- and sidechain-mediated gRNA pairing. intra-chain and chain-solvent interactions. Our results have broad implications for interactions of unfolded proteins in crowded milieus, for folding-unfolding Platform: Intrinsically Disordered Proteins (IDP) transitions, and for tilting the balance toward heterogeneous ensembles as is the and Aggregates I case with intrinsically disordered proteins.

1549-Plat 1551-Plat Resolving the Controversy between SAXS and FRET Measurements on Biophysical Studies of the Interaction between Optimized Peptides and Unfolded Proteins Amyloid-Beta Elucidate a Completely Novel Binding Mode 1 2 1 1 1 2 3 Tamar Ziehm , Antonia Klein , Janine Kutzsche , Dieter Willbold . Wenwei Zheng , Alessandro Borgia , Alexander Grishaev , 1 2 Benjamin Schuler2, Robert B. Best1. ICS-6, Forschungszentrum Juelich, Juelich, Germany, Department of 1Laboratory of Chemical Physics, NIDDK, NIH, Bethesda, MD, USA, Ecosystem & Public Health, University of Calgary, Calgary, AB, Canada. 2Department of Biochemistry, University of Zurich, Winterthurerstrasse, Amyloid-beta (Ab) is an intrinsically disordered peptide, which is formed Switzerland, 3National Institute of Standards and Technology and the throughout our lifetime. Its aggregation triggers the onset and progression of Institute for Bioscience and Biotechnology Research, Rockville, MD, USA. Alzheimer’s disease. Previously, we selected the Ab-targeting peptide D3 as Chemical denaturants are the most commonly used agents for unfolding pro- a lead compound for a causative and disease modifying treatment of Alz- teins. An expansion of unfolded chains with increasing denaturant concen- heimer’s disease. This lead compound was systematically optimized using pep- tration is expected due to improved solvation. However, experiments have tide microarrays resulting in novel compounds with improved affinity. Hence, yielded qualitatively different results. Studies using Fo¨rster resonance en- the efficacy to stabilize non-toxic Ab monomers and eliminate toxic Ab olig- ergy transfer (FRET) and other methods found an increase in radius of gy- omers is highly enhanced in vitro. In the current approach, the interactions between Ab and the novel compounds ration (Rg) with denaturant concentration, but most small-angle X-ray scattering (SAXS) studies found no change. This discrepancy challenges were studied in detail using methods like surface plasmon resonance (SPR), our understanding of denaturation mechanism and the accuracy of these ex- isothermal titrationcalorimetry (ITC) and biolayer interferometry (BLI).Thereby, periments on intrinsically disordered proteins. We use all-atom molecular the binding kinetics, affinities, stoichiometries, and epitopes were investigated. simulations to investigate the effect of urea and guanidinium chloride on The current study elucidated that some of the novel compounds have gained an the intrinsically disordered protein ACTR, which can be studied by both entropy-driven hydrophobic binding mode in comparison to the entirely FRET and SAXS over a wide range of denaturant concentration. Using a enthalpy-driven electrostatic interaction of the lead compound, resulting in a carefully calibrated denaturant model, we find that the chain expands with highly robust complex formation with Ab. As a consequence, the already increasing denaturant concentration. This is due to the favorable association demonstrated therapeutic potential of the lead candidate in transgenic animal of urea or guanidinium chloride with the backbone of all residues and with models might be further enhanced for the new compounds. We will present the side-chains of most residues. The denaturant-water transfer free energies new data on the efficacy of the most promising drug candidates, which are inferred from this association are in reasonable agreement with experiment. perfectly suited for further in vivo investigations with regard to the develop- By computing FRET efficiencies and SAXS intensities, we showed that the ment towards a curative therapy for Alzheimer’s disease. simulations are in accord with both experiments on this protein, demon- 1552-Plat strating that there is no fundamental inconsistency between the two experi- Single Molecule FRET Investigation of the Dimensions and Dynamics in mental methods. In addition, our simulations suggest that some potential Highly Cooperative Sic1-WD40 Binding artefacts are unlikely, i.e. the commonly used FRET chromophores do not Gregory Gomes1, Veronika Csizmok2, JIanhui Song3, Hue-Sun Chan3, qualitatively alter the results and that SAXS data are not distorted by pref- Julie Forman-Kay2, Claudiu C. Gradinaru1. erential solvent partitioning into the interior of the chain. A careful analysis 1Physics, University of Toronto, Toronto, ON, Canada, 2Molecular Structure of the experimental data suggests that the discrepancy is largely related to and Function, Hospital for Sick Children, Toronto, ON, Canada, the challenging inverse problem of inferring Rg from ensemble-averaged 3Biochemistry, University of Toronto, Toronto, ON, Canada. data. We show how this can be overcome by fitting the data to explicit mo- Sic1 is a cyclin-dependent kinase inhibitor which must be phosphorylated on at lecular ensembles. least six sites (termed Cdc4 phosphodegrons, CPDs) to allow its recognition by

BPJ 7813_7824 316a Tuesday, February 14, 2017 the WD40 binding domain of Cdc4. The highly-cooperative switch-like depen- We synthesized and characterized three TOAC-peptides (TOAC1 -3) and dence on the number of phosphorylated sites on Sic1 cannot be accounted for one reference peptide (EZ). We show that the EPR rotation correlation times by traditional thermodynamic models of cooperativity. Further experimental differ depending on TOAC position. Only one of the TOAC variants (peptide attention is necessary to determine the physicochemical/mechanistic basis of TOAC2) showed aggregation by CD signatures similar to[1], same as the non- its highly cooperative binding. TOAC containing EZ peptide. Changes in room temperature EPR spectra sug- We used single molecule fluorescence techniques to study the dimensions and gestive of amyloid aggregation were found in TOAC2, but not in the other dynamics of Sic1’s N-terminal targeting region (residues 1-90, henceforth TOAC variants. Sic1), phosphorylated Sic1 (pSic1), and the pSic1-WD40 dynamic complex. Peptide sequences: Previous single molecule Frster Resonance Energy Transfer (smFRET) mea- Peptides: TOAC1: KVKVT*GDVIEV; TOAC2: T*KVKVLGDVIEVG; surements [Liu, 2014] observed end-to-end reconfiguration on timescales TOAC3: KVKVLGDVIEVT*G; EZ: KVKVLGDVIEVGG, where T* stands larger than ~1ms; resulting in FRET histograms with multiple conformational for TOAC. sub-ensembles. Sic1, pSic1, and the pSic1-WD40 complex are examined using Results smFRET to study the dynamics and dimensions of the various sub-ensembles. TOAC2 and EZ acquire b sheet character under aggregation conditions In a refinement to the conventional approaches for inferring dimensions from (in H2O, shaking at 1000 rpm at 310 K). Rotation correlation times (tr)by smFRET experiments, we use distance distributions from Monte Carlo simula- continuous wave EPR at 9 GHz are: TOAC1: 0.398 5 0.018 ns; TOAC2: tions which extensively sample coarse-grained protein conformations. The 0.122 5 0.005 ns; TOAC3: 0.316 5 0.028 ns. For TOAC2 tr increases to application of polymer physics theory/simulation towards smFRET data inter- 0.162 5 0.007 ns upon aggregation. pretation, and towards IDP binding, contributes to the growing toolkit for Conclusions: understanding the diverse behaviours of IDPs. We propose that for rigid spin labels such as TOAC, the spin label position and the peptide host are crucial and that the TOAC label may interfere with peptide 1553-Plat properties. The results are essential to design spin-labeled constructs for the Measuring the (Good) Solvent Quality of Water for Disordered Proteins investigation of intrinsically disordered proteins. from a Single SAXS Measurement [1] Laganowsky et al., Science 335 (2012) 1228. Joshua A. Riback1, Micayla A. Bowman2, Adam Zmyslowski3, Catherine R. Knoverek2, John Jumper4, James Hinshaw3, Emily B. Kaye2, 1555-Plat Karl F. Freed4, Patricia L. Clark2, Tobin R. Sosnick3. Examination of the Oligomerization Mechanism of SOD1 In Vitro and in 1Graduate Program in the Biophysical Sciences, University of Chicago, Live Cells Chicago, IL, USA, 2Department of Chemistry and Biochemistry, University Brian C. Mackness, Noah R. Cohen, C. Robert Matthews, Jill A. Zitzewitz, of Notre Dame, Notre Dame, IN, USA, 3Department of Biochemistry and Osman Bilsel. Molecular Biology, University of Chicago, Chicago, IL, USA, 4Department Biochemistry and Molecular Pharmacology, University of Massachusetts of Chemistry, University of Chicago, Chicago, IL, USA. Medical School, Worcester, MA, USA. Significance: Proteins adopt non-native geometries during their synthesis, The dimeric b-barrel protein Cu, Zn superoxide dismutase (SOD1) is an folding, transport, and turnover and a significant fraction of the proteome is optimal model system for investigating the molecular basis for toxicity in intrinsically disordered. The characterization of disordered proteins is chal- ALS. Mutations throughout the b-barrel sequence are causative for ALS lenging in part due to difficulties in the interpretation of techniques including and implicated in the gain-of-function toxicity. The occurrence of mutations SAXS and FRET. We have developed a method to extract the dimensions at dozens of sites suggests a common mechanism of toxicity for SOD1 dis- and solvent quality from a single SAXS measurement. We apply it to intrinsi- ease causing variants. Previous biophysical and structural data on SOD1 cally disordered proteins (IDPs), finding them to be expanded in water, which and its variants have established a fairly detailed understanding of its behaves as a good solvent. folding energy landscape. A key finding is that ALS-variants of SOD1 Abstract: SAXS has been used to measure the properties of denatured proteins lead to enhanced populations of both folded and unfolded monomeric forms and IDPs. Many ensembles are expanded in water, with little dependence on of SOD1. An unresolved question, however, is the mechanism of oligomer- denaturant. These findings appear at odds with FRET studies, suggesting issues ization and toxicity of SOD1 variants. To address this question, we present with data interpretation. We conduct SAXS measurements on IDPs up to 334 an aggregation assay that matches physiological conditions as much as residues using in-line size-exclusion chromatography. The IDPs are expanded possible, avoiding denaturants, fluorinated alcohols and agitation. Our re- and the Flory exponent, describing the solvent quality and a chain’s collapse sults, based on the analysis of the WT protein and several variants, suggest propensity, is n~0.53. This value is between the ideal self-avoiding random that the oligomerization rate is correlated with the population of the walk (n=0.59) and the theta solvent limit (n=½) where intra-chain attraction unfolded monomer. Furthermore, although a secondary nucleation mecha- exactly counterbalances excluded volume terms. Addition of denaturants re- nism has been proposed as a key step in SOD1 aggregation our results, ob- sults in a mild expansion, most noticeable in longer sequences, and an increase tained without agitation, are best fitted with a crystallization-like nucleation in n to 0.59. Simulations generate a molecular form factor applicable to IDPs model without fragmentation. We test the consistency of in vitro and live that can be used to determine the Rg and n from a single measurement. The cell data by performing fluorescence correlation spectroscopy (FCS) mea- methodology is robust (hetero- or homopolymers, energy function, Ramachan- surements on live HEK293 cells expressing EGFP fusion constructs of dran maps). This new analysis procedure and the dependence on chain length, WT and disease causing SOD1 variants. Preliminary quantitative modeling along with recent FRET studies, largely resolves the controversy concerning of the FCS data suggest consistency in the propagation rates between in vi- the dimensions disordered proteins in water. These studies reaffirm that an tro and live cell data. The implication of the unfolded monomer of SOD1 early stable collapse often is not the first step in folding of single domain pro- suggests that small-molecules stabilizing the dimeric native state can be teins upon dilution of denaturant. used as a potential therapeutic strategy. This research was supported by NIH grant GM54836. 1554-Plat Rigid Spin Labels for Improved Distance and Dynamics in Intrinsically 1556-Plat Disordered Proteins and Peptides Exploring the Functional and Structural Impact of Disease-Associated Enrico Zurlo1, Nico J. Meeuwenoord2, Dmitri V. Filippov2, Martina Huber1. Mutants of Tau 1Leiden University, Department of Physics, Huygens-Kamerlingh Onnes Ana M. Melo, Elizabeth Rhoades. Lab, Leiden, Netherlands, 2Leiden University, Leiden Institute of Chemistry, Department of Chemistry, University of Pennsylvania, Philadelphia, Leiden, Netherlands. PA, USA. Spin-labeling and Electron Paramagnetic Resonance spectroscopy (EPR) have Tau is an intrinsically disordered microtubule associated protein, whose path- become powerful tools for structure determination in proteins and bio- ological self-association is linked to several tauopathies, including Alzheimer’s macromolecules. Usual spin labels, such as the disulfide-linked MTSL labels disease. A number of lines of evidence support tau aggregation as well as loss are flexible, owing to multiple single bonds separating the nitroxide from the of its native interactions with microtubules (MTs) as contributing to pathology. protein backbone. We investigate spin labels rigidly linked to the backbone Prior work from our lab showed that both disease-relevant as well as designed in a series of peptides derived from the KVKVLGDVIEV peptide[1], with point mutations to tau’s microtubule binding region (MTBR) enhance binding the 4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-4-carboxylic acid (TOAC). of tau to soluble tubulin. This observation indicates the mechanism of MT These peptides mimic amyloid-aggregation proteins. They were found to destabilization in disease may arise, in part, from an altered distribution of form stable oligomers that are cell-toxic, and enabled X-ray crystallography tubulin- and microtubule-bound tau. Here we determine whether the increase to determine their structures[1]. in affinity observed previously generally holds for disease-relevant tau mutants.

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Specifically, we characterize variants within all four repeats in the MTBR and differential effects of protein dynamics on the mechanism of action in at both N- (R5L and R5H) and C-termini (R406W). To investigate this, we used different species by performing a comparative analysis over the UPPS super- fluorescence correlation spectroscopy (FCS) to quantify the binding of tau var- family, Cis-Isoprenyl Pyrophosphate Synthase (Cis-IPPS) using protein iants to soluble tubulin. Changes in the affinity and function were correlated dynamics and pharmacophore modeling. We used MOE and Ligand Scout with the topological features of each tau mutant in solution or upon interaction to compute protein structure and ligand-based pharmacophore models of 14 with soluble tubulin by using single-molecule FRET. Together, our work al- UPPS protein structures. Molecular dynamics simulation and an ensemble- lows to identify domain/region specific conformational changes in tau relevant based statistical mechanics model are used to quantify mobility and stability to tauopathies and to explore the effect on its intrinsically disordered features. of UPPS in the apo form, and with bound substrates. We find that quantitative This provides a structural basis for understanding the impact of tau disease mu- stability flexibility relationships (QSFR) identify conserved flexibly corre- tations in the tau-tubulin complex. lated motifs. We find that the binding event affects backbone flexibility over an extended range, suggesting a mechanism of allostery. The most Platform: Protein Dynamics and Allostery II conserved regions in the QSFR motifs include the active site and hydrophobic tunnel, which reflects evolutionary conservation. However, variations are sub- 1557-Plat stantial at the interface, particularly in the coiled-coil arm that glues the two Predicting Residues that Increase Antibiotic Resistance of CTX-M9 monomers together. Interestingly, we observe slight differences in structure- Enzymes using Molecular Simulation and Statistical Learning based pharamacophore features across species. We corroborate results ob- George A. Cortina, Malgorzata J. Latallo, Peter M. Kasson. tained from evolutionary trees, pharmacophore modeling, characterization Biomedical Engineering, University of Virginia, Charlottesville, VA, USA. of dynamics and stability to reveal the mechanism of binding selectively. Over 2 million people annually contract a serious antibiotic resistant bacterial With 14 proteins studied in detail thus far, our results suggest a critical flex- infection. Beta-lactamases contribute to antibiotic resistance by hydrolyzing ibly correlated motion spans both domains of the UPPS homodimer, which beta-lactam antibiotics, the most commonly used class of antibiotics, and en- creates an allosteric pathway responsible for modulating binding selectively. zymes such as CTX-M9 confer resistance to nearly all commonly used outpa- tient beta-lactams. We seek to understand how point mutations of these 1560-Plat enzymes, both within and away from the drug-binding pocket, alter drug resis- Dynamic Flexibility Index Sheds Light on Pin1 Allostery tance. We have performed molecular dynamics simulations of CTX-M9 and a Paul Campitelli1, Huan-Xiang Zhou2, Giovanna Ghirlanda3, number of point mutants that increase drug resistance. We then analyzed these S. Banu Ozkan1. with information theoretic methods to (1) prospectively identify residues that 1Department of Physics, Arizona State University, Tempe, AZ, USA, alter drug specificity and (2) explain the effect of previously identified gain of 2Department of Biophysics, Florida State University, Tallahasse, FL, USA, function mutations distant from the pocket. In order to globally identify res- 3Department of Chemistry and Biochemistry, Arizona State University, idues contributing to drug activity, we employed positional mutual informa- Tempe, AZ, USA. tion, a non-linear measure of atom association based on movement, to Allostery, which is regulation from distant sites, plays a major role in biology. detect residues most associated with the drug. We tested these predictions Pin1 is a modular protein containing a WW domain and a larger peptidyl experimentally via alanine mutagenesis. High-ranking residues had a signifi- prolyl isomarase domain (PPIase) that isomerizes phospho-serine/threonine- cantly greater loss of activity than low-ranking ones, suggesting that this proline (pS/T-P) motifs, which are critical for signaling within intrinsically metric can be used to identify residues important for drug resistance. We disordered loops of cell cycle proteins. Pin1 utilizes allosteric regulations also utilized machine learning methods in the form of mutual-information- for its function, and binds (pS/T-P) motifs in both domains. The WW domain guided feature selection and decision trees to understand how the effect of serves as a docking module, whereas catalysis solely takes place within the distant mutations is transmitted to the drug-binding pocket. Using this tech- PPIase domain. However, enzymatic activity gets enhanced when WW is in nique, we were able to identify a small set of atoms in the binding pocket the bound form, highlighting PIN1’s allosteric regulation. Previous work us- where particular positional changes identify enhanced drug resistance with ing NMR and molecular dynamics analysis has shown that binding induced >90% accuracy. quenching of fast local motions and strengthening of the interaction between two domains, indicated in particular by decrease in flexibility of catalytic 1558-Plat loops. Here we present a novel method, the dynamic flexibility index (DFI) Modeling Proteins’ Hidden Conformations to Predict Antibiotic analysis, for characterizing the underlying allosteric communications between Resistance two domains. DFI measures the resilience of a given position to the perturba- Gregory Bowman. tions that occur at different parts of the protein, using linear response theory. Washington University in St. Louis, St Louis, MO, USA. This index captures multi-dimensional effects when the protein is displaced TEM b-lactamase confers bacteria with resistance to many antibiotics and out of equilibrium. Moreover, we can also identify the allosteric response rapidly evolves activity against new drugs. However, functional changes are in dynamic flexibility based on the perturbation response fluctuation profile not easily explained by differences in crystal structures. We employ Markov of the PPIase domain upon WW binding and distinguish the positions that state models (MSMs) to identify hidden conformations and explore their role contribute the most. Finally we also explore the mechanistic link between in determining TEM’s specificity. We integrate these models with existing conformational dynamics and co-evolution to identify mutational positions drug-design tools to create a new technique, called Boltzmann docking, which to alter enzymatic function. better predicts TEM specificity by accounting for conformational heterogene- ity. Using our MSMs, we identify hidden states whose populations correlate with activity against cefotaxime. To experimentally detect our predicted hidden 1561-Plat states, we use rapid mass spectrometricfootprinting and confirm our models’ Intrinsic Disordered Controls Transcription of Bacterial Toxin-Antitoixn prediction that increased cefotaxime activity correlates with reduced U-loop Modules flexibility. Finally, we design novel variants to stabilize the hidden cefo- Abel Garcia-Pino. taximase states, and find their populations predict activity against cefotaxime Biologie Structurale et Biophysique, Universite´ Libre de Bruxelles, in vitro and in vivo. Therefore, we expect this framework to have numerous Gosselies, Belgium. applications in drug and protein design. Intrinsic disorder is highly prevalent in type II bacterial antitoxins. It has long being assumed their presence is linked to the rapid turnover of these antitoxins, 1559-Plat however recent evidence suggest they provide unparalleled features that allow Revealing the Mechanism of Binding Selectivity in Undecaprenyl moonlighting between toxin neutralization and allosteric control of transcrip- Diphosphate Synthase tion. Conditional cooperativity the a common mechanism at play for transcrip- Fareeha Kanwal1, Donald Jacobs2,3. tion regulation of type II toxin-antitoxin operons and is intricately related to 1Bioinformatics and Genomics, University of North Carolina, Charlotte, NC, persistence. Conditional cooperativity allows the toxin component of toxin- USA, 2Physics and Optical Science, University of North Carolina, Charlotte, antitoxin modules to act as a co-repressor at low toxin dose respect to the anti- NC, USA, 3Center for Biomedical Engineering and Science, University of toxin or de-repressor when the toxin level exceeds a certain threshold. It has North Carolina, Charlotte, NC, USA. been suggested that the presence of a disordered region in toxin-antitoxin sys- Undecaprenyl Pyrophosphate Synthase (UPPS) represents an excellent drug tem may play a central role in their transcription regulation.To address how the target to combat bacterial virulence because it disrupts the glycan biosynthesis antitoxin IDR is involved in transcription regulation we studied the phd/doc pathway when inhibited. Binding of a substrate causes the activity in UPPS operon as model transcription regulation system. We provide evidence that from different species to respond differently, making it potentially possible the intrinsically disordered region of Phd is a bona fide entropic barrier that pre- to develop a suite of narrow-spectrum antibiotics. Here, we investigate the cludes full operon repression in the absence of Doc. Binding of the Doc toxin to

BPJ 7813_7824 318a Tuesday, February 14, 2017

Phd results in a cooperativity switch and consequent strong operon repression, functional conformational change. This solvent slaving of the long range vibra- enabling context-specific modulation of the transcription output to the regula- tions potentially impacts protein structural stability and vulnerability to struc- tory process.The regulation of transcription of TA modules by conditional co- tural disorder. This work was supported by NSF (DBI 1556359 and MCB operativity has strong implications in the formation and frequency of persister 1616529), and DOE DE-SC0016317. 1. Doster,W., et al. Phys.Rev.Lett., cells. Variations of this theme are likely a common mechanism in the auto- 2010.104(9):098101. 2. Niessen,K., et al. Biophys.Rev., 2015.7,201. 3. Ac- regulation of bacterial operons that involve intrinsically disordered regions. bas,G., et al. Nat.Commun., 2014.5,3076.

1562-Plat 1564-Plat Integrated View of Internal Friction in Unfolded Proteins from Single- Facilitator Models of Weak Binding in Protein-Protein Interactions Molecule FRET, Contact Quenching, Theory, and Simulations Seyit Kale, Madeleine Strickland, Alan Peterkofsky, Nico Tjandra, Jian Liu. Andrea Soranno1,2, Andrea Holla2, Fabian Dingfelder2, Daniel Nettels2, NHLBI, National Institutes of Health, Bethesda, MD, USA. Dmitrii E. Makarov3, Benjamin Schuler2. Pairwise interactions are intuitive to our understanding on protein-ligand bind- 1Department of Biochemistry and Molecular Biophysics, Washington ing; however in vivo this is rarely true. Most intracellular proteins operate in a University in St Louis, St Louis, MO, USA, 2Biochemistry Department, highly cooperative manner to perform tasks ranging from metabolic turnover to University of Zurich, Zurich, Switzerland, 3Department of Chemistry and intricate signaling regulation. In some cases, one substrate needs to simulta- Biochemistry and Institute for Computational Engineering and Sciences, neously interact with more than one binding partner to carry out faithful signal University of Texas at Austin, Austin, TX, USA. transductions. While one of these binding partners is the determinant of such The dynamics of proteins, which are essential for both folding and function, signal transduction, it can share very similar tertiary structure with the other are known to be strongly dependent on solvent viscosity or friction. How- but might differ in functional role and abundance. Motivated by this observa- ever, an increasing number of experiments have demonstrated the impor- tion, we explore the physical consequences of the mere steric presence of a non- tance of a contribution to protein dynamics independent of solvent specific ligand, the ‘‘competitor’’, crowding the surface of a ‘‘target’’ ligand. friction. Such ‘internal friction’ has been detected at all stages along the The specific interaction occurs between the protein and the ‘‘target’’ ligand, folding reaction. Even in unfolded and intrinsically disordered proteins, in- which explores the same surface as well, albeit for its unique binding site. A ternal friction has a large influence, as demonstrated with several experi- simple lattice model incorporating these elements along with the natural rules mental techniques and in simulations. However, these methods probe of exclusion and hopping reveals the regimes for when recruitment (turnover) different length- and timescales and have thus been used to illuminate or residence (transition state stabilization) are favored. Exploration of the different facets of internal friction in diverse molecular systems. To obtain search dynamics of the two ligands along the protein surface provides further an integrated and quantitative understanding, we apply the combination of insight. two complementary experimental techniques, theory, and simulations to one system, unfolded protein L. We use single-molecule Fo¨rster resonance Platform: Membrane Structure I: Cholesterol energy transfer (FRET) to measure the global reconfiguration dynamics of 1565-Plat the chain, and photoinduced electron transfer (PET), a contact-based Polyphenol Alkyl Ester Inhibits Membrane Cholesterol Domain Forma- method, to quantify the rate of loop formation between two residues. This tion Through an Antioxidant Mechanism Based, in Nonlinear Fashion, combination enables us to probe unfolded-state dynamics on different length on Chain Length scales, corresponding to different parts of the intramolecular distance distri- Samuel Sherratt1, Pierre Villeneuve2, Robert Jacob1, Erwann Durand2, butions. Both FRET and PET measurements indicate that internal friction R. Preston Mason1,3. dominates unfolded-state dynamics at low denaturant concentration, and 1Elucida Research, Beverly, MA, USA, 2CIRAD, UMRIATE, Montpellier, both are in remarkably good agreement with recent large-scale molecular dy- France, 3Brigham and Women‘s Hospital, Harvard Medical School, Boston, namics simulations employing a new water model. The simulations indicate MA, USA. that both hydrogen bond formation and dihedral angle rotation are correlated Under conditions of oxidative stress, cholesterol aggregates into discrete with the presence of internal friction. Theoretical models of polymer dy- membrane bilayer domains that precipitate the formation of extracellular crys- namics allow us to quantitatively relate the contribution of internal friction tals, a hallmark feature of the advanced atheroma in cardiovascular disease. in the two types of experiments with simulations and thus provide a coherent Molecular intervention using membrane-directed antioxidants, such as poly- picture of internal friction in unfolded proteins. phenolic esters, alkylated to increase their lipophilicity and bioavailability, may reduce cholesterol domain formation and associated pathology. In this 1563-Plat study, we tested the effects of rosmarinic acid (R0) and rosmarinic esters, Escaping the Water Cage: Protein Intramolecular Vibrations and the with alkyl chain lengths ranging from 4 to 16 carbons (R4-R16), on mem- Dynamical Transition brane lipid oxidation and cholesterol domain formation. Model membranes Mengyang Xu, Katherine A. Niessen, Yanting Deng, Nigel S. Michki, were prepared as binary mixtures of dilinoleoylphosphatidylcholine and Andrea G. Markelz. cholesterol (at a cholesterol-to-phospholipid mole ratio of 0.6:1), in the Department of Physics, University at Buffalo, SUNY, Buffalo, NY, USA. absence or presence of each of the various rosmarinic compounds, and The protein dynamical transition is the remarkable increase in the average exposed to oxidative conditions for up to 72 hr. Changes in lipid hydroperox- atomic root mean squared displacement (RMSD) in the 180-220 K range. ide (LOOH) and cholesterol domain formation were measured using iodomet- The effect is associated with the onset of anharmonic motions critical to biolog- ric and small angle x-ray diffraction approaches, respectively. Rosmarinic ical function. However evidence of the actual biological relevance of the acid and the various esters were observed to have differential effects on dynamical transition is inconsistent. While for some proteins, function ceases LOOH formation based on alkyl chain length. R8 had the greatest antioxidant below the dynamical transition, for other proteins the dynamical transition ap- effect, reducing LOOH levels by 60 5 18% as compared to vehicle. R8 also pears to have no effect. Here we report measurements that suggest the differ- inhibited cholesterol domain formation. By contrast, R0 and R16 failed to ence arises from the dependence of function on large scale conformational inhibit LOOH formation (6 5 19% reduction, 5 5 13% increase compared change, and specifically the reliance on long range vibrations to access these to vehicle, respectively), resulting in cholesterol domain formation. These structural changes. The dynamical transition has been extensively observed us- data indicate that the membrane antioxidant potential of rosmarinic acid esters ing X-ray, neutron scattering, NMR and terahertz absorption spectroscopy is dependent, in a nonlinear manner, on alkyl chain length. The mechanism for [1,2], with the results indicating it arises from thermally activated solvent mo- this effect is attributed to the influence of alkyl chain length on the optimal tions. Those techniques measure all motions contributing to the RMSD depth of the polyphenols into the lipid bilayer. These findings provide insight including both localized motions and intramolecular vibrations. To isolate into novel atheroprotective benefits of polyphenol esters that are dependent on the vibrations and examine how the dynamical transition impacts them, we their membrane location. use a new technique, anisotropy terahertz microscopy (ATM) [3]. This unique method suppresses the background from the localized motions giving unprec- 1566-Plat edented access to the long range motions that enable large scale conformational Is the Site of Influenza Virus Assembly and Budding Enriched with Choles- changes. ATM measurements of lysozyme anisotropic optical absorbance in terol and Sphingolipids? the 150-300 K temperature range show that the resonant vibrational bands Mary L. Kraft1, Ashley N. Yeager1, Peter K. Weber2, Joshua Zimmerberg3. rapidly increase in intensity at the dynamical transition, and surprisingly blue 1Chem & Biomol Engr, University of Illinois, Urbana, IL, USA, 2Glenn T. shift with increasing temperature, in contrast to the expected anharmonicity. Seaborg Institute, Lawrence Livermore National Laboratory, Livermore, CA, The measurements demonstrate that the surrounding solvent below the dynam- USA, 3Program in Physical Biology, Eunice Kennedy Shriver National ical transition acts as a frozen cage preventing the vibrations necessary for Institute of Child Health and Human Development, Bethesda, MD, USA.

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The observation that the influenza viral envelope is enriched with cholesterol reorganize lipid rafts. To investigate this model, we compared lipid bilayers and sphingolipids contributed to the hypothesis that the influenza virus assem- composed of PDPC (1-palmitoyl-2-docosahexaenoylphosphatidylcholine) bles and bud from cholesterol- and sphingolipid-enriched domains in the or POPC (1-palmitoyl-2-oleoylphosphatidylcholine) in mixtures with raft- plasma membrane. The formation of infectious influenza virions and the clus- forming, SM (sphingomyelin) and cholesterol (1:1:1 mol). A combination tering of influenza virus proteins in the plasma membrane during virus assem- of solid state 2H NMR, confocal imaging and coarse grain MD simulations bly are reportedly reduced by depletion of cellular cholesterol. Though this were employed. In the PDPC mixture, individual NMR signals assigned to cholesterol sensitivity certainly suggests that cholesterol plays a role in influ- deuterated analogs of each lipid in ordered raft-like domains and disordered enza virus infection, whether the plasma membrane domain from which the non-raft domains were resolved in the spectra recorded for aqueous multila- influenza virus buds is enriched with cholesterol and sphingolipids has not mellar dispersions. In the POPC mixture, by contrast, separate signals were been decisively determined. We have addressed this question by using not resolved. Our interpretation is that PDPC promotes the formation of high-resolution secondary ion mass spectrometry (SIMS) to image the distri- larger domains between which the diffusion-mediated exchange of lipids is butions of stable isotope-labeled lipids and immunolabeled influenza virus slow, whereas the domains are smaller with POPC and the exchange of lipids proteins in the plasma membranes of virus-infected cells with ~100 nm lateral between them is fast. This interpretation is supported by imaging studies of resolution. In these experiments, we metabolically incorporated the distinct GUV (giant unilamellar vesicles) and MD simulations. Controlling the size stable isotopes 15N and 18O into the sphingolipids and cholesterol, respec- of lipid rafts may be, we propose, an underlying role for PUFA in the plasma tively, in living MDCK cells. We infected these MDCK cells with an membrane. H3N2 strain of the influenza virus for 24 h, and immunolabeled the influenza envelope protein, hemagglutinin, to permit the detection of budding virus. 1569-Plat Finally, we imaged the metabolically incorporated 15N-sphingolipids and Azobenzene-Cholesterol as a Photoactivator in Biomimetic Membranes: 2. 18O-cholesterol and immunolabeled influenza hemagglutinin on the surfaces Membrane Structure 1 2 3 1 of the influenza-infected MDCK cells, and assessed co-localization between Chen Shen , Jorge de la Serna , Bernd Struth , Beate Klo¨sgen . 1Department of Physics, Chemistry, and Pharmacy, University of Southern these components. The results of these experiments and there implications 2 will be presented. Denmark, Odense M, Denmark, John Raddcliffe Hospital, University of Oxford, Oxford, United Kingdom, 3Deutsches Elektronen-Synchrotron, 1567-Plat Hamburg, Germany. Lipid-Protein Interactions in Fiber Cell Plasma Membrane Isolated From Properties of biomembranes are modified in presence of additives such as Human and Porcine Eye Lenses cholesterol (chol) or proteins. Azobenzene-cholesterol (azo-chol) is a pho- Marija Raguz1, Laxman Mainali2, William J. O‘Brien3, toactive variety of chol since its azo-headgroup exhibits a reversible Witold Karol Subczynski4. change of the conformation upon illumination (365nm: trans to cis; 1Department of Medical Physics and Biophysics, School of Medicine 455nm: cis to trans). Grazing incidence X-ray diffraction (GIXD) was University of Split, Split, Croatia, 2Department of Biophysics, applied to resolve the lateral structure within biomimetic monolayers con- Medical College of Wisconsin, Milwaukee, WI, USA, 3Department of sisting of a 1,2-dipalmitoyl-sn-phosphatidyl-choline (DPPC) matrix with Ophthalmology, Medical College of Wisconsin, Milwaukee, WI, USA, embedded azo-chol in either of the two conformational states. A pure 4Department of Biophysics, Medical College of Wisconsin, Milwaukee, DPPC monolayer and also a DPPC monolayer with inactive chol were WI, USA. used as control systems. The results report on the symmetry of the 2D-lat- The protein content in human lens fiber cell plasma membranes is tice and the inter-chain distance dxy. The tilt angle of the lipid chains was extremely high, increases with age, and is higher in the nucleus than in cor- calculated from the lattice parameters and the geometrical boundary condi- tex. This strongly affects the organization and physical properties of the tions. The lattice parameters are modified when azo-chol in the host mem- lipid bilayer portion of intact fiber cell plasma membranes including the brane is in all-trans state as compared to the control systems. With all-cis alkyl chain order, fluidity, hydrophobicity, and oxygen transport parameter. azo-chol, the structure is broken. The findings show the capacity of azo- Investigated membranes were isolated from the eye lenses of pigs and hu- chol for switching the lateral structure of the host lipid membrane though man donors. They were studied using electron paramagnetic resonance photoactivation. spin-labeling method that provides an opportunity to discriminate coexist- ing lipid domains and to evaluate the relative amounts of phospholipids 1570-Plat and cholesterol in them. Fiber-cell membranes were found to contain three Submicrometric Lipid Domains Play Key Roles in Erythrocyte Deforma- distinct lipid environments: bulk, boundary and trapped domains that appear tion: From Membrane Bending to Shape Restoration Catherine Leonard1, Marie-Paule Mingeot-Leclercq1, Donatienne Tyteca2. due to the presence of membrane proteins. In nuclear membranes the 1 2 amount of boundary and trapped phospholipids as well as the amount of LDRI-FACM, Ucl, Bruxelles, Belgium, DDUV-CELL, Ucl, Bruxelles, cholesterol in trapped lipid domains was greater than that in cortical mem- Belgium. branes. Cholesterol was to a large degree excluded from trapped lipid do- Although direct evidence for lipid domains in living plasma membranes mains in cortical membranes. The difference between the amounts of have been recently provided, their existence is still up for debate as their lipids in domains uniquely formed due to the presence of membrane pro- functions remain unclear. Vital imaging of the smooth erythrocyte plasma teins increased with the donors‘ age. The rigidity of nuclear membranes membrane allowed us to highlight two distinct populations of submicromet- was greater than that of cortical membranes for all age groups. The nuclear ric domains with differential cholesterol and sphingomyelin enrichment. We fiber cell plasma membranes were less permeable to oxygen than cortical here report direct evidence for the implication of these two domains in key membranes and both of them become less permeable to oxygen with age. steps of erythrocyte deformation, combining erythrocyte models exhibiting In nuclear porcine membranes the amounts of lipids in domains created differential membrane curvature (healthy erythrocytes vs erythrocytes from due to the presence of membrane proteins were greater than those in patients with elliptocytosis and spherocytosis, two erythrocyte membrane cortical membranes and the differences were larger than the differences fragility diseases), (de)stretching biophysical experiments and the decoration observed for human membranes. Lipids in porcine nuclear fiber cell mem- of endogenous cholesterol and sphingomyelin by specific fluorescent toxin branes were more rigid and less permeable to oxygen than in human nu- fragments. Whereas cholesterol-enriched domains were implicated in the clear membranes. generation/maintenance of high membrane curvature areas needed for mem- brane bending, sphingomyelin-enriched domains were involved in calcium 1568-Plat efflux during erythrocyte shape restoration. Our results indicate that choles- Raft-Like Domains are Driven Together by Pufa terol- and sphingomyelin-enriched domains play distinct key roles in eryth- Jacob J. Kinnun1, Xiaoling Leng1, Dylan Johnson2, Edward R. Pennington2, rocyte deformation, as confirmed by the specific domain loss upon Andrew J. Meador1, Saame Raza Shaikh2, Stephen R. Wassall1. erythrocyte aging and erythrocyte deformability impairment upon domain 1Department of Physics, Indiana University-Purdue University Indianapolis, abrogation by pharmacological approaches. Thus, whereas cholesterol- and Indianapolis, IN, USA, 2Department of Biochemistry and Molecular Biology sphingomyelin-enriched domains were so far highly associated, our findings and East Carolina Diabetes and Obesity Institute, East Carolina University, give direct evidence for differential biophysical properties but also distinct Greenville, NC, USA. physiopathological roles: (i) membrane bending modulators and/or stabi- Fish oils rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as lizers (cholesterol-enriched domains), (ii) platforms for protein sorting docosahexaenoic acid (DHA, 22:6), have a wide variety of health benefits and/or activation (e.g. calcium efflux pump; sphingomyelin-enriched do- but a complete molecular mechanism is yet to be elucidated. One model mains), and (iii) preferential fragility sites for membrane vesiculation postulates that n-3 PUFAs are incorporated into the plasma membrane and (both domains).

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1571-Plat Nicotinic acetylcholine receptors are ligand gated ion channels that mediate Membrane Crowding and Complexity: Interplay between Protein-Lipid fast chemical neurotransmission at the neuromuscular junction and play Interactions, Clustering and Diffusion diverse signaling roles in the central nervous system. Here we describe a Anna L. Duncan, Heidi Koldsø, Tyler J. Reddy, Jean He´lie, biochemical approach for characterization of subunit stoichiometry in het- Mark S.P. Sansom. eromeric membrane proteins and present the first X-ray crystal structure Department of Biochemistry, University of Oxford, Oxford, United of a nicotinic receptor. The a4b2 nicotinic receptor is the most abundant re- Kingdom. ceptor subtype in the brain, is the principal target in nicotine addiction and It is well-understood that cell membranes are crowded and complex environ- its dysfunction is associated with familial epilepsy. The structure of the re- ments, containing up to 50 % protein by mass [1] and comprised of myriad ceptor in complex with the agonist nicotine reveals principles of ligand types of lipid species [2]. Less well understood is the molecular detail of the selectivity among different classes of subunit interfaces in the heteropenta- effects of complexity and crowding on membrane organisation and dynamics. meric assembly. The receptor is stabilized by nicotine in a non-conducting, Advances in coarse-grained force fields and computational power mean that desensitized conformation. The constriction point in the permeation large-scale coarse-grained (CG) molecular dynamics (MD) simulations are pathway is formed at the selectivity filter, located at the cytosolic end of increasingly being used to gain such understanding [3]. the pore. The desensitized state of this channel provides a distinct structural To investigate the role of protein crowding and lipid complexity on the reference point in the allosteric gating cycle of the larger Cys-loop receptor organisation and dynamics of mammalian cell membranes, and partic- superfamily. ularly the effect of protein-lipid interactions on protein and lipid diffusion in crowded membranes, we have performed large CG MD simulations 1574-Plat containing over 100 inward rectifier potassium (Kir) channels, which have Photoaffinity Labeling of A4B2 Nicotinic Acetylcholine Receptor using [3H]-Labeled Positive Allosteric Modulators specific lipid interactions with PIP2, other anionic lipid interactions and cholesterol. Gordon Ang1, Farah Deba1, Akash Pandhare2, Michael P. Blanton2, Jonathan B. Cohen3, Ayman K. Hamouda1. Lipid complexity has a marked effect on the clustering behaviour of channels, 1 underlying which are the diffusive properties of different lipid mixtures. Sub- Pharmaceutical Sciences, Texas A&M Health Science Center, Kingsville, TX, USA, 2Pharmacology and Neurosciences, Texas Tech University Health diffusion of lipids tightly interacting with Kir channels is also observed, and 3 this subdiffusion is further modulated by protein crowding. By simulating Kir Science Center, Lubbock, TX, USA, Neurobiology, Harvard Medical channels in a complex lipid mixture, we can also examine the interplay be- School, Boston, MA, USA. The a4b2 nicotinic acetylcholine receptor (nAChR) is a potential drug target tween PIP2, other anionic lipids and cholesterol interactions. In simulating systems of >100 nm in dimension whilst retaining membrane complexity at for treating neuropathological and tobacco use disorders. Positive allosteric a molecular level, we start to move further towards the use of simulation as modulators (PAMs), which bind o site(s) distinct from the ACh (agonist) a computational microscope, and can assess the possibilities using this binding sites, may provide the required specificity by binding to unique approach. site(s) present only in the a4b2 nAChR. However, the molecular pharma- 1. Dupuy and Engelman. Proc Natl Acad Sci USA, 2008 105 (8): 2848–52 cology of this class of compounds is unclear, and the diversity of PAM bind- ing site(s) have not been determined. In previous work, two radiolabeled 2. Coskun and Simons. Structure, 2011 19(11): 1543–48. 3 3. Chavent, Duncan and Sansom. Curr Op Struct Biol, 2016. 40: 8–16 a4b2 nAChR PAMs, desformylflustrabromine; ([ H]dFBr, 76 Ci/mmol) and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H- pyrrazol-4-yl) 3 1572-Plat isoxazole ([ H]CMPI, 16 Ci/mmol) have been prepared and their merits as The Effect of Propofol on Plasma Membrane Ultrastructure in the Intact photoprobes established using Torpedo nAChR as a model (Hamouda Cells et al. 2015, Mol. Pharmacol. 88:1; Hamouda et al. 2016, Mol. Pharmacol. Weixiang Jin, Arnd Pralle. 89: 575). Here we expand these studies to a4b2 nAChR. Using membrane Physics, University at Buffalo, Buffalo, NY, USA. fractions isolated from a HEK-293 cell line stably expressing a4b2nAChR Despite many observed effects of anesthetic drugs, the mechanism of general (HEK-a4b2 nAChR), we are examining the effects of orthosteric ligands, ion channel blockers and other PAMs on the equilibrium binding of [3H] anesthesia is still unknown.One well-studied drug used for human anesthesia, 3 propofol, has been shown to interact with some ligand gated ion-channels. dFBr and [ H]CMPI. In addition, we purified a4b2 nAChRs from HEK- a4b2 nAChR membranes using a bromo-acetylcholine bromide affinity However, propofol also easily dissolves in the lipid bilayer and alters mem- 3 3 brane fluidity. Which mechanism dominates in anesthesia or even how anes- column and performed initial [ H]dFBr and [ H]CMPI analytical photolab- thesia arises are unclear. Here, we study the influence of propofol on plasma elings. When ~100 mgofaffinity-purifieda4b2 nAChR and 20 mCi of [3H]dFBr (76 Ci/mmol) or [3H]CMPI (16 Ci/mmol) were used, photolysis membrane (PM) ultrastructure in intact cells, which affect cell signaling. In 3 3 the PM, transient submicroscopic nano-domains form by interactions between at 312 nm for 5 min ([ H]dFBr) or 3 min ([ H]CMPI) resulted in photolab- eling efficiency of (pmol of 3H incorporated/pmol of a4b2 nAChR) of lipid-acyl-chains or lipid head groups, stabilized by cholesterol. In addition, 3 3 membrane cytoskeleton may further regulate these nano-domains. These do- ~0.05% and 0.7%, respectively. Additional analytical [ H]dFBr and [ H] mains regulate receptor interactions and signaling.We study transient propofol CMPI photolabeling in the presence of other nAChR ligands and preparative affects on these domains from low to clinically relevant propofol concentra- photolabeling experiments are in progress to identify amino acids contrib- tions by analyzing diffusion of GFP-tagged outer leaflet/inner leaflet membrane uting to dFBr and CMPI binding sites. proteins.Using bimFCS we measure diffusion on multiple length scales simul- 1575-Plat taneously. We observe that at lower propofol concentrations (up to 2yM), the Structural Mechanisms Underlying PUFA Modulation in Pentameric cholesterol nano-domains trap the GPI-mGFP less, which is consistent with Ligand Gated Ion Channels the recent studies showing that propofoldecreases the phase transition temper- Yvonne W. Gicheru, Sandip Basak, Sudha Chakrapani. ature of plasma membrane derived vesicles. Interestingly, at higher concentra- Physiology and Biophysics, Case Western Reserve University, Cleveland, tions of propofol (20yM to 150yM), the nanodomains trap the GPI-mGFP more OH, USA. strongly. This is only observed at physiological temperatures (37 C). By inhib- Pentameric ligand gated ion channels (pLGICs) mediate fast neurotransmis- iting myosin activity or actin filaments (de-)polymerization, we find that the ac- sion in the central and peripheral nervous systems. Modulators such as tivity of actin filaments further alters the behavior of cholesterol nano-domains lipids, alcohols and general anesthetics allosterically modify the conforma- due to propofol. We compare the effect of propofol and its analog confirming tions of pentameric ligand gated ion channels (pLGICs) via the transmem- its specific effect as anesthetic drug. These results suggest that in intact cells, brane domain. However, the molecular mechanisms underlying modulation propofol induced cholesterol nano-domains changes are temperature depen- are still unclear. GLIC is a prokaryotic homologue that has an overall dent, regulated by cytoskeleton activities, and the effect is specific comparing conserved architecture and high sensitivity to clinically relevant compounds to its analogs. as the eukaryotic members of the family. Our study probed the functional and structural effect of acyl chain length and degree of unsaturation of poly- Platform: Ligand-gated Channels I unsaturated fatty acids (PUFAs) on GLIC function. A novel binding site for PUFAs on GLIC and the effect of perturbations on this site were investi- 1573-Plat gated. By using a combination of electrophysiology, EPR spectroscopy, Structure and Mechanism of Neuronal Nicotinic Acetylcholine Receptors and X-ray crystallography we show a possible mechanism of PUFA modu- Claudio L. Morales-Perez, Colleen M. Noviello, Ryan E. Hibbs. lation of pLGIC function through coupling of the outermost M4 helix with Neuroscience and Biophysics, UTSW Medical Center, Dallas, TX, USA. the channel pore.

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1576-Plat residues in the ECD and TMD that have close contacts with BrEtOH in Direct Visualization of Conformational Changes Related to Pentameric crystal structures. Functional electrophysiology assays on Xenopus laevis Receptor Ion Channel GLIC Gating oocytes show that the ECD mutants, even tagged with 4-(chloromercuri) Yi Ruan1, Pierre-Jean Corringer2, Simon Scheuring1. benzenesulfonic acid to mimic alcohol binding in an ECD binding site, 1Department of Anesthesiology, Weill Cornell Medecine, New York, NY, have almost the same functional responses to ethanol and BrEtOH as USA, 2Channel-Receptors Unit, Institut Pasteur, Paris, France. wild-type ELIC. In contrast, the mutation to the pore binding site signif- GLIC, a proton-gated cation-selective channel, is a prokaryotic homologue icantly alters channel responses to alcohols. The data suggest that of the pentameric Cys-loop receptor ligand-gated ion channel family. The alcohol binding to the pore, rather than to the ECD, dominates the inhib- conformational changes in the extracellular domain (ECD) and transmem- itory action on ELIC. The finding is further supported by the distinctly brane domain (TMD) during gating of this receptor channel family were in- different functional modulations of ELIC and ELIC-a1b3GABAARby ferred from X-ray structures at pH4 (open) and pH7 (closed), but remain ethanol and BrEtOH. Replacing the ELIC TMD with the TMD of elusive under native conditions. Here, we used high-speed atomic force mi- a1b3GABAAR in the ELIC-a1b3GABAAR chimera turned alcohol inhibi- croscopy (HS-AFM) combined with a buffer exchange system to directly tion into potentiation that has been observed on a1b3GABAAR. The func- study receptor channel gating on the single molecule level in the membrane tional results along with the X-ray structures support a pore-blocking in physiological buffer and at ambient temperature and pressure. We show mechanism for alcohol inhibition of pLGICs. Research supported by first real-time real-space conformational changes of single GLIC channels NIH funds. as a function of environmental pH. At pH4 the ECDs form a large funnel opening that collapsed at pH6.5 and adopted a tight conformation at 1579-Plat pH7.5. Conformational transitions in the TMD region occurred at pH6.5, Structural Rearrangements in GABA visibly disrupting molecular contacts. Concomitantly, the intermolecular Namrta Purwar1, Sana A. Shaikh2, Vasanthi Jayaraman2, distances between densely packed GLICs reporting a widening of the Cynthia Czajkowski1. 1Neuroscience, University of Wisconsin - Madison, Madison, WI, USA, external TMD helices. These structural changes were fully reversible. The 2 direct HS-AFM analysis of membrane-embedded single channels under Biochemistry and Molecular Biology, University of Texas Health Science native conditions suggest that the conformational changes may be more Center at Houston, Houston, TX, USA. important and different than what X-ray structures suggested, notably the GABAA receptors (GABARs) are allosteric proteins that convert a chemical amplitude of the ECD rearrangements exceed strongly what is observed in signal (GABA binding) to an electrical signal (opening of an integral ion- the crystals. Our work highlights the power of HS-AFM for the elucidation conducting channel). They mediate inhibition in the brain and are the thera- of mechanistic structure/function relationships at the single membrane pro- peutic targets for a variety of drugs such as benzodiazepines (BZDs), tein level. barbiturates, neuroactive steroids, volatile and intravenous anesthetics. For these receptors, binding of neurotransmitter in the extracellular ligand- 1577-Plat binding domain triggers opening of an intrinsic ion channel more than 50A˚ Uncommon Features of a Novel g-Proteobacterial Pentameric Ligand- away in the transmembrane domain of the receptor. While recent structures Gated Ion Channel Revealed by its Crystal Structure of prokaryotic and eukaryotic pLGICs solved in detergent micelles have HaiDai Hu1, Akos Nemecz2, Zeineb Fourati1, Pierre-Jean Corringer2, shed light on their molecular architecture, a full understanding of pLGIC Marc Delarue1. function requires knowledge of the protein motions that govern their confor- 1Structural Biology and Biological Chemistry, Institut Pasteur, Paris, France, mational transitions in physiological lipids. Comparing closed versus open 2Neuroscience, Institut Pasteur, Paris, France. channel structures of prokaryotic and eukaryotic pLGICs predict that Pentameric ligand-gated ion channels (pLGICs) control synaptic neurotrans- agonist-mediated channel opening is associated with a quaternary twisting mission in higher eukaryotes via an allosteric mechanism, where agonist and a radial un-blooming (inward tilting) of the extracellular binding domain binding in the extra-cellular domain triggers the opening of an ion- (ECD). Using a luminescence resonance energy transfer (LRET) approach conducting pore in the transmembrane domain. Here we present the x-ray that allows us to measure structural motions in full length heteropentameric structure at 2.6 A˚ resolution of a novel bacterial homologue bound to an GABARs in live cells, we are examining if, and how, the ECD moves in agonist, an amino acid analogue, in an apparently open conformation. This response to agonist-dependent gating transitions and allosteric drug binding. new structure presents unique features compared to the other pLGICs struc- We found that GABA decreased the distances between fluorescent probes tures described to date: the presence of a constriction ring formed by 5 ar- attached to the ECD of both the alpha and beta GABAR subunits, whereas ginines that partially obstruct the vestibule; and an open channel pore with binding of a competitive antagonist had no effect consistent with receptor an extra large diameter. Moreover, the agonist doesn’t bind the normal activation promoting a global inward tilting of the ECD. Surprisingly, a agonist site located under loop C at the interface of two subunits. Instead, BZD positive modulator that binds in the ECD but does not open the ion chan- it is found to bind at a site previously described as an allosteric site for other nel induced similar decreases in LRET-based distances as GABA raising the bacterial receptors. In addition, the structure clearly shows several monova- intriguing idea that the inward motion may reflect a low affinity to high affin- lent cations in the ion channel pore. Overall, this new structure is most ity binding site transition in the ECD versus a closed to open channel closely related to ELIC structure, also from a gamma-proteobacteria, but transition. its open pore differs markedly from the one seen in ELIC. We will discuss the implications of this new structure for the evolution and function of bac- 1580-Plat terial and eukaryotic pLGICs. Crosslinking-Mass Spectrometry of Targeted Single Cys Mutants to Refine Allostery and Model Building in the Glycine Receptor 1578-Plat Kayce A. Tomcho, Rathna J. Veeramachaneni, David J. Lapinsky, Structural Basis of Alcohol Inhibition of the Pentameric Ligand-Gated Ion Jeffry Madura, Michael Cascio. Channel ELIC Chemistry and Biochemistry, Duquesne University, Pittsburgh, Qiang Chen1, Marta M. Wells1, Tommy S. Tillman1, Monica N. Kinde1, PA, USA. Aina Cohen2, Yan Xu1, Pei Tang1. Studies regarding glycine receptor (GlyR) allostery can help provide 1Department of Anesthesiology, University of Pittsburgh School of insight into receptor function and allow us to develop novel therapeutics Medicine, Pittsburgh, PA, USA, 2Stanford Synchrotron Radiation to modulate its activity. To conduct these studies, single active thiols are Lightsource, Menlo Park, CA, USA. introduced into a Cys null background (C41S/C290A/C345S) or in the Neuronal pentameric ligand-gated ion channels (pLGICs) are molecular same background with a double mutation (F207G/A288G) that causes targets of alcohols. The structural basis for alcohol modulation of pLGICs, GlyR to be activated in a non-desensitizing manner to ivermectin (IVM). however, remains unclear. Multiple alcohol binding sites have been pro- The crosslinker, MTS-Benzophenone, is attached via disulfide linkage to posed previously in all three domains of pLGICs: the extracellular a single active Cys and the receptor is enriched in an allosteric state, resting (ECD), transmembrane (TMD), and intracellular (ICD) domains. In this (no ligand), desensitized (excess glycine) or open (F207G/A288G þ iver- study, we co-crystallized ELIC, a pLGIC from Erwinia chrysanthemi, mectin), before photoactivation and generation of non-specific crosslinks with the ethanol analog 2-bromoethanol (BrEtOH) in both the absence that examine the local topography near the sites of attachment. The cross- and presence of an agonist. We found BrEtOH bound to the pore near linked peptides, generated by trypsin digestion of monomeric and oligo- T237(60) and three different locations in the ECD of each subunit. In order meric bands, are analyzed using crosslinking mass spectrometry (CX-MS) to determine functional relevance of individual binding sites, we mutated and tandem mass spectrometry (MSMS) to sensitively identify sites of

BPJ 7813_7824 322a Tuesday, February 14, 2017 intra- and intermolecular crosslinking, respectively. In this study, we 1583-Plat examine the network of interactions generated by three mutations, intro- Insights into the Dynamics of the HIV-1 Env Glycan Shield duced singly at A41C, H419C or M287C in the resting, open and desen- Thomas Lemmin1, Cinque Soto2, Peter D. Kwong3. sitized states. Recent studies using M287C, a position near the top of 1UCSF, San Francisco, CA, USA, 2Vanderbilt University, Nashville, TN, TM3, potentially provides information on how this helix moves and how USA, 3NIH, Bethesda, MD, USA. the membrane regions are involved in the opening and closing of the The glycan shield of the HIV-1 envelope glycoprotein (Env) presents a chal- channel. lenge for the adaptive immune system by effectively shielding the Env- protein surface from potentially neutralizing antibody. HIV-1 Env is one of Platform: Molecular Dynamics II the most heavily glycosylated proteins known with roughly half of its mass con- sisting of host derived N-linked glycans. Little is known about the structural 1581-Plat dynamics of these N-linked glycans and how they coordinate together to shield Molecular Effects of Cardiac Troponin DCM Mutations on Calcium Sensi- the protein surface. tivity and Myofilament Activation - an Integrated Multi-Scale Modeling We carried out a 2us molecular dynamics simulation of a fully glycosylated Study atomistic model of the HIV-1 Env trimer. We observed a scissoring movement Sukriti Dewan1, Kimberly McCabe1, Michael Regnier2, Andrew McCulloch1, that lead to an asymmetry in the Env trimer structure. More detailed analysis Steffen Lindert3. involving the interactions between glycans using small-world network analysis 1University of California at San Diego, San Diego, CA, USA, 2University showed that highly conserved glycans regulate the accessibility of the CD4 of Washington, Seattle, WA, USA, 3Ohio State University, Columbus, binding site. We also observed the formation of microdomains by patches of OH, USA. glycans that remained stable throughout the most of simulation. Mutations in cTnC (D75Y, E59D and G159D), a key regulatory protein of In conclusion, the molecular dynamics simulation showed that HIV-1 trimer is myofilament contraction, have been associated with dilated cardiomyopathy an asymmetrical structure and that at any given time the protein surface asso- (DCM). Despite reports of altered myofilament function in these mutants, the ciated with only one CD4 binding site is freely accessible. This accessibility underlying molecular alterations caused by these mutations remain elusive. and scissoring may play a role in enabling initial contact of CD4 with Env Here we investigate in silico the intra-molecular mechanisms (both structure trimer, which occurs at an asymmetric stoichiometry (1 CD4 per trimer). Over- and dynamics) by which these mutations affect myofilament contraction (i.e. all, the collective behaviour of glycans at the surface of the HIV trimer is likely function). Based on the location of cTnC mutations, we tested the hypothesis a factor that needs to be considered for both CD4 interaction, as well as with that intra-molecular effects can explain the altered myofilament calcium potentially neutralizing antibody. sensitivity of force development for D75Y and E59D cTnC, whereas altered 1584-Plat cTnC-cTnI interaction contributes to the reported contractile effects of the We Can Predict the Effects of Kinase Domain Mutations using Molecular G159D mutation. We employed a multi-scale approach combining molecular Dynamics and Machine Learning dynamics (MD) and Brownian dynamics (BD) simulations to estimate cTnC E. Joseph Jordan1, Ravi Radhakrishnan2. calcium association and hydrophobic patch opening. We then integrated 1Biochemistry and Molecular Biophysics, The University of Pennsylvania, these parameters into a Markov model of myofilament activation to compute Philadelphia, PA, USA, 2Bioengineering, The University of Pennsylvania, the steady-state force-pCa relationship. The analysis showed that myofila- Philadelphia, PA, USA. ment calcium sensitivity with D75Y and E59D can be explained by changes Protein kinases, which are known to be involved in cell proliferation, differ- in calcium binding affinity of cTnC and the rate of hydrophobic patch open- entiation, and survival, have been shown to also play a key role in cancer ing, if a partial cTnC interhelical opening angle (110 ) is sufficient for cTnI formation. Numerous cancer therapies have been developed that target switch peptide association to cTnC. In contrast, interactions between cTnC over-active kinases, but the ease of sequencing cancer genomes has shown and cTnI within the cardiac troponin complex must also be accounted for that kinase proteins can be mutated at virtually any residue of the kinase cat- to explain contractile alterations due to G159D. In conclusion, this is the first alytic domain. This raises the question of which patients should get which multi-scale in silico study to elucidate how direct molecular effects of ge- treatments, as these therapies generally target only over-active kinases, but netic mutations in cTnC translate to altered myofilament contractile it is generally not known whether each new kinase domain mutation is active function. without conducting laborious experiments. This has created a strong desire, both in the clinic and in the lab, to be able to computationally predict the ef- 1582-Plat fects of kinase domain mutations. Here, we report on a method which utilizes Spatial Heat Maps from Fast Information Matching of Fast and Slow a convergence between standard molecular dynamics, free energy calcula- Degrees of Freedom in Molecular Dynamics Simulations tions, and machine learning approaches in order to predict the effects of ki- 1 2 2 Willy Wriggers , Federica Castellani , P. Thomas Vernier , nase domain mutations. This method has been shown to give few false 1 Julio A. Kovacs . negatives and very few false positives across dozens of mutations in several 1Dept. of Mechanical and Aerospace Engineering, Old Dominion University, 2 different kinases, from both the tyrosine and serine/threonine families, and Norfolk, VA, USA, Frank Reidy Research Center for Bioelectrics, Old in both transmembrane receptor as well as soluble kinases. This method is Dominion University, Norfolk, VA, USA. much more accurate, sensitive, and specific than widely used mutation assess- We describe a novel Fast Information Matching (FIM) method for trans- ment algorithms which are either not mechanistic, not kinase specific, or are forming time domain data into spatial images through handshaking between not either. As such, kinase specific mechanistic models could have a role in fast and slow degrees of freedom. The analytics takes advantage of the the clinic for directing patient care and the insights gained from these models detailed time series available from biomolecular computer simulations, could also be leveraged to design new generations of kinase specific and it yields spatial heat maps that can be visualized on 3D molecular struc- inhibitors. tures or in the form of interaction networks. The analytics is applied to the detection of hinge-bending and contact breaking hot spots in folding and un- 1585-Plat folding simulations of proteins. We demonstrate that the FIM method is su- Large Scale Molecular Dynamics Simulations of Three Classical Cadher- perior to linear cross-correlation for the detection of nonlinear dependence in ins Involved in Cell Adhesion challenging situations where measures for the global protein dynamics Brandon Neel, Collin Nisler, Raul Araya-Secchi, Marcos Sotomayor. diverge. We are also extending the analysis for the first time to fluids and Chemistry & Biochemistry, The Ohio State University, Columbus, OH, fluid-like systems such as lipid bilayers and solvent. The application takes USA. advantage of our earlier graph-based activity functions by accounting for Cadherins are a family of proteins responsible for intercellular adhesion be- the contact forming and breaking activity of the water hydrogen-bond tween epithelial cells and for maintaining tissue integrity. E-cadherin, found network and of lipids in the bilayer. Our novel analysis of lipid interaction in adherens junctions, and desmogleins and desmocollins, found in desmo- networks under periodic boundary conditions shows that the disruption of somes, are three classical-type cadherins that interact through their extracel- the bilayer, as measured by the breaking activity, is associated with the lular domains. These have five extracellular cadherin (EC) repeats that link externally imposed pore formation. As a result of our analysis, relatively adjacent cells through a tip-to-tip interaction mediated by an exchange of short simulations of electroporation can provide clues on the nucleation conserved tryptophans in their distal extracellular repeats. Here we present events relevant for bioelectrics experiments on much longer time scales. large-scale molecular dynamics simulations that model the complete extracel- The FIM implementation is freely disseminated with our open-source pack- lular domains of available crystal structures, with systems encompassing up to age, TimeScapes. 5 million atoms. Equilibrium and steered molecular dynamics simulations of

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E-cadherin lattices predict a two-phase elastic response in which E-cadherin conformational transitions and pathways to folded states, since simulations complexes first straighten and then unbind without unfolding. Cis- are always at equilibrium. interactions are predicted to play a minor role in the mechanical response of these complexes. Simulations of the desmoglein/desmocollin systems 1588-Plat show that forces required to unbind both the homodimer of desmoglein-2 Temperature Dynamics of Single Molecular Antifreeze Protein 1,2 3,4 3,4 1 and a canonical heterodimer of desmoglein-2 and desmocollin-1 are similar, Rio Okada , Tatsuya Arai , Daichi Fukami , Yuhuku Matsushita , 1 5 5 6 even in the presence of a salt-bridge shown to be important for the hetero- Jae-won Chang , Hiroshi Sekiguchi , Noboru Ohta , Tadashi Mori , 7 8 8 1 dimer interaction. As with the E-cadherin system, both the desmoglein dimer Masaki Nishijima , Keisuke Miyazawa , Takeshi Fukuma , Keigo Ikezaki , Sakae Tsuda3,4, Yuji C. Sasaki1,5. and desmoglein/desmocollin systems exhibited unbinding after an initial 1 straightening of both molecules, with no unfolding. Overall, our simulations Graduate School of Frontier Science, The University of Tokyo, Chiba, Japan, 2National Institute of Advanced Industrial Science and Technology provide insights into the molecular mechanics of adherens junctions and 3 desmosomes. (AIST), OPERANDO-OIL, Tokyo, Japan, Graduate School of Life Science, The University of Hokkaido, Hokkaido, Japan, 4Bioproduction Research 1586-Plat Institute, National Institute of Advanced Industrial Science and Technology Towards a Rational Design of Macrolide Antibiotics in Order to Combat (AIST), Hokkaido, Japan, 5Research & Utilization Division, Japan Bacterial Resistance Synchrotron Radiation Research Institute, SPring-8, Hyogo, Japan, 6Graduate Anna Pavlova1, Jerry M. Parks2, Adegboyega K. Oyelere3, School of Engineering, The University of Osaka, Osaka, Japan, 7Office for James C. Gumbart1. University-Industry Collaboration, The University of Osaka, Osaka, Japan, 1Physics, Georgia Institue of Technology, Atlanta, GA, USA, 2Center of 8School of Electrical and Computer Engineering, The University of Molecular Biophysics, Oak Ridge National Laboratory, Oak Ridge, TN, Kanazawa, Kanazawa, Japan. USA, 3Chemistry & Biochemistry, Georgia Institue of Technology, Atlanta, AntiFreeze Proteins (=AFPs) bind to a surface of ice crystals and inhibit their GA, USA. growth. Some living organisms; fishes, insects and fungus, at low-temperature Macrolides, which bind in the protein exit tunnel of the bacterial ribosome, are environment have several different types of AFPs. AFPs protect their body one of the most prescribed classes of antibiotics.However, macrolide resistance from freezing damages. To clarify the antifreeze effect of lpAFP (isolated caused by ribosomal modifications at base A2058 in segment 23S is a growing from longsnout poacher, a fish living in the sea of Okhotsk) at single molec- problem. It has been foundthat attachment of aromatic moieties can increase- ular level, we performed the single molecular observation using diffracted macrolide activity against both wild-type and macrolide-resistant ribosomes X-ray tracking (=DXT). DXT is the method to observe single molecular mo- due to additional aromatic interactions with rRNA, resulting in new antibiotics, tions using X-rays and gold nanocrystals as probe. Gold nanocrystals are e.g., telithromycin. Our previous simulations of the stalling peptideSecM in the labeled on AFPs, and when irradiated with X-rays, diffracted spots from ribosome revealed stable aromatic interactions between residue W155 in SecM gold nanocrystals can be observed. We can observe the motion of the AFPs and base A751 in segment23S, inspiring the development of azithromycin by tracking these spots. We performed the DXT experiments at SPring-8 derivatives containing indole-analog moieties that could mimic these interac- BL40XU, and the spatial and time resolutions are the order of milli-radian tions. Several of these derivatives showed improved activity against wild-type- and micro-second. In this study, we used lpAFP isolated from longsnout Escherichia coli compared to azithromycin, although, a better understanding of poacher, and observed the temperature dependency of the lpAFP’s single mo- the structure-activity relationship for the different moietiesis needed for further lecular brownian motion. As a result, lpAFP’s motion increased at 5 C. Next, improvement. to ensure the relationship between the adsorption property and the maximum Here, we used molecular dynamics simulations to study erythromycin and azi- brownian motion, we observed the temperature dependency of the adsorption thromycin in wild-type and A2058-modified, E. coli ribosomes. The ribo- affinity to AgI thin film as ice surface. As a result, the adsorption amount to somal modifications resulted in less favorable interactions between base AgI of lpAFP was the largest at 2C. These two experiments suggest that there 2058 and the desosamine sugar of the macrolides as well as a greater displace- is a strong correlation between lpAFP’s molecular motion and adsorption ment of the macrolides, explaining the causes for resistance. Additionally, property. We acquired the temperature dependency of lpAFP using other two of the azithromycin derivatives noted above were simulated in the experiments; circular dichroism (CD), atomic force microscopy (AFM), wild-type ribosome. We found that the added indole-analog moieties adopted dynamic light scattering (DLS), and small angle X-ray scattering (SAXS, different geometries when interacting with base A751, which could explain performed at SPring-8 BL40B2). In poster session, we discuss the detail the differences in their activity. Our results illustrate the utility of MD simu- experimental procedure and results. lations in the design of a new generation of macrolides that can overcome bac- terial resistance. Platform: Mitochondria in Cell Life and Death

1587-Plat 1589-Plat Fast Forward Protein Folding Charcot-Marie-Tooth Type2A Mfn2 Domain-Specific Mutants Deferen- Maxwell I. Zimmerman, Gregory R. Bowman. tially Alter Mitochondrial Fusion Dynamics and Motility Biochemistry and Molecular Biophysics, Washington University in St. Louis, Veronica Eisner1, Diego Troncoso1, Pamela Rojas1, Josefa Vial1, St. Louis, MO, USA. Mauricio Castro1, Sergio Henrı´quez1, Rita Horvath2. Molecular dynamics simulations are increasingly used to understand complex 1Universidad Cato´lica de Chile, Santiago, Chile, 2Newcastle University, phenomenon such as protein folding; simulations can provide full atomistic Newcastle, United Kingdom. details on a level that is currently unavailable to many experimental ap- Mitochondrial function relays on the balance between fusion and fission proaches. Despite this utility, computational resources often limit the time- events that determine mitochondrial communication. Mutations of outer mito- scales accessible to simulations and prevent the observation of protein chondrial membrane (OMM) fusion protein Mfn2, cause motor neuron degen- folding events (i.e. even the fastest folding events occur in the ms-ms regime, eration disease Charcot-Marie-Tooth Type 2 (CMT2). Also, Mfn2 plays a yet typical desktop computers only simulate a few ns per day). In lieu of controversial role in the ER-mitochondrial communication. Thus, it is un- increasing computer power by orders of magnitude, better algorithms for known if Mfn2 CMT2-associated domain-specific mutations alter mitochon- reaching these timescales must be developed. Adaptive sampling algorithms drial fusion dynamics, ER/mitochondria communication, or both. Here we reduce required simulation time by taking advantage of many simulations study mitochondrial fusion dynamics in human fibroblasts isolated from run in parallel, for which simulations can be pieced together with the con- skin samples of control and CMT2-confirmed patients from the Centre for struction of a Markov State Model. Adaptive sampling schemes can be Neuromuscular Diseases Biobank, Newcastle. The cells were transfected described as an undirected approach that follow four main steps: 1) run sim- mitochondrial matrix-targeted mtDsRed and photo-switchable protein mtPA- ulations, 2) build a Markov State Model from current data, 3) analyze the GFP. By means of 408 nm mediated photo-conversion and time series discovered states, and 4) start new simulations from structures that have confocal imaging, we studied mitochondrial continuity and fusion events been analyzed. Structures that are used to restart simulations are traditionally (f.e.) frequency. We evaluated two domain-specific Mfn2 mutants: L248H chosen to optimize some statistical ranking. Here, we present a new sampling (GTPase domain) and M376V (GTPase to HR1 connector). Mfn2-L248H car- scheme that additionally ranks states based on physical traits and can follow rying fibroblasts display hyperfused mitochondria and f.e. frequency inhibi- gradients in conformational space, which we demonstrate reduces the simula- tion 0.42 f.e./min as opposed to 1.2 f.e./min in control fibroblasts. tion time required to observe protein folding events. In contrast to simulation Moreover, Mfn2-M378V fibroblasts showed inhibition of both mitochondrial techniques that perturb energy landscapes, such as steered molecular dy- continuity and f.e. frequency (0.45 f.e./min). In addition, the ultrastructure of namics, this new sampling scheme provides thermodynamically accurate control and Mfn2-M378V fibroblasts, evaluated by Transmission Electron

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Microscopy, showed two populations of mutant mitochondria: high frequency and JNK inhibitor SP600125 prevented mitochondrial superoxide formation of small round-shaped organelles, consistent with inhibition of mitochondrial and mitochondrial dysfunction. fusion, and a group of enlarged mitochondria displaying partially empty Conclusion: Antagonists of VDAC-tubulin interaction promote mitochondrial cristae. Finally, mitochondrial motility was significantly elevated in Mfn2- metabolism and increase mitochondrial ROS formation, which in turn leads to M378V fibroblasts, suggesting that Mfn2 GTPase to HR1 connector may JNK activation, mitochondrial dysfunction and selective death of cancer cells have a role in the interaction with the mitochondrial motility machinery. that is prevented by antioxidants and JNK inhibition. Thus, CMT2A-causing Mfn2 specific-domain mutations display differential defects in human mitochondrial ultrastructure and fusion dynamics. Funding 1592-Plat FONDECYT 1150677 to VE. Manipulation of Amyloid Precursor Protein Processing Impacts Brain Bioenergetics and Glucose Metabolism 1590-Plat John A. Findlay1, D. Lee Hamilton2, Sarmi Sri1, Mariana Vargas-Caballero1, Yeast VDAC2 has Pore Forming Activity: Functional Consequences Michael L.J. Ashford3, Peter J.S. Smith1. Maria Carmela Di Rosa, Andrea Magri, Simona Reina, Vito De Pinto. 1Institute for Life Sciences, University of Southampton, Southampton, Biometec, University of Catania, Catania, Italy. United Kingdom, 2Health and Exercise Sciences, University of Stirling, The pore-forming proteins Voltage Dependent Anion Channel (VDAC) on Stirling, United Kingdom, 3Molecular & Clinical Medicine Division, the mitochondrial outer membrane (MOM) mediate the metabolic exchanges University of Dundee, Dundee, United Kingdom. between cytosol and mitochondria. The pore has been characterized in eu- The amyloid precursor protein (APP) and one of its terminal cleavage prod- karyotes spanning from insects [1,2] to mammals [3]. In S. cerevisiae,the ucts, amyloid beta (Ab) are proposed to play a central role in the pathogen- por1 gene encodes for the main porin yVDAC1, with electrophysiological esis of Alzheimer’s disease (AD). These proteins have also been shown to features similar to the human homologous [4]. yVDAC1 regulates yeast co-localise within mitochondria with Ab directly inhibiting the electron viability in non-fermentable conditions since the genetic inactivation of transport chain. Cleavage of APP occurs down distinct pathways. Initial por1 (Dpor1 strain) reduces dramatically the yeast growth on glycerol [4]. cleavage of the full-length protein is mediated by either a-orb-secretase In yeast genome a por1 paralog, por2, was found, but the putative porin (BACE1). Subsequent cleavage by g-secretase releases p3 or Ab protein yVDAC2 was definitely less characterized than VDAC1, despite 50% of fragments into the extracellular milieu. Whilst these interactions with mito- sequence identity. Many evidences have questioned the ability of yVDAC2 chondria have been noted, the impact of manipulating APP processing by to act as a pore on the MOM. E.g., the por2 inactivation does not affect yeast these distinct pathways on cellular metabolism and bioenergetics has yet growth on glycerol; furthermore, no pore-forming activity of yVDAC2 was to be elucidated. We utilised the human SH-SY5Y neuronal cell line stably noticed so far in electrophysiological systems. Nevertheless, if overex- overexpressing BACE1 and cortical slices from mice bearing inducible over- pressed, yVDAC2 can functionally complement the absence of yVDAC1 in expression of APP harbouring the Swedish and Indiana mutations, favouring Dpor1 [4]. This was recently confirmed by our work, showing that por2 over- Ab production. Protein expression and enzyme activity were determined via expression, a consequence of human SOD1 expression, completely restores western blotting and enzyme-linked immunosorbance assays (ELISA). A Dpor1 yeast growth on glycerol, as well as the mitochondrial functionality Seahorse extracellular flux analyser and radio-labelled substrate assays [5]. In this work, we have characterized the electrophysiology of recombinant were used to determine cellular bioenergetics in real-time. All data are ex- yVDAC2 and its features in model cell. The influence of the cysteine oxida- pressed as mean 5 standard error of the mean and statistical significance tion was investigated following recent achievements in other eukaryotic determined by Student’s t-test. Manipulation of APP cleavage resulted in al- VDAC isoforms [6-7]. terations of 2-deoxyglucose uptake into cells. Chronic elevation in BACE1 1. De Pinto V et al, Biochim Biophys Acta (1989) 987:1-7 resulted in impaired functioning of a key fuel-partitioning enzyme, pyruvate 2.Aiello et al, J Biol Chem (2004) 279:25364-73 dehydrogenase (PDH, activity reduced to 69 5 8%, p < 0.05, n=6). This 3. Menzel VA et al, Biosc Rep (2009) 29:351-62 reduced substrate delivery to the mitochondria and increased reliance upon 4. Blachly-Dyson E et al, Mol Cell Biol (1997) 17:5727-38. aerobic glycolysis for ATP generation (oxygen consumption rate (OCR) 5. Magrı` A et al, Biochim Biophys Acta (2016) 1857:789-98. reduced to 65 5 9%, p < 0.01, n=7 and extracellular acidification rate 6. Reina S et al, Oncotarget (2016) 7:2249-68 (ECAR) increased to 165 5 16%, p < 0.01, n=7). Thus BACE1 overexpres- 7. De Pinto V et al, Biochim Biophys Acta (2016) 987:1-7 sion impairs neuronal glucose oxidation resulting in an increased reliance upon aerobic glycolysis for ATP generation. Acute cortical overexpression 1591-Plat of APP favouring amyloidogenic processing resulted in a significant reduc- Oxidative Stress Induced by Vdac Opening in Cancer Cells Depends on tion in PDH protein expression (reduced to 62 5 6%, p < 0.01, n = 9-10) Cytosolic Free Tubulin and is Blocked by ROS Scavenging and Suppres- and activity of key tricarboxylic acid cycle enzymes, isocitrate dehydroge- sion of Superoxide Formation by Complex III nase and alpha-ketoglutarate dehydrogenase (reduced to 62 5 7%, p < 1 1 1 1 Diana Fang , Kareem Heslop , Morgan Morris , David DeHart , 0.01, n=6 and 88 5 2%, p < 0.001, n = 6). Therefore, manipulation of 1 1,2 1 Monika Beck Gooz , John J. Lemasters , Eduardo N. Maldonado . APP processing towards Ab production in cells and tissue significantly im- 1Drug Discovey & Biomedical Sciences, Medical University of South 2 pacts cellular metabolic pathways at the level of glucose uptake, substrate Carolina, Charleston, SC, USA, Institute of Theoretical and Experimental entry to the mitochondria and the TCA cycle. Biophysics, Pushchino, Russian Federation. Background: Voltage dependent anion channels (VDAC) control flux of me- 1593-Plat tabolites into mitochondria. Free a,b-tubulin decreases VDAC conductance Mitochondrial NM23-H4/NDPK-D and OPA1: Partners in Shaping Mito- and high free tubulin in cancer cells decreases mitochondrial membrane poten- chondria and Initiating Mitophagy? tial (DJ). Erastin antagonizes the inhibitory effect of tubulin on VDAC. Here, Uwe Schlattner1,Ce´line Desbourdes1,Ma1gorzata Tokarska-Schlattner1, we hypothesized that erastin and erastin-like compounds (X1 and X2) discov- Valerian E. Kagan2. ered during high-throughput screening of a small molecule library, increase 1Laboratory of Fundamental and Applied Bioenergetics (LBFA), Univ. mitochondrial formation of reactive oxygen species (ROS) and activate c-jun Grenoble Alpes, Grenoble Cedex 9, France, 2Department of Environmental N-terminal kinase (JNK), culminating in mitochondrial dysfunction and death and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA. of cancer cells. Our AIM was to evaluate the effects of N-acetyl-L-cysteine The well-established function of the mitochondrial intermembrane space (NAC), S3QEL-3 and paclitaxel (PTX) on mitochondrial dysfunction induced protein NM23-H4/NDPK-D is phosphotransfer activity as a nucleoside by erastin/erastin-like compounds. diphosphate kinase (NDPK). However, recent data have revealed a second Methods: Confocal fluorescence microscopy assessed DJ (TMRM) and ROS function in lipid signaling that triggers mitophagy, a critical process for (cmDCF; MitoSOX Red). JNK was assessed by Western blotting and cell cell homeostasis (1,2). This latter function involves NM23-H4-mediated killing by propidium iodide fluorometry. intermembrane transfer of cardiolipin (CL) from the mitochondrial inner Results: In HepG2 and Huh 7 hepatocellular carcinoma cells, erastin and X1 membrane to the mitochondrial surface. Interestingly, both functions seem increased DJ and NAD(P)H and activated JNK with maximal phosphorylation to involve an interaction of NM23-H4 with OPA1, a dynamin-like GTPase within 1-2 h. Initial increases of DJ were followed by mitochondrial depolar- of the mitochondrial inner membrane. First, NM23-H4 directly fuels ization occurring 1-2 h after X1 and X2 and 3-4 h after erastin. X1 did not alter OPA1 with GTP via its NDPK bioenergetic function (3). In addition, also free/polymerized tubulin. The microtubule stabilizer PTX (10 mM) depleted the CL transfer activity of NM23-H4 seems to depend on OPA1, since cytosolic free tubulin and prevented depolarization induced by X1. The antiox- knock-down of OPA1 in HeLa cells reduces CL transfer in NM23-H4 WT idant NAC (100 mM) blocked mitochondrial dysfunction and cell death induced expressing cells as compared to those expressing CL-transfer incompetent by X1. S3QEL-3 (100 mM), a suppressor of superoxide production at site IIIQo NM23-H4 mutants. Thus, OPA1 seems to be a negative regulator of the

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CL transfer function of NM23-H4. Our current model suggests that NM23- of muscle fibers derived from the ALS G93A mice. Mitochondria are the ma- H4/OPA1 complexes exist in healthy mitochondria at the inner membrane to jor source of ROS production. In order to detect early changes in ROS-related maintain OPA1 functions in membrane fusion and dynamics. Upon OPA1 mitochondrial metabolic function, we generated double transgenic mice cleavage, an early step during mitophagy, NM23-H4 may be released (G93A/cpYFP) that carry human ALS mutation SOD1G93A and mt-cpYFP from these complexes, allowing simultaneous interaction of the hexameric transgenes, in which mt-cpYFP monitors dynamic changes of ROS-related NM23-H4 complex with inner and outer mitochondrial membrane and CL mitoflash events at the single mitochondrion level. Remarkably, G93A muscle transfer. cells show early and disease stage-dependent changes in mitoflash events. The (1) Schlattner et al. (2015) Naunyn Schmiedebergs Arch. Pharmacol. 388, mitoflash activity at the age of 2 months (before ALS symptom onset) is 271-8. marked by an increased flashing area in G93A muscle fibers, while their ki- (2) Kagan et al. (2016) Cell Death Diff. 23, 1140-51. netics properties remain unchanged. After ALS onset (3-month old), there (3) Boissan et al. (2014) Science 344, 1510-5. are drastic changes in the kinetics of mitoflash signal with prolonged FDHM (full duration at half maximum) of the mitoflash signal. Thus, mito- 1594-Plat flash provides a sensitive and quantitative biomarker for evaluating ROS- Emerging Role of the Mitochondrial Outer Membrane Translocator related mitochondrial dysfunction. It is known that uncontrolled opening of Protein (TSPO) in Heart Failure and Mitochondrai Quality Control mitochondrial permeability transition pore (mPTP) is a key step to promote Phung N. Thai1, Daniel Daugherty1, Bert J. Frederich2, Samuel Galice1, mitochondrial ROS production. While the molecular composition of mPTP Wenbin Deng1, Donald M. Bers1, Saul Schaefer1, Elena N. Dedkova1. is still incompletely understood, studies have shown that cyclophilin D 1 2 University of California, Davis, CA, USA, University of California, San (CypD) promotes the opening of mPTP and phosphorylated form of GSK- Francisco, CA, USA. 3b participates in maintaining mPTP in the closed state. We used subcellular Introduction: Mitochondrial dysfunction is a hallmark of heart failure (HF), fractionation to isolate mitochondria from skeletal muscle of G93A mice, and resulting in decreased energy production and impaired mitochondrial quality found an increased CypD expression level in muscle mitochondria, while the control (mitophagy) with resultant contractile dysfunction and cell death. phosphorylated form of GSK-3b was significantly reduced in mitochondria of The 18-kDa mitochondrial translocator protein (TSPO) has been considered G93A muscle. Thus, both may act as triggering factors for mPTP opening in a part of the mitochondrial permeability transition pore (mPTP) for a long G93A muscle. time; however, its role in mPTP regulation is now debatable and the precise role of the TSPO in cardiac physiology and HF remains poorly understood. 1596-Plat Objective: Determine the role of TSPO in a murine pressure-overload model An Alternative Splice Variant of Chloride Intracellular Channel 5 Protein, of HF. (CLIC5B) Regulates Cardiac Mitochondrial Localization and Function of Methods and Results: Conditional, cardiac-specific TSPO knockout (KO) CLIC5 mice were generated using the Cre-loxP system. Pressure overload by trans- Devasena Ponnalagu, Ahmed Tafsirul Hussain, Shubha Gururaja Rao, verse aortic constriction (TAC) for 8 weeks significantly increased TSPO Harpreet Singh. expression in wild type (WT) mice, but not in KO mice. While WT TAC Pharmacology and Physiology, Drexel University College of Medicine, mice showed a marked reduction in systolic function, KO TAC mice did not Philadelphia, PA, USA. have a significant reduction in ejection fraction, and also exhibited fewer clin- Mitochondrial bioenergetics is critical for the cell survival. Mitochondrial ical HF signs (less cardiac dilation and fibrosis). TAC reduced mitochondrial ion channels like BKCa and KATP play a significant role in cardio- calcium uptake and enhanced mPTP opening in permeabilized myocytes protection from ischemia-reperfusion (IR) injury possibly by modulating from WT animals. Calcium uptake was restored in TAC KO mice without sig- the mitochondrial physiology. Interestingly, inner mitochondrial membrane nificant effect on mPTP activity. The restoration of the mitochondrial calcium (IMM) channels lack canonical mitochondrial targeting sequence (MTS) uptake coincided with reduced oxidative stress, improved oxygen consumption but localize to mitochondria. In case of mitoBKCa, a c-terminal splice rate, and enhanced ATP generation and mitochondrial reserve capacity. In WT variant, DEC, (BKCa-DEC) was reported to target BKCa to mitochondria TAC, elevated mitochondrial accumulation of mCherry-Parkin1 was not of adult cardiomyocytes. Recently, we reported chloride intracellular chan- coupled to the increase in LC3-autophagosomes formation indicating impaired nel (CLIC) proteins, specifically, CLIC4 and CLIC5 are enriched in the mitophagy. In contrast, mitophagy was normalized in TSPO KO. outer and inner membrane of rat cardiac mitochondria, respectively, in Conclusions: These data suggest a novel mechanism to prevent HF at the spite of lacking any of the classical MTS. Surprisingly, two distinct bands cellular level via normalization of TSPO levels which leads to preservation of CLIC5 at ~30 kDa (CLIC5A) and ~50 kDa, were observed in the rat car- of mitochondrial calcium uptake, oxidative phosphorylation, and mitochon- diac mitochondrial lysates which could be attributed to the presence of drial mt-QC resulting in preservation of mitochondrial integrity and cell another isoform (~50 kDa, CLIC5B). Our recent findings further demon- survival. strate that variants CLIC5A and CLIC5B are present in the heart, but lungs and kidneys lack CLIC5B. Also, CLIC5 localized to the Percoll-purified 1595-Plat mitochondria isolated from rat heart (69.13 5 12.2%, n=3) and rat cardi- Dysregulation of Mitochondrial Permeability Transition Pore (MPTP) is omyocyte cells, H9C2 (55.12 5 7.1%) but not in mouse lung epithelial Associated with Enhanced ROS Production in Skeletal Muscle of an (11 5 2.1 %) cells which lacked CLIC5B. Thus, suggesting that the splice ALS Mouse Model variant of CLIC5, promotes its localization to the IMM. Also, we observed Yajuan Xiao1,2, Jianxun Yi1, Chehade Karam2, Xuejun Li1, Kamal Dhakal1, that cardiac mitochondria isolated from clic5-/- mice exhibited increased Dosuk Yoon1, Jingsong Zhou1,2. reactive oxygen species (ROS, p< 0.05, n=3) production whereas the 1Kansas City University of Medicine and Biosciences, Kansas City, MO, clic5-/- lung mitochondria did not show any change, supporting the role USA, 2Molecular Biophysics and Physiology, Rush University, Chicago, IL, of CLIC5B in regulating the mitochondrial function. Our study further es- USA. tablishes splice variation as a conserved mechanism for targeting ion chan- Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease. Oxida- nels to mitochondria. As CLICs are cardioprotective, understanding tive stress is implicated in ALS pathophysiology. Using ROS BriteÔ 570, a mitochondrial localization of CLIC5 will further aid in development of cytosolic ROS detector, we found a significant increase in ROS production novel therapeutics.

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Platform: General Protein-Lipid Interactions II mediated transfer of the intrinsically fluorescent sterol, dehydroergosterol (DHE). Using the method, we have studied how point mutations in the binding 1597-Plat pocket of PITPa and the yeast Sec14-like PtdIns transfer protein, Sfh3, affect Lipid Perturbation by Membrane Proteins and the Lipophobic Effect PC/PI and DHE binding and transfer respectively. The advantages of using Jean-Pierre Duneau, Jon Khao, James N. Sturgis. fluorescently labeled lipids to measure protein mediated transfer includes LISM - CNRS / Aix-Marseille Univ., Marseille, France. time-resolved data acquisition and greater sensitivity compared to other estab- Understanding how membrane proteins interact with their environment is lished methods. The possible artefacts introduced by the pyrene label can, how- fundamental to the understanding how the fold and interact with each other. ever, not be dismissed. To address this, we have used a well-established in vitro We report coarse grain molecular dynamics simulations on a series of mem- radiolabeled transfer assay to verify and validate the results obtained using the brane proteins in a membrane environment and describe how the lipids are per- fluorescence assay. These results will help to increase the understanding of how turbed by the presence of proteins. We analyze these perturbations in terms of cells utilize PITPs in regulating lipid signaling cascades. elastic membrane deformations and local lipid protein interactions. 1600-Plat However these two factors are insufficient to describe the variety of effects that Pi(4,5)P2 Clustering by the Ebola Virus Matrix Protein VP40 we observe and the changes caused by membranes proteins to the structure and Jeevan B. GC, Bernard S. Gerstman, Prem P. Chapagain. dynamics of their lipid environment. Other factors that change the conforma- Physics, Florida International University, Miami, FL, USA. tion available to lipid molecules are evident and are able to modify lipid struc- The Ebola virus protein VP40 is a peripheral protein that is trafficked to the lower ture far from the protein surface, and thus mediate long-range interactions leaflet of the plasma membrane. Upon membrane localization in its dimeric between membrane proteins. We suggest that these multiple modifications to form, VP40 undergoes major structural rearrangements to form hexamers which lipid behaviour are responsible, at the molecular level, for the lipophobic effect further assemble into filaments and form the viral matrix that provides the major we have proposed to account for our observations of membrane protein structure for the Ebola virus particles. VP40 hexamers and larger filaments have organization. been shown to be stabilized by PI(4,5)P2 in the plasma membrane inner leaflet. Reduction in the plasma membrane levels of PI(4,5)P significantly reduce the 1598-Plat 2 formation of VP40 oligomers and virus-like particles. We computationally inves- The Myristoyl Modification of C-SRC Alters the Lateral Organization of tigated the lipid-protein interactions in VP40 hexamers at the plasma membrane Host Heterogenous Raft-Like Membranes and quantified lipid-lipid self-clustering by calculating the fractional interaction Mridula Dwivedi1, Tom Mejuch2, Herbert Waldmann2, Roland Winter1. matrix. We found that the VP40 hexamer significantly enhances the PI(4,5)P 1Department of Chemical Biology and Physical Chemistry, Technical 2 clustering. The radial pair distribution functions suggest a strong interaction be- University Dortmund, Dortmund, Germany, 2Department of Chemical tween PI(4,5)P and the VP40 hexamer. The cationic side chains are found to Biology, Max Planck Institute of Molecular Physiology, Dortmund, 2 mediate the PI(4,5)P clustering around the protein, with cholesterol filling the Germany. 2 space between the interacting PI(4,5)P molecules. These computational studies Lipidated motifs of membrane associated peripheral proteins play a crucial role 2 support recent experimental data and provide new insights into the mechanisms in targeting signalling modules on the plasma membrane and trafficking by which VP40 assembles at the plasma membrane inner leaflet, alters mem- biochemical signalling pathways. Membrane bound c-Src (from Rous sarcoma brane curvature, and forms new virus-like particles. virus) tyrosine kinase nonreceptor, unlike other acyl modified lipid anchored proteins, anchors to the membrane via a myristoyl chain along with a polybasic 1601-Plat residue stretch, which notably is much shorter in chain length than its host Interaction of PH Domains with Phosphatidylinositol Phosphates: membrane. The packing defect arising from this mismatched chain length of Structures and Energetics by Simulation the host and the anchor lipid chain significantly affects the lateral organization Fiona B. Naughton1, Antreas C. Kalli2, Mark S.P. Sansom1. of phase separated raft-like heterogeneous membranes. In the present work, we 1Department of Biochemistry, University of Oxford, Oxford, United reveal the mixing of phase domains and formation of novel nanoscale-clusters Kingdom, 2Faculty of Medicine and Health, University of Leeds, Leeds, upon binding of the Myr-Src (2-9) peptide. High resolution images were ob- United Kingdom. tained using atomic force microscopy. PIE-FCCS was used to explore the na- Pleckstrin Homology (PH) domains are among the most prevalent domains in ture of these clusters. It was found that Myr-Src (2-9) does not self-cluster or the human proteome. Many have been shown to associate with phosphatidyli- oligomerize, but rather forms lipid platforms, by some kind of sorting mecha- nositol phosphate (PIP) lipids, mediating interactions between peripheral pro- nisms, around itself. Further, confocal fluorescence microscopy was used to teins and cell membranes in a range of vital cellular processes. Different PH visualize the binding of the peptide to giant unilamellar vesicles, and partition domains bind to different PIP species with a range of specificities and affinities. coefficients of the peptide could be obtained for neutral and anionic lipid mix- The exact molecular and energetic details behind these interactions are not tures. Remarkably, we found that anionic lipids were able to shift the partition always clear, partly because of the complexity of these interactions. Through coefficient towards the lo phase. use of coarse-grained molecular dynamics simulations combined with an um- brella sampling methodology to generate a potential of mean force profile, 1599-Plat the binding of PH domains to PIP-containing model membranes may be quan- The Use of Fluorescently Labeled Lipids to Determine Dynamics of Protein tified and investigated on a molecular scale. This approach has previously been In Vitro Mediated Lipid Transfer applied to the PH domain of GRP1. In this study, we expand this analysis to Max Lo¨nnfors1, Aby Grabon1,2, Kaitlyn R. McGrath3, Ashutosh Tripathi1, compare 12 PH domains known to associate with PIPs, obtaining free energy Marta G. Lete1, Pentti Somerharju4, Vytas A. Bankaitis1,5. profiles for binding of each to PIP3 and PIP2. Favourable binding was observed 1Department of Molecular and Cellular Medicine, Texas A&M Health 2 in all cases. PH-PIP interactions formed while bound at the membrane were Science Center, College Station, TX, USA, Lineberger Comprehensive consistent with structural data. The presence of an additional ‘atypical’ PIP Cancer Center, University of North Carolina School of Medicine, Chapel binding site (which may be involved in coincidence detection) adjacent to Hill, NC, USA, 3Department of Biochemistry and Biophysics, Texas A&M 4 the ‘canonical’ site was demonstrated, including identification of atypical- University, College Station, TX, USA, Department of Biochemistry and site interactions in domains for which previously only canonical binding had Developmental Biology, University of Helsinki, Helsinki, Finland, 5 been reported. Domains exhibiting initial atypical-site interactions show in Department of Chemistry, Texas A&M University, College Station, general less favourable binding. This work demonstrates how coarse-grained TX, USA. umbrella-sampling simulations may be used to investigate and compare the In recent years, phosphatidylinositol (PtdIns) transfer proteins (PITPs) have energy landscape and molecular details of protein-lipid interactions within a been established as essential regulators of phosphoinositide signaling outcomes family of molecular recognition proteins. in eukaryotic cells. The key function of these proteins is to mediate a PtdIns/ phosphatidylcholine (PtdCho) exchange reaction. This function is central to 1602-Plat their abilities to regulate lipid signaling. Despite its importance the mechanistic Running Against Time - A Kinetic Limit for Endophilin Action during details of this lipid exchange remain largely undetermined. In order to decipher Rapid Endocytosis some aspects of the exchange reaction, we have modified an in vitro lipid Kumud Raj Poudel1, Hang Yu2, Klaus Schulten2, Jihong Bai1,3. transfer method first described by Somerharju et al. (Biochemistry, 1984). 1Fred Hutchinson Cancer Research Center, Seattle, WA, USA, 2University of By utilizing pyrene-labeled PtdCho and PtdIns lipid species the membrane Illinois Urbana-Champaign, Urbana, IL, USA, 3University of Washington, extraction and lipid transfer mediated by the mammalian StART-like PITP, Seattle, WA, USA. PITPa (and other lipid transfer proteins) has been fluorescently monitored. Time is a limiting factor for many cellular processes. Neurons are particularly We have also further modified the method to include the measuring of protein sensitive to time as they are specialized for rapid communication. For instance,

BPJ 7825_7838 Tuesday, February 14, 2017 327a synaptic vesicle endocytosis could occur within 50 milliseconds to recycle membrane surfaces, amplifying steric pressure. More generally, our findings vesicle membranes from the plasma membrane. By contrast, endocytosis in suggest that under crowded conditions, any membrane-bound protein, regard- non-neuronal cells occurs slowly, often taking minutes to complete. While a less of structure, can contribute to membrane fission. In line with this reasoning similar set of protein machinery is involved in both slow and rapid endocytosis, we find that intrinsically disordered proteins, which have large hydrodynamic it is unknown whether the difference in time duration impacts the action of radii yet lack a defined structure, drive fission with substantially greater po- these proteins. To address this issue, we have investigated the dynamic action tency than the structured ENTH domain. of endophilin. Endophilin is a critical protein for both rapid and slow endocy- tosis. Biochemical studies have shown that endophilin converts flat membranes Platform: Membrane Protein Dynamics into bilayer tubules and vesicles in vitro. To examine how endophilin acts within physiological time windows to remodel membranes, we have developed 1605-Plat kinetic assays to monitor how endophilin binds membranes, induces local Modulated Dynamics of Pam-a7 nAChR From X-Ray Single Molecular membrane deformation, and produces large-scale changes of membrane Observations morphology. We find that endophilin undergoes a sensing-to-bending transi- Tomoyuki Baba1,2, Tai Kubo2,3, Sumiko Ohashi3, Keigo Ikezaki1,2, tion, which takes about 30 milliseconds. These results show that endophilin Kazuhiro Mio2,3, Hiroshi Sekiguchi4, Yuji C. Sasaki1,2. 1 does not immediately deform membranes upon its arrival. The bending func- Graduate School of Frontier Sciences, The University of Tokyo, Chiba, 2 tion is activated only after two amphipathic helices become ready for mem- Japan, National Institute of Advanced Industrial Science and Technology 3 brane insertion. Surprisingly, we find that endophilin is fairly slow in (AIST), OPERANDO-OIL, Tokyo, Japan, National Institute of Advanced producing large-scale membrane changes. While local membrane deformation Industrial Science and Technology (AIST), Molecular Profiling Research 4 occurs rapidly, it takes minutes for endophilin to induce the extensive morpho- Center for Drug Discovery, Tokyo, Japan, Research & Utilization Division, logical changes for membrane tubulation and vesiculation. Therefore, we pro- Japan Synchrotron Radiation Research Institute, SPring-8, Hyogo, Japan. pose that endophilin’s action depends on how much time it has on endocytic The alpha 7 nicotinic acetylcholine receptor (a7 nAChR) is a ligand-gated ion membranes. In other words, it is the kinetic property, rather than the thermody- channel mainly localized in the central nervous system. Binding of ligands like namic equilibrium, that dominates endophilin action during rapid endocytosis. acetylcholine (ACh) or nicotine induces ion flux through the channel, then it leads to the excitement of the neuronal cells and triggers the following 1603-Plat signaling cascades. As a7 nAChR plays an important role in the cholinergic Optogenetic Control of Membrane Tethering and Inter-Membrane Com- neuro-signal transduction, it is one of the drug targets for therapeutics of Alz- munications heimer disease and cognitive impairment associated with schizophrenia. For Ji Jing, Lian He, Yubin Zhou. drug development, however, structural information especially those on the mo- Texas A&M Health Science Center, Houston, TX, USA. lecular dynamics of a7 nAChR is to be revealed. Here, we adopted Diffracted Owing to its unsurpassable flexibility and spatiotemporal precision to manipu- X-ray Tracking (DXT) method for the molecular dynamic study of the a7 late biochemical pathways, the field of non-opsin-based optogenetics has been nAChR and its ligands interaction. DXT is a method to track the X-ray diffrac- gaining rapid momentum in cell biology over the past decade. By installing tion spots from the gold nanocrystal labeled on an individual single protein with light sensitivities into a series of polybasic domains (PB) that mediate phos- high-speed real time (ms) and high-accuracy real space (pm). The ligand ACh phoinositide binding in mammalian cells, we report herein the development induced both tilting and twisting motions in AChBP, while in a7 it was not of a set of single-component optogenetic tools to control protein translocation obvious, or rather calm than a ligand-free state. This may represent that the (termed ‘‘optoPB’’) toward the plasma membrane, and to further manipulate a7 receptor fell into the ‘‘desensitization’’ state. We also measured the effect membrane contact sites between the endoplasmic reticulum (ER) and the of Positive Allosteric Modulators (PAMs) of type I (Ivermectin) and type II plasma membrane (PM). We demonstrated the use of these tools to i) perturb (PNU-120596). The PAMs are electrophysiologically shown that pretreatment phosphoinositide metabolism and cell signaling on the plasma membrane; ii) of the receptor with the PAMs leads to substantial potentiation of the ACh- reversibly control the formation of ER-PM junctions with high spatial and tem- induced inward currents. The DXT measurements revealed that the binding poral precision; iii) aid the dissection of molecular determinants that govern of the type I PAM alone could induce tilting and twisting motions in the recep- protein-phosphoinositide interactions; and iv) photo-tune the distance between tor. Furthermore, we observed that ACh enhanced further the PAM-activated ER-PM junctions to restrict protein diffusion on the plasma membrane. These molecular motions in the a7 nAChR. In this presentation, the dynamics of noninvasive optical tools will likely open new possibilities for cell biologists a7 nAChR with PAMs will be discussed. to quantitatively examine the effect of chemical or protein modulators that regulate the dynamics of ER-PM junctions at defined spatiotemporal resolution 1606-Plat in living cells. (This work was supported by grants from the NIH The Role of G-Protein-Coupled Receptor Activation by Conformational (R01GM112003 to Y.Z.) and the Rober A. Welch Foundation (BE-1913 to Selection as Revealed by Single-Molecule Fluorescence 1,2 1 1,2 1,2 Y.Z.), the China Scholarship Council (to J.J.) ) Dennis D. Fernandes , Libin Ye , Yuchong Li , Zhenfu Zhang , Gregory-Neal Gomes1,2, R. Scott Prosser1,3, Claudiu C. Gradinaru1,2. 1604-Plat 1Deparment of Chemical and Physical Sciences, University of Toronto, Membrane Fission by Protein Crowding Mississauga, ON, Canada, 2Department of Physics, University of Toronto, Wilton Snead1, Carl Hayden1, Avinash Gadok1, Padmini Rangamani2, Toronto, ON, Canada, 3Department of Chemistry, University of Toronto, Jeanne Stachowiak1. Toronto, ON, Canada. 1University of Texas at Austin, Austin, TX, USA, 2University of California, G-protein-coupled receptors (GPCRs) are the largest class of transmembrane San Diego, La Jolla, CA, USA. proteins, making them an attractive target for therapeutics. At the heart of Membrane fission, which facilitates compartmentalization of biological pro- drug discovery is understanding the structural implications that ligands pose cesses into discrete, membrane-bound volumes, is essential for cellular life. on the activation states of receptors. Two models are typically used to explain Proteins with specific structural features including constricting rings, helical the mechanistic basis of ligand-activation: induced-fit and conformational se- scaffolds, and hydrophobic membrane insertions are thought to be the primary lection. Recent evidence from our groups and others (Ye, L. et al., Nature drivers of fission. In contrast, here we report a mechanism of fission that is 533,265-268, May 2016) incline towards the second model, suggesting the independent of protein structure - steric pressure among membrane-bound pro- coexistence of multiple receptor states, which, depending on the nature of teins. In particular, random collisions among crowded proteins generate sub- the ligand bound, i.e., full, partial or inverse agonist, will be more or less popu- stantial pressure, which if unbalanced on the opposite membrane surface, can lated. We acquired single-molecule fluorescence (SMF) lifetime and anisotropy dramatically increase membrane curvature leading to fission. Using the N-ter- data of Alexa488-labelled A2A adenosine receptors (A2AR) in order to probe minal homology domain of the endocytic protein epsin1 (ENTH), previously local chain-reconfiguration dynamics of the TM1, TM5, TM6, and H8 domains thought to drive fission by hydrophobic insertion, our results show that mem- of A2AR. We designed functional single-cysteine mutants for dye attachment in brane coverage correlates equally with fission regardless of the hydrophobicity close proximity to native amino-acid quenchers (i.e., histidine, tryptophan, and of insertions. Specifically, combining FRET-based measurements of membrane tyrosine). The global analysis of the intensity and anisotropy decay curves show coverage with multiple, independent measurements of membrane vesiculation that the basal state of A2AR can be described as a superposition of three states, reveals that fission becomes spontaneous as steric pressure increases, in agree- whose relative abundance shift upon binding of the full agonist, NECA, the ment with a simple physical model. Further, fission efficiency remains equally partial agonist, LUF5834, or the inverse agonist, ZM241385. To study the potent when helices are replaced by synthetic membrane-binding motifs. These implications of this model on the binding of G proteins, a peptide mimic of data challenge the view that hydrophobic insertions drive membrane fission, the a-helical domain of the Gas subunit, a374-394, was labelled with a red suggesting instead that the role of insertions is to anchor proteins strongly to fluorophore, Alexa647. Time-resolved single-molecule Fo¨rster resonance

BPJ 7825_7838 328a Tuesday, February 14, 2017 energy transfer (FRET) was performed on surface-immobilized receptors in the an algorithm to predict the smallest set of residue pairs for spin labelling that presence of the peptide. The data provided information on the binding dy- best capture the slowest dynamics in a membrane protein using molecular namics and on the variation in peptide-efficacy toward A2AR in the presence dynamics (MD) simulation datasets and a hyperparameter optimization strat- of full, partial, or inverse agonists. egy for a Markov state model (MSM). MSMs describe the transitions between the conformational states of proteins using transition probability matrices 1607-Plat obtained from MD simulation data. We use a genetic algorithm to guide MinDE Membrane Patch Oscillations Observed by High-Speed AFM the choice of residue pairs, then construct an MSM and assign it a score. Atsushi Miyagi1, Simon Scheuring1, Beatrice Ramm2, Petra Schwille2. 1 2 The score is based on the variational principle of conformational dynamics Weill Cornell Medicine, New York, NY, USA, Max Planck Institute of using a machine learning protocol. A higher score indicates that a set of dis- Biochemistry, Martinsried, Germany. tance restraints better describes the slowest processes in the dynamics and Cell division is one of the most important processes in living organisms. The hence considered as an optimal choice for experiments. We predict the Min system composed of MinC, MinD and MinE is used for definition of optimal residue pairs for DEER in ß2AR and PepTSo that capture slow confor- the cell division midpoint in E. coli. To define the midcell for cell division, mational dynamics of the proteins. Further, our choices are ranked higher the Min system makes oscillatory patterns moving from one cell pole to the than the restraints used in DEER experiments. Hence, the predicted DEER re- other. The oscillation pattern was reconstituted in vitro with fluorescence- straint choices determined from our method are an improvement over random labeled MinD and MinE, and has been observed by fluorescent microcopy, choices. showing that these two proteins are necessary and sufficient for pattern formation. The precise oscillation mechanism remains however unknown. 1610-Plat In this study we used high-speed atomic force microscopy (HS-AFM) to Multiscale Dynamics of Flavivirus Fusion Peptides - Membrane Inter- address the association and dissociation dynamics of MinDE at the nano- actions via Simulation and Experiments meter scale. We observed MinDE association and dissociation dynamics Jan K. Marzinek1,2, Nirmalya Bag2, Roland G. Huber1, as a function of three variables; these are MinD concentration, MinE concen- Daniel A. Holdbrook1, Thorsten Wohland2, Chandra Verma1,2, tration and membrane patches of variable size. We detect the association and Peter J. Bond1,2. dissociation of proteins by measuring the height of the membrane patches 1Bioinformatics Institute, A*STAR, Singapore, Singapore, 2Department of that change from 5nm, without protein (Off state), to 10nm, with protein Biological Sciences, National University of Singapore, Singapore, Singapore. (On state). Characterizing the durations of the Off- and On-states and the A primary causative agent of infectious disease is the positive single-stranded association and dissocation processes allows us to determine the detailed RNA flavivirus genus. Members of this family include Dengue (DENV), function mechanism of the MinDE cycle. Furthermore, we observed out- tick-borne encephalitis, west nile virus (WNV), japanese encephalitis, yellow of-phase On-Off cycle of patches as close together as 30nm from each other, fever and zika virus (ZIKV). During receptor-mediated endocytosis and indicating that each patch represents an individual oscillation system despite exposure to lowered pH, the envelope (E) proteins undergo major structural being exposed to the same bulk solution. Based on all our results, we build a changes and a dimeric-to-trimeric transition. This structural rearrangement model of the MinDE system that allows us to compute oscillation cycles in a leads to exposure of the hydrophobic tips - fusion peptides (FPs) which fully automated manner. interact with the endosomal bilayer, subsequently initiating fusion of the viral 1608-Plat and host membranes. The FP sequence is highly conserved among all known Structural Dynamics of Single Metabotropic Glutamate Receptors in flaviviruses. Here, a multiscale molecular dynamics (MD) simulation Solution approach has been employed to investigate the interaction of all FP sequences Anne-Marinette Cao1, Fataneh Fatemi1, Linnea Oloffson1, Suren Felekyan2, with lipid membranes, the results of which were compared with biophysical Claus Seidel2, Philippe Rondard3, Jean-Philippe Pin3, Emmanuel Margeat1. experiments. Following extensive validation using all-atom simulations with 1Centre de Biochimie Structurale, CNRS, Montpellier, France, 2Heinrich- a range of sampling methods and force fields, coarse-grained models of the Heine-Universitat, Dusseldorf, Germany, 3UMR 5203 CNRS, Institut de FP were derived in order to follow the long-timescale process of FP/lipid Genomique Fonctionnelle, Montpellier, France. bilayer assembly. Atomic-resolution free energy profiles were calculated as Metabotropic glutamate receptors (mGluRs) are multidomain proteins a function of insertion depth within lipid bilayers composed of a physiolog- belonging to class C G-protein coupled receptors (GPCR). There are essential ically realistic endosomal membrane model. These profiles were correlated in controlling synaptic transmission, and as such are important drug targets for with peptide structural and lipid-interaction data, and validated by compari- the treatment of several disorders including pain, Parkinson’s disease, schizo- son with fluorescence spectroscopy and NMR data. A mutant (W101A) pre- phrenia etc. viously shown to abolish flavivirus membrane fusion was found to possess We recently introduced the use of single molecule FRET combined with Multi- twice as low affinity towards host lipid bilayers in comparison with the parameter Fluorescence Detection (MFD) and Pulsed Interleaved Excitation wild-type FP. The binding affinity was similarly reduced for wild-type FP (PIE) to investigate the allosteric transitions associated with mGluR activation. interaction with pure zwitterionic membranes in comparison with realistic We demonstrated that the isolated ligand binding (VFT) domain oscillates be- endosomal membrane models containing anionic lipids. Novel insights into tween a resting and an active state in a time range of 50-100 ms*. Here, we the structural and thermodynamic basis for flavivirus virus infection process extend these investigation to the full length receptor solubilized in detergent obtained here should prove useful in future rational antiviral therapeutic and reconstituted in liposomes. Our result confirm the general mechanism development. observed for the VFT domain, and decipher the role of the transmembrane domain, that slightly slows down the receptor dynamics, while stabilizing its 1611-Plat active state solely in the presence of a full agonist. Enhanced Simulations and Drug Discovery of a Muscarinic G-Protein- *Olofsson et al., Nature Comm., 2015 Coupled Receptor Yinglong Miao1, J. Andrew McCammon2. 1609-Plat 1Department of Pharmacology, Howard Hughes Medical Institute, University Optimal Probes: An Efficient Method to Select Deer Distance Restraints of California San Diego, La Jolla, CA, USA, 2Department of Pharmacology, using Machine Learning Department of Chemistry and Biochemistry, Howard Hughes Medical Shriyaa Mittal1, Diwakar Shukla1,2. Institute, University of California San Diego, La Jolla, CA, USA. 1Center for Biophysics and Quantitative Biology, University of Illinois at G-protein-coupled receptors (GPCRs) represent primary targets of about one Urbana-Champaign, Urbana, IL, USA, 2Department of Chemical and third of currently marketed drugs. However, GPCR drug discovery has suffered Biomolecular Engineering, University of Illinois at Urbana-Champaign, from major challenges, including the highly flexible nature of the receptors and Urbana, IL, USA. the toxicity of agonist or antagonist drugs that are designed to bind the Double electron-electron resonance (DEER) spectroscopy is an important conserved ‘‘orthosteric’’ site. Here, we have performed all-atom enhanced sim- biophysical technique to determine distance distributions between pairs of ulations using the robust Gaussian accelerated molecular dynamics (GaMD) spin-labelled cysteine residues in membrane proteins, a common target for method to investigate the ligand-dependent structural dynamics of the M2 DEER experiments. Crucial to the technique is the choice of residue pairs muscarinic GPCR. The GaMD simulations revealed distinct structural flexi- to label which is not so obvious and is generally based only on rational intu- bility and free energy profiles that depict graded activation of the GPCR. ition. For a 500 residue protein one must choose a set of labelled residues Both dissociation and binding of an orthosteric ligand were captured in a single pairs from ~125000, which leads to combinatorial choices counting to a all-atom GPCR simulation(1). In addition, we have implemented a unique billion, that can represent the essential conformational dynamics. We propose structure-based approach to design allosteric modulators as selective drug

BPJ 7825_7838 Tuesday, February 14, 2017 329a leads of the M2 muscarinic receptor. Through iterative molecular docking and the hallmarks of misfolding. The focus of these studies is human peripheral experimental testing, half of our 38 computationally selected lead compounds myelin protein 22 (PMP22), a tetraspan membrane protein for which mutations were validated as effective allosteric modulators of the M2 receptor. Our result in the peripheral neuropathy, Charcot-Marie-Tooth Disease. method successfully identified both positive and negative allosteric modulators of the M2 muscarinic receptor with unprecedented chemical diversity and 1614-Symp outstanding potential for further structure-activity relationship studies(2). The Assembly of Beta-Barrel Proteins into Bacterial Outer Membranes This approach shall be of wide applicability for drug discovery of many other Trevor Lithgow. GPCRs(3). Biomedicine Discovery Institute, Monash University, Melbourne, Australia. References: 1.Miao, Y., and J. A. McCammon. 2016. Graded activation and In order to cause disease, bacterial pathogens must (i) avoid immune detection, free energy landscapes of a muscarinic G-protein-coupled receptor. Proc. (ii) inhibit immune responses, and (iii) induce pathology favoring bacterial Natl. Acad. Sci. U. S. A. In press, DOI: 10.1073/pnas.1614538113. replication. In gram-negative bacteria, pathogenesis depend on structures 2.Miao, Y., D. Goldfeld, E. V. Moo, P. M. Sexton, A. Christopoulos, J. A. assembled in the bacterial outer membrane. We have been working to under- McCammon, and C. Valant. 2016. Accelerated structure-based design of stand the mechanism by which beta-barrel and other integral membrane pro- chemically diverse allosteric modulators of a muscarinic G protein-coupled teins are assembled into bacterial outer membranes. receptor. Proc. Natl. Acad. Sci. U. S. A. Early Edition, DOI:10.1073/pnas. The BAM complex is the core machinery for the assembly of membrane pro- 1612353113. teins, and inhibition of BAM complex activity is lethal to bacteria. In Escher- 3.Miao, Y., and J. A. McCammon. 2016. G-protein coupled receptors: ad- ichia coli, the BAM coplex is formed from four lipoproteins (BamB, BamC, vances in simulation and drug discovery. Curr. Opin. Struct. Biol. 41:83-89. BamD and BamE) attached to the integral membrane protein BamA to modu- late its function, with the BamCDE module spanning the outer membrane. An 1612-Plat additional, enigmatic module of the beta-barrel membrane protein assembly is Multiscale GPCR Activation in Lipid Membranes Probed by Solid-State the translocation and assembly module (the TAM). Composed of an outer NMR and Scattering Methods membrane protein (TamA) and an integral inner membrane protein (TamB), Xiaolin Xu1, Andrey V. Struts2,3, Trivikram R. Molugu2, the TAM is not essential for cell viability under laboratory conditions. How- Suchithranga M.D.C. Perera2, Udeep Chawla2, Soohyun K. Lee2, ever, the tamA and tamB genes are often hits in genetic screens of virulence, Rami Musharrafieh2, Amanda M. Johnson2, Annie Huang2, and have been well-conserved through evolution suggesting selection for an Thomas A. Knowles2, Michael F. Brown2,4. important function. Recent work demonstrating the reconstitution of active 1University of Arizona, Tucson, AZ, USA, 2Chemistry and Biochemistry, forms of the BAM complex and the TAM, and new assays for the assembly University of Arizona, Tucson, AZ, USA, 3Laboratory of Biomolecular of a diverse array of outer membrane protein substrates will be discussed. NMR, St. Petersburg State University, St. Petersburg, Russian Federation, Comparative genome analysis will be presented that demonstrates evolu- 4Physics, University of Arizona, Tucson, AZ, USA. tionary tinkering in the interplay between the various modules (BamAB, G-protein-coupled receptors (GPCRs) are the targets of the majority of phar- BamCDE and TamAB) of the beta-barrel membrane protein assembly ma- maceuticals used worldwide. Here we address the multiscale activation chinery. Finally, new data from neutron imaging and super-resolution micro- mechanism of the Rhodopsin class of GPCRs in proteolipid membranes. scopy will be presented which details the molecular organization of assembly Solid-state 2H NMR lineshapes are used to determine the average conforma- precincts in the outer membrane: specialized clusters of BAM complexes that tion and orientation of the retinal cofactor for active Meta-II rhodopsin in suggest current models for the mechanism of beta-barrel assembly need aligned membranes. Comparison between our NMR and crystal structures revision. informs us how crystal packing forces affect the results. Besides the retinal 1615-Symp cofactor, a peptide agonist (high-affinity C-terminal peptide of transducin Mechanistic Insights into Outer Membrane Protein Folding alpha-subunit) is studied alone and bound to rhodopsin in a membrane lipid Sheena Radford. environment. Our combined 2H solid-state NMR order parameter and relax- Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, ation measurements of the high-affinity peptide show similar behavior of the United Kingdom. isoleucine methyl groups, and that their motions are restricted regardless of The beta-barrel outer membrane proteins (OMPs) of Gram-negative bacteria the environment. Moreover, large-scale protein conformation changes are are essential for cell viability and survival, but the molecular mechanisms of studied by wide-angle X-ray diffraction with a femtosecond X-ray laser their biogenesis remain poorly understood. OMPs are synthesised in the cyto- and quasi-elastic neutron scattering methods. We describe how scattering plasm, translocated across the inner membrane and transported across the peri- methods reveal a volumetric expansion of the protein during the receptor plasm by the molecular chaperones Skp or SurA, before being inserted into the activation (protein quake). Notably, the protein environment within the OM by the essential beta-barrel assembly machinery (BAM) complex (a 203 membrane plays a key role. Removal of water by osmolytes yields striking kDa heteropentameric membrane protein complex). The mechanisms by which changes in lipid order parameters, due to a smaller area per lipid at the Skp and SurA recognise OMPs and successfully deliver their cargos to BAM aqueous interface. The lipid-protein interactions entail a flexible surface remain poorly understood. Questions about these mechanisms are especially model (FSM) which illuminates the role of soft matter (membrane lipids interesting since the periplasm is devoid of ATP, and it is completely unknown plus water) in signaling. Frustration of the monolayer spontaneous curvature how substrate binding and release are controlled and coordinated. Moreover, of the lipids is counterbalanced by the solvation energy of the proteolipid whilst several structures of the BAM complex have been solved recently, interface. Protein conformational changes take place within the curvature how OMPs are delivered to BAM from Skp/SurA and folded into the crowded stress field of the lipid bilayer allowing energetic coupling to the membrane. outer membrane remain mysterious. We have been exploiting a range of bio- Our combined biophysical approach gives us an overall description of how physical methods to gain new insights into the mechanisms of OMP folding GPCR activation occurs within lipid membranes resulting in their key and assembly, and its assistance by chaperones and BAM. In this lecture I signaling functions. will describe our recent results using a variety of OMPs of different sizes com- bined with analysis using fluorescence kinetic assays, cryo-EM and non- Symposium: Membrane Protein Biogenesis covalent mass spectrometry. The results have revealed a new mechanism of Skp action and new insights into how BAM facilitates OMP folding into the 1613-Symp bacterial outer membrane. References: The transition state for the folding of The Scarlet Letter: Cellular Recognition of Misfolding-Prone Membrane an outer membrane protein, Huysmans, G.H.M., Baldwin, S.A., Brockwell, Proteins D.J. & Radford, S.E. (2010) Proc. Natl. Acad. Sci. USA 107, 4099-4104 Skp Charles R. Sanders. is a multivalent chaperone of outer membrane proteins, Schiffrin, R., Calabr- Center for Structural Biology and Departments of Biochemistry and ese, A.N., Devine, P.W.A., Harris, S.A., Ashcroft, A.E., Brockwell, D.J. & Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA. Radford, S.E. (2016) Nat. Struct. Mol. Biol., in press Mutations that alter the sequence of membrane proteins are a frequent cause of, or risk factor for human disease. Many such mutations result in membrane pro- 1616-Symp tein misfolding, causing loss of function and, sometimes, formation of toxic ag- Solving the Membrane Protein Expression Problem gregates. This is so even though the endoplasmic reticulum has an elaborate Bil Clemons. quality control system for surveilling and managing the misfolding of mem- Caltech, Pasadena, CA, USA. brane proteins. In this work we probe the structural and biophysical nature of The expression and purification of integral membrane proteins remains a the defects in membrane proteins that are recognized by quality control as major bottleneck in the characterization of this important class. Expression

BPJ 7825_7838 330a Tuesday, February 14, 2017 levels are currently unpredictable, which renders the pursuit of these targets hesive dynamics simulations, we show how stable rolling adhesion in shear challenging and highly inefficient. Evidence demonstrates that small flow emerges roughly at the transition from the trophozoite to the schizont changes in the nucleotide or amino-acid sequence can dramatically affect stage. membrane protein biogenesis; yet these observations have not resulted in generalizable approaches to improve expression. In this talk, I will present 1619-Symp two different but related approaches we have used to address this issue. For Measuring and Modelling Host Cell Subversion by the Malaria Parasite 1 2 2 2 3 the first, using as a model system the protein TatC, we monitor the effect of Leann Tilley , Boyin Liu , Oliver Looker , Emma McHugh , Yao Zhang , Paul McMillan4, Eric Hanssen5, Sulin Zhang3, Matt Dixon2. sequence changes on experimentally observed expression levels demon- 1 2 strating a strong correlation with the simulated integration efficiency ob- University of Melbourne, Australia, Parkville, Australia, Biochemistry and tained from coarse-grained modeling, which is directly confirmed using Molecular Biology, University of Melbourne, Australia, Parkville, Australia, 3Department of Engineering Science and Mechanics, The Pennsylvania State an in vivo assay. For the second, we develop a computational model that 4 predicts membrane protein expression in E. coli directly from sequence. University, State College, PA, USA, Biological Optical Microscopy Platform, University of Melbourne, Australia, Parkville, Australia, The model, trained on experimental data, combines a set of sequence- 5 derived variables resulting in a score that predicts the likelihood of expres- Advanced Microscopy Facility Bio21 Molecular Science and Biotechnology sion. The overall outcome are tools that will enable further investigation of Institute, University of Melbourne, Australia, Parkville, Australia. these difficult to study proteins. New microscopy techniques are providing amazing views of the cellular landscape. We have used 3D Structured Illumination Microscopy (SIM), Symposium: Biophysics of Malaria Parasites direct Stochastic Optical Reconstruction Microscopy (dSTORM), 3D-Elec- tron Tomography and Block-Face Scanning EM to explore the sub-cellular 1617-Symp topography of the malaria parasite, Plasmodium falciparum. P. falciparum Cryo-Em Structure of the Plasmodium Proteasome in the Search of New is the most virulent of malaria parasites, causing ~480,000 deaths per Antimalarials year. Efforts to control malaria need to target both asexual multiplication Paula da Fonseca. in red blood cells (RBCs), which causes disease, and sexual development, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom. which is responsible for transmission. We have probed the changes to the The Plasmodium falciparum proteasome has been suggested as possible target host RBC membrane skeleton that mediate rigidity changes and imaged for new generation antimalarials. However, the proteasome is essential in all the virulence complex that the parasite establishes at the RBC surface, which eukaryotes and a proteasome inhibitor with potential to be developed as anti- mediates adhesion to blood vessel walls. We have explored the changes in malarial must specifically target the Plasmodium proteasome, while evading the parasite and host cytoskeletal structures that underpin the remarkable cross-reactivity with the human counterpart. A high resolution structure of reversible morphology changes that permit sexual blood stages to survive the Plasmodium proteasome was needed in order to validate it as a drug target, in the circulation ready for transfer to the mosquito vector. We have devel- to understand the molecular basis for its specific inhibition and to directly guide oped a computationally-efficient coarse-grained model of the RBC mem- candidate drug discovery and development. While X-ray crystallography has brane and show that a global increase in the rigidity of the bilayer/ been the method of choice for such protein/ligand studies, the small amounts membrane skeleton composite, mediated by skeletal stretching and enhanced of proteasome that can be realistically purified from cultured parasites impair vertical interactions, underlies the observed changes in deformability in both its crystallisation. trophozoites and mid-stage gametocytes. We hypothesize that circulating Recently cryo-electron microscopy (cryo-EM) has seen an enormous trans- late stage gametocytes adopt an elongated shape to facilitate passage through formation. It is now possible to use cryo-EM and single particle analysis the sinusoidal slits in the spleen, thereby avoiding host surveillance to determine protein structures at resolutions that used to be achievable mechanisms. only by crystallography or NMR methods. Our initial cryo-EM structure of an inhibitor-bound human 20S proteasome, at a resolution of around Platform: Biosensors 3.5A˚ , served as proof of principle that cryo-EM is emerging as a realistic approach for structural studies of protein/ligand interactions, with its own 1620-Plat advantages. Cryo-EM requires significantly less amounts of purified protein Single-Molecule Protein Fingerprinting with Nanopores 1 2 3 than other methods and allows structure determination in closer to physio- Laura Restrepo-Perez , Misha Soskine , Shalini John , Aleksei Aksimentiev3, Giovanni Maglia2, Chirlmin Joo1, Cees Dekker1. logical conditions, where active site specificities can be preserved. Within 1 this context, we used cryo-EM to determine the structure of the Plasmodium Department of Bionanoscience, Delft University of Technology, Delft, Netherlands, 2Groningen Biomolecular Sciences and Biotechnology Institute, falciparum 20S proteasome bound to a newly designed specific inhibitor. 3 This structure, and its comparison with that of the human complex, reveals University of Groningen, Groningen, Netherlands, Department of Physics, the molecular basis for the Plasmodium proteasome specific inhibition, University of Illinois at Urbana-Champaign, Urbana - Champaign, IL, USA. therefore validating it as a realistic antimalarial target. Furthermore, the Proteins are the most abundant biomolecules in the human body. Prote- cryo-EM structure of the Plasmodium proteasome can guide the develop- omics is instrumental to understand and detect diseases. Despite the impor- ment of the bound prototype inhibitor towards potential next-generation tance of protein analysis, only a handful of techniques are available to antimalarials. determine protein sequences and these face limitations, for example in the sizeable sample volume needed. During the last few decades, nanopore 1618-Symp technology has emerged as one of the most promising techniques to The Role of Cell Adhesion in the Malaria Life Cycle: From Gliding Sporo- sequence DNA. Sequencing proteins at the single-molecule level is a clear zoites to Rolling Adhesion of Infected Red Blood Cells next step. This however presents even greater challenges than DNA Ulrich S. Schwarz1,2, Friedrich Frischknecht3, Michael Lanzer3, sequencing, due to the three-dimensional folded structure of proteins and Anna Battista1,2, Christine Lansche3, Anil Kumar Dasanna1,2. the presence of 20 different amino acids to be identified. We present a 1Institute for Theoretical Physics, Heidelberg University, Heidelberg, new scheme for protein sequencing in which we simplify the identification Germany, 2BioQuant, Heidelberg University, Heidelberg, Germany, of proteins by using a fingerprinting method. We present the first steps to- 3Parasitology - Center for Infectious Diseases, Heidelberg University, wards protein fingerprinting by exploring ways to denature and translocate Heidelberg, Germany. unfolded proteins and peptides. we observe distinctive current blockades for During its journey through the human host, the malaria parasite makes the native and denatured proteins. Additionally, we show that specific moi- strong use of adhesive interactions with its environment. We use biophys- eties in peptides or proteins can be recognized, and additive chemical side ical assays and mathematical models to investigate the underlying mecha- groups can be distinguished. Finally, we explore ways to denature and nisms. After release into the host skin during a mosquito blood meal, the translocate unfolded proteins and peptides through nanopores. We use malaria sporozoite quickly moves through the connective tissue using SDS (sodium dodecyl sulfate), an anionic detergent used routinely in mo- gliding motility. On a flat substrate, single cells follow circular trajectories lecular biology, to denature proteins into a linear chain as well as to induce with stick-and-slip motion. Using pillar arrays and agent-based computer a negative charge to them. When combined with SDS, proteins move simulations, we show how this circular motion is converted into different through solid-state nanopores in the direction prescribed by the transmem- motion patterns that depend on the geometrical properties of the environ- brane bias voltage regardless of their native charge. We observe distinctive ment. During the blood stage, malaria parasites rebuild the infected red current blockades for the native and SDS-denatured proteins. These first blood cells so that they start adhering to the vascular endothelium, in order steps demonstrate the clear potential for single-molecule protein finger- to avoid clearance by the spleen. Using flow chamber experiments and ad- printing with nanopores.

BPJ 7825_7838 Tuesday, February 14, 2017 331a

1621-Plat enable non-invasive visualization of biological processes at different scales, Novel Local RYR1 Junctional Calcium Responses and Dynamics by from super-resolution microscopy to in vivo imaging, using the same probes. Improved Calcium Sensor Catcher Jenny Yang1, Florence Reddish1, Cassie Miller1, Rakshya Gorkali1, 1624-Plat Susan Treves2, Francesco Zorzato2. The Investigation of Dynamic Changes of the Particle Surface Charge with 1Georgia State University, Atlanta, GA, USA, 2University of Ferrara, Ferrara, Resistive-Pulse Technique Italy. Yinghua Qiu1, Anna Dawid2, Preston Hinkle1, Yunfei Chen3, 2þ Zuzanna Siwy1. The precise spatio-temporal characteristics of Ca transient are critical for the 1 physiological regulation of Ca2þ-dependent signaling processes. Because Department of Physics and Astronomy, University of California, Irvine, CA, 2þ USA, 2College of Inter-Faculty Individual Studies in Mathematics and determining Ca dynamics occurring in the myoplasm and sarcoplasmic retic- 3 ulum (SR) under normal conditions is essential in order to fully understand Natural Sciences, University of Warsaw, Warsaw, Poland, School of changes that may occur under stressful or pathological conditions, we designed Mechanical Engineering, Southeast University, Nanjing, China. a new generation of Ca2þ sensors. Here, we report the optimization of a In the resistive-pulse technique, individual particles passing through a pore genetically-encoded Ca2þ sensor called ‘‘CatchER’’ which exhibits rapid ki- cause transient changes of the transmembrane current, and the amplitude of netics and specific targeting to the endoplasmic reticulum (ER). We also the current change corresponds to the object size. This method has been applied describe the characteristics of CatchER-T’, a sensor variant with significantly to detect cells, viruses, molecules (e.g. DNA and proteins) and particles. With improved specificities, including enhanced fluorescence at 37C and a greater the special structure of track-etched polycarbonate pores, i.e. narrower en- calcium dependent dynamic range. CatchER-T’ is targeted to the ER via the trances and cylindrical interior, the events of highly carboxylated charged poly- signal peptide of calreticulin and the ER retention sequence KDEL. Differential styrene particles can show a current decrease when a particle enters the pore, a drug induced Ca2þ releases and ER refilling are observed for various cell types. flat region in the center part, and a current increase when the particle exits the Furthermore in electroporated mouse flexor digitorum brevis (FDB) muscle fi- pore. The current increase results from concentration polarization as well as bers, targeting CatchER-T’ close to the RyR1 channel via a sequence obtained ionic sourcing effects, which depend on the particle surface charge density. from the junctional SR protein JP-45, revealed that local Ca2þ release via elec- Thus, the characteristics of the obtained resistive-pulse signal carry information trical stimulation is significantly greater and faster than that occurring in the SR not only on the size of the objects, but also on dynamic changes of the particle lumen as assessed using CatchER-T’, i.e. the probe targeted to the lumen of the surface charge via protonation/deprotonation of surface carboxyl groups. Ex- longitudinal SR. The impact of local ER/SR calcium dynamics in controlling periments were performed with a pH gradient set across the pore, so that the cellular signaling and excitation contraction coupling will be presented. particles while translocating would change their effective surface charge due to the dependence of protonation of carboxyl groups on the local proton con- 1622-Plat centration. Analysis of the current increase at the end of the event revealed High-Throughput Spectral and Lifetime-Based FRET Screening in Living that the process of deprotonation/protonation required longer time than the Cells to Identify Small-Molecule Effectors of SERCA transit time. This finding was unexpected, because the transit times were tens Tory M. Schaaf1, Kurt C. Peterson2, Benjamin D. Grant2, of milliseconds thus significantly longer then the chemical reaction time esti- Samantha L. Yuen1, Prachi Bawaskar1,JiLi1, Joesph M. Muretta1, mated based on proton diffusion. Our experiments also indicated that a longer Gregory D. Gillispie2, David D. Thomas1. pore and a higher pH gradient led to a more complete protonation/deprotona- 1Biochemsitry, Molecular Biology, and Biophysics, University of Minnesota, tion in the pore during transit. Using our finding, we designed a protocol to Minneapolis, MN, USA, 2Fluorescence Innovations, Minneapolis, MN, USA. trap individual particles in a pore for as long as 3 seconds. The experiments A robust high-throughput screening (HTS) strategy has been developed to are important for developing new methods which can be used to in situ charac- discover small-molecule effectors targeting the sarco/endoplasmic reticulum terize and precisely control single molecules and particles. calcium ATPase (SERCA), based on a fluorescence microplate reader that re- 1625-Plat cords both the nanosecond decay waveform (lifetime mode) and the complete Electrostatic Control of DNA Hydridization Kinetics Studied with the emission spectrum (spectral mode), with high precision and speed. This spec- Single-Molecule Field-Effect Transistor tral unmixing plate reader (SUPR) was used to screen libraries of small mole- Sefi Vernick, Scott M. Trocchia, Steven B. Warren, Erik F. Young, cules with a fluorescence resonance energy transfer (FRET) biosensor Delphine Bouilly, Ruben L. Gonzalez Jr., Colin Nuckolls, expressed in living cells. Ligand binding was detected by FRET associated Kenneth L. Shepard. with structural rearrangements of green (GFP, donor) and red (RFP, acceptor) Columbia university, New York, NY, USA. fluorescent proteins fused to the cardiac-specific SERCA2a isoform. The re- Single-molecule studies generally rely on fluorescence-based reporting with sults demonstrate accurate quantitation of FRET along with high precision of signal levels limited by photon emission from single optical reporters to effective hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct current levels in optical detectors of much less than 1 fA. Bioelectronic detection structural states, providing a method to classify small-molecule chemotypes with a point-functionalized carbon nanotube transistor, known as the single- on the basis of their structural effect on the target. The spectral data also was molecule field-effect transistor (smFET), in contrast offers signal levels that applied to flag interference by fluorescent compounds. FRET hits were further are more than 106 times higher. In our case, point functionalization is achieved evaluated for functional effects on SERCA’s ATPase activity via both a with a nano-confined diazonium attachment chemistry. We previously used coupled-enzyme assay and a FRET-based calcium sensor. Concentration- smFETs to investigate DNA hybridization kinetics, yielding rate constants, response curves indicated excellent correlation between FRET and function. melting curves and activation energies for different oligonucleotides. Temporal These complementary spectral and lifetime FRET detection methods offer an analysis of association and dissociation reaction rate constants with temperature attractive combination of precision, speed, and resolution for HTS. This allows both target cDNA concentrations and free energies for hybridization to be work was supported by NIH grants (GM27906, HL129814, AR07612, and determined. Here we show that hybridization kinetics are strongly affected by DA037622). bias between the smFET device and the surrounding electrolyte, allowing bias to act as a proxy for temperature. We identify various concentrations of 20- 1623-Plat mer target sequences from the Ebola Zaire nucleoprotein gene through smFET Bright Monomeric Near-Infrared Fluorescent Proteins for Multiscale detection. Electrostatic modulation enables the detection of single base mis- Imaging matches due to significantly altered kinetics under applied potential. Daria Shcherbakova, Mikhail Baloban, Vladislav Verkhusha. Albert Einstein College of Medicine, Bronx, NY, USA. 1626-Plat Monomeric near-infrared (NIR) fluorescent proteins (FPs) and biosensors are in Locked Nucleic Acid Thymine Monomer Probe Identifies Four Single- high demand as additional colors for microscopy and, particularly, for deep- Nucleotide Variants by Melting Temperature tissue imaging. We engineered three bright and spectrally distinct monomeric Judy M. Obliosca1, Sara Y. Cheng2, Yu-An Chen1, Mariana F. Llanos3, NIR FPs, called miRFPs, miRFPs are 2-5-fold brighter in mammalian cells than Yen-Liang Liu1, Darren M. Imphean1, David Bell1, Jeffrey T. Petty3, the only available monomeric NIR FP and perform well in protein fusions, al- Pengyu Ren1, Tim Yeh1. lowing multicolor structured illumination microscopy. Starting from miRFPs, 1Department of Biomedical Engineering, University of Texas at Austin, we engineered the first monomeric multicolor fluorescence complementation AUSTIN, TX, USA, 2Department of Physics, University of Texas at Austin, reporters for protein-protein interactions and RNA labeling. We then designed AUSTIN, TX, USA, 3Department of Chemistry, Furman University, NIR IkBa signaling reporter for canonical NF-kB signaling in both cells and Greenville, SC, USA. animals. We further developed NIR cell cycle biosensor for detection of prolif- High-resolution melting (HRM) analysis of DNA is a closed-tube SNP eration status of individual cells and cell populations. For the first time, miRFPs detection method that has shown many advantages in clinical laboratory,

BPJ 7825_7838 332a Tuesday, February 14, 2017 point-of-care diagnostics, and personalized medicine. While recently devel- suggesting the presence of unique drug-bound channel conformational states. oped melting probes have significantly improved the discrimination of mis- To differentiate the effects of retigabine binding in the pore from possible matched (mutant) alleles from matched (wild-type) alleles, no one attempts coupled effects on the voltage sensor, we co-expressed a voltage sensitive to design a simple probe that can reliably distinguish mismatched alleles phosphatase (CiVSP) to deplete membrane PIP2 that is essential for func- among themselves. Such a new probe could facilitate the discovery of tional domain coupling. CiVSP-mediated PIP2 depletion abolishes ionic cur- rare genetic mutations at lower cost. Here we demonstrate that a melting rents, along with retigabine effects on the voltage sensor fluorescence signal, probe incorporated with a locked nucleic acid (LNA) thymine monomer suggesting that PIP2-mediated VSD-PD coupling is important for retigabine (tL) can reliably differentiate the four SNP alleles by four distinct melting effects on voltage-dependent gating. In a complimentary set of experiments, temperatures (termed the ‘‘4Tm probe’’). This miraculous discriminatory we have identified an essential cluster of positively charged C-terminal resi- power comes from the decreased melting temperature of the tL.C mis- dues likely required for PIP2-mediated domain coupling. Neutralization of matched hybrid as compared to that of the t.C mismatched hybrid. Not this cluster strongly diminishes retigabine effects on voltage sensor fluores- only in the HRM experiments, we have also observed this decreased stabil- cence. Our findings highlight the role of PIP2 as a coupling element that trans- ity in our molecular dynamics simulation. We emphasize that there is no duces retigabine binding in the KCNQ pore to changes in voltage sensor other melting probe at this moment that can achieve the same SNP differen- conformation. tiation results as those demonstrated in this report. Our findings are not only important to the HRM community but also important to the DNA origami 1629-Plat and DNA-metal interaction communities as researchers in these commu- Conformational Changes Occurring in the Acidic Pocket during ASIC nities are always searching for new ways to fine tune the affinity between Activity strands and fine tune the nucleobase environment around DNA-bound metal Sabrina Vullo, Gaetano Bonifacio, Stephan Kellenberger. atoms. University of Lausanne, Lausanne, Switzerland. Acid-Sensing Ion Channels (ASICs) are Hþ-gated and Naþ-conducting chan- 1627-Plat nels. They are widely expressed throughout the central and peripheral nervous Detection of DNA Methylation with Aerolysin Nanopore systems where they play important roles in several physiological and patholog- Jie Yu, Chan Cao, Jie Yang, Yi-tao Long. ical processes. The crystal structure of chicken ASIC1 has been determined in East China University of Science and Technology, ShangHai, China. the desensitized and open states; the conformation of the closed state is how- DNA methylation is an important epigenetic modification in mammals, ever not known. The acidic pocket is the binding site of several animal toxins, which plays an essential role in regulating cell growth, gene expression and, since it contains diverse negatively charged amino acids, it is likely that it and genetic diseases. Nanopore technique has been utilized as a rapid, pre- contributes to proton sensing. cise and label-free method for detection of methylated DNA at single- In this project we investigated the conformational changes occurring in the molecule level. Recently, we found that wild-type (WT) aerolysin nanopore acidic pocket during ASIC activity. In order to detect changes in fluores- performed a higher current sensitivity for discrimination of oligonucleotides cence, we generated Cysteine-Tryptophan pairs; the engineered Cys was compared with the mostly used biological nanopores such as a-hemolysin. used as docking site for the specific labeling with the fluorophore and the Herein, we explored the ability of WT aerolysin for identification of meth- Trp as a quencher of the fluorescence. The mutants were expressed in ylated cytosine (mC) in a single-strand DNA. It has shown that aerolysin Xenopus laevis oocytes and the voltage-clamp fluorometry technique was nanopore could not only directly distinguished the mC from unmethylated used to simultaneously measure current and fluorescence signals. Changes cytosine (C) by different amplitude of residual currents, but also obtain a in fluorescence intensity reflect changes in distance between Cys and Trp perfect separation (~ 98%) in the mixture of C and mC sample at voltage residues, providing clues about the structural rearrangements occurring in of þ 60 mV. The scatter plots clearly indicated that mC located at 14.7 this region. A negative fluorescence signal would indicate an approaching pA while C was concentrated at 15.1 pA. The duration time of mC traverse of the two residues, and a positive suggests that Cys and Trp amino acids aerolysin pore (6.68 ms) was longer than that of C (4.05 ms), which provided move away from each other. Interestingly, some of the tested mutants an additional parameter to improve the accuracy of separation. Furthermore, showed a composite fluorescence signal containing a rapid and a slow we measured the mC in real sample of human serum to explore the possibil- component. Comparison of the kinetics of current and fluorescence signals ity of aerolysin nanopore for clinical diagnosis application. The statistical re- indicates that some observed movements are specifically related to the chan- sults revealed that mC could be obviously distinguished from C in such nel opening and others to the desensitization. Together these experiments complex sample. Therefore, aerolysin protein pore will be a promising allow us to predict the conformational changes in the acidic pocket occurring candidate for DNA methylation detection and apply to the relevant disease during ASIC activity and provide the basis for the design of new drugs diagnosis in the future. acting on these channels.

Platform: Ion Channels, Pharmacology, and 1630-Plat Disease Novel Human Eag Channel Antagonists from Spider Venoms Linlin Ma1, Yanni K. Chin1, Zoltan Dekan1, Maria Ikonomopoulou2, 1628-Plat Volker Herzig1, Chun Y. Chow1, Paul Alewood1, Glenn F. King1. Dissecting KCNQ Channel Pharmacology using Voltage Clamp 1Institute for Molecular Bioscience, The University of Queensland, Brisbane, Fluorometry Australia, 2QIMR Berghofer Medical Research Institute, Brisbane, Australia. Robin Y. Kim1, Stephan A. Pless2, Harley T. Kurata3. Kv10.1 (hEAG) is a member of the large family of voltage-gated potassium 1Anesthesiology Pharmacology and Therapeutics, University of British channels. Functional channels are homotetramers, with each monomer Columbia, Vancouver, BC, Canada, 2Department of Drug Design and comprised of six transmembrane segments. hEAG channels are distinctive in Pharmacology, University of Copenhagen, Copenhagen, Denmark, that they are almost exclusively expressed in the central nervous system in 3Department of Pharmacology, University of Alberta, Edmonton, AB, healthy people, with gain-of-function mutations leading to epilepsy. Remark- Canada. ably, however, hEAG is overexpressed in 70% of human tumors of various tis- Compounds that activate KCNQ voltage-gated potassium channels are an sue origin, and this overexpression has been demonstrated to be causative for emerging class of therapeutics for disorders of cellular electrical excitability, tumorigenesis. Thus, selective antagonists of the channel are much sought after but fundamental questions regarding their effects on channel function remain both as pharmacological tools for studying channel function and as potential unanswered. For instance - how/why do some drugs specifically modulate leads for anti-cancer and anti-epileptic drugs. We recently identified and puri- channel open probability, while others impact voltage sensitivity, or both? fied two hEAG inhibitors from the venoms of tarantula spiders. As revealed by We have employed voltage clamp fluorometry (VCF) to measure retigabine a combination of MALDI-TOF mass spectrometry, Edman sequencing and effects on voltage sensor movements in KCNQ3 channels. Fluorescence re- carboxypeptidase Y digestion, both toxins are C-terminally amidated peptides ports from channels labelled at position Q218C (in the S3-S4 linker) indicate composed of 36 amino acid residues. Their structures solved by NMR spectros- tight coupling between the voltage sensor domain and pore in KCNQ3 chan- copy revealed classical inhibitor cystine knot folds in which the peptides nels, with both processes displaying similar voltage dependence and kinetics. are cross-braced by three disulfide bonds. These hEAG inhibitors do not In response to retigabine, VSD conformational changes and pore gating block the channel pore; rather, they act as gating modifiers that affect channel are equally shifted to hyperpolarized potentials, reflecting the ability of reti- activation and inactivation, presumably by interacting with the channel‘s gabine to stabilize the channel active state. Furthermore, retigabine increases voltage-sensor domain. We show that these peptides specifically inhibit the the magnitude of the fluorescence change induced by channel activation, proliferation of certain cancer cell lines, but not others.

BPJ 7825_7838 Tuesday, February 14, 2017 333a

1631-Plat all subtypes of breast cancer (Luminal A, Luminal B, Her2, and ‘‘triple nega- A Novel Membrane Potential Assay to Identify Nav1.7-Selective Blockers tive’’) using 12 different cancer cell lines for the presence of the hHV1 protein 1 1 2 2 Tania Chernov-Rogan , Tianbo Li , Henry Verschoof , Kuldip Khakh , in membrane fractions by western blotting. Significant hHV1 expression was Steven Jones1, Steve McKerrall1, David H. Hackos1, Dan Sutherlin1, found only in 3 of the 6 triple-negative cell lines tested. Charles J. Cohen2, Jun Chen1. Immunostaining of MDA-MB-231 cells demonstrated that the channel resided 1Genentech, South San Francisco, CA, USA, 2Xenon, Burnaby, BC, Canada. in the plasma membrane, with little or no expression in the cytosol. CRIPSR/ The voltage-gated sodium channel Nav1.7 is one of the most validated pain tar- Cas9-mediated deletion of the hHV1 gene did not detectably change the recov- gets, as gain-of-function mutations cause excessive pain, whereas loss-of- ery of MDA-MB-231 cells from an acid load. In contrast, 100 mMZn2þ pro- function mutations produce insensitivity to pain. Targeting Nav1.7 holds great foundly slowed recovery. Na/Hþ exchange appeared to be the dominant promise in treating pain, yet presents a tantalizing challenge, as few Nav1.7 se- mechanism in cytosolic pH recovery. Nevertheless, deletion of hHV1 resulted lective chemical scaffolds exist. So far millions of compounds have been in multiple changes in protein expression, assessed by reverse phase protein screened by using fluorescence membrane potential (MP) assays, but very analysis, including increased COX2 and lowered CD171 expression in few selective hits have been identified. Here we show that the conventional MDA-MB-231 cells in three independent clones, as compared to wild-type MP approach, which uses Nav1.7 WT channel and the activator veratridine, and Cas9-expressing controls. is intrinsically flawed: it is biased toward non-selective pore blockers which These data suggest a mechanism outside of pH regulation for hHV1 in breast compete with veratridine, and fails to detect highly potent, selective com- cancer cells and caution when using Zn2þ as a pharmacological agent to test pounds such as the aryl sulfonamide PF-771. By using a mutant channel and for hHV1 function. an alternative activator, we developed a novel method that not only robustly de- Supported by NIH-GM102336, Bears Care, and Gavers Community Cancer tects known Nav1.7 blockers, but also decreases the number of non-selective Fund. pore blocker hits. We conducted a high-throughput screen using this methodol- ogy and identified novel Nav1.7-selective blockers. 1634-Plat BK Channel Activation in Chronic Vasodilation by Thiazide-Like 1632-Plat Diuretics: Role of the Beta-1 Auxiliary Subunit High Diastolic Sodium Influx in Heart Failure has Drug-Sensitivities Pedro Martı´n, Agustı´n Asuaje, Valentina Pastore, Vero´nica Milesi. Like Late Sodium Current, but Produces Inward Current at Diastolic Instituto de estudios Inmunolo´gicos y Fisiopatolo´gicos (IIFP Potentials UNLP-CONICET), La Plata, Argentina. Zhandi Liao1, Kenneth S. Ginsburg1, Daniel C. Bartos1, Yanyan Jiang1, Thiazide-like diuretics are still recommended as first-line antihypertensive Sanda Despa2, Donald M. Bers1. therapy, based on their chronic vasodilatory effects. Previous studies suggest 1Pharmacology, University of California Davis, Davis, CA, USA, activation of the large conductance voltage- and Ca2þ- dependent Kþ channel 2Pharmacology and Nutritional Sciences, University of Kentucky, Lexington, (BK channel) expressed in vascular smooth muscle cells (SMCs), as respon- KY, USA. sible for this vasodilator effect, but electrophysiological evidence supporting Heart failure (HF) affects over 6 million people, enhances arrhythmogenesis this is lacking. BK can be accompanied by accessory b-subunits, which confer and is associated with elevated intracellular [Na] ([Na]i), which impacts Ca specific pharmacological characteristics to the channel. The b1 subunit is handling via Na/Ca exchange. HF myocytes exhibit increased late Na current mainly expressed in SMCs. (INaL), typically insufficient to explain the rise in [Na]i. However, in HF there We measured the effect of hydrochlorothiazide (HCTZ) on BK channel activity is also an increase in diastolic Na influx (INaDiast) that is tetrodotoxin (TTX) using the patch-clamp technique in SMCs from human umbilical artery sensitive. INaL is known to be activated by CaMKII and blocked by the selective (HUASMCs) and in HEK293 T expressing the BK channel (with and without INaL inhibitors ranolazine and GS967. To test whether the elevated INaDiast in the beta-1 subunit). HF has the same sensitivities to CaMKII and INaL inhibitors, we studied freshly In HUASMCs, HCTZ (10 mM) caused significant activation of the BK current isolated ventricular myocytes from HF and control rabbits. We measured [Na]i in the whole-cell configuration (528 5 215 to 1379 5 132 pA at þ40mV, n: 4, and unidirectional Na-influx rate upon acute Na/K-ATPase blockade. Ranola- p <0.05), while, it did not produce any changes in BK channel unitary conduc- zine (10 mM) inhibited INaDiast in HF myocytes (by 58.5%), consistent with tance nor open probability in the inside-out configuration (NPo at þ 40mV: INaDiast in HF being mediated via an INaL-like current. GS967, tested in control 0.0114 5 0.0015 (control) vs 0.0135 5 0.0037 (HCTZ), n: 4, P> 0.05), sug- myocytes, also inhibited INaDiast. Application of CaMKII blocker, KN-93 gesting an indirect activation mechanism. In HEK cells expressing the BK (1 mM) reduced INaDiast in HF, but not control myocytes. We also measured channel (with and without b1-subunit), HCTZ only activates BK channel in INaDiast under voltage clamp, as shifts in holding current (Ihold) at diastolic the presence of the b1-subunit. This activation was concentration-dependent Vm upon acute addition of INaL blockers. In control myocytes TTX induced with an EC50 of 28 mM (pD2=4.546 5 0.211, n: 5-8). Membrane potential 7.5pA IHold, but in HF myocytes the TTX-sensitive IHold was 25.4 pA at did not influence the concentration relationship on HCTZ-induced BK channel 80mV. This agrees with INaDiast measured using Na dyes. INaDiast could be activation. Consistently, HCTZ did not change the BK channel activity when it via a window INa. However, the V1/2 of the INa steady state activation and inac- was evaluated in HEK cells expressing the b1-subunit in the inside-out config- tivation curves were shifted in HF by 3.8mV and 7.2mV, respectively, and uration, where cell integrity is lost. intermediate INa inactivation was also enhanced in HF. Both of these effects These results suggest that the vasodilatory effects of HCTZ could be due to an make window INa current unlikely to mediate INaDiast.INaL measured in AP- indirect activation of the BK channel depending on the beta-1 subunit clamp was increased by ~50% in HF. We suggest that INaDiast in HF has expression. drug sensitivities like INaL during the AP, but produces inward current at dia- 1635-Plat stolic potentials that can explain the rise in [Na]i. Differential KD Channels Expression in ‘‘Classically’’ and ‘‘Alterna- 1633-Plat tively’’ Activated Microglia Voltage Gated Proton Channel Expression and Function in Breast Cancer Hai M. Nguyen1, Eva M. Gro¨ssinger1, Makoto Horiuchi2, Kyle W. Davis2, Cells Lee-Way Jin2, Izumi Maezawa2, Heike Wulff1. Dan Bare1, Vladimir V. Cherny1, Abde M. Abukhdeir2, 1Pharmacology, University of California, Davis, Davis, CA, USA, Thomas E. DeCoursey1, Deri Morgan1. 2Pathology and Laboratory Medicine, M.I.N.D. Institute, University of 1Molecular Biophysics, Rush University Medical Center, Chicago, IL, USA, California, Davis, Sacramento, CA, USA. 2Medicine and Pharmacology, Rush University Medical Center, Chicago, As CNS resident immune cells, microglia are highly active and can assume IL, USA. different phenotypes in response to microenvironmental signals. Lipopolysac- Immunohistochemical evidence for voltage gated proton channels (hHV1) was charide (LPS) and interferon-g (IFN-g) promote differentiation into classically recently reported in breast cancer cells, but not healthy breast tissue. Higher activated M1-like microglia, which produce high levels of pro-inflammatory levels of hHV1 expression were correlated with advanced stages of the disease, cytokines and nitric oxide and are thought to contribute to neurological damage worse prognosis, and decreased survival [Wang et al, 2012, J.Biol.Chem. in ischemic stroke and Alzheimer’s disease. IL-4 in contrast induces a 287:13877]. However, its function and mechanism of action have yet to be phenotype associated with anti-inflammatory effects and tissue repair. We elucidated. Here, we examine a number of breast cancer cell lines for expres- here investigated whether these microglia subsets vary in their Kþ channel sion of hHV1 and explored the consequences of hHV1 genetic knockout. expression by differentiating neonatal mouse microglia into M(LPS) and þ Using patch-clamp, we demonstrated that MDA-MB-231 cells have hHV1 cur- M(IL-4) microglia and studying their K channel expression by whole-cell rents. The activation threshold, gating kinetics, selectivity, and pH dependence patch-clamp, quantitative PCR and immunohistochemistry. We identified three of the channel were similar to those in other human cells. We screened across major types of Kþ channels based on their biophysical and pharmacological

BPJ 7825_7838 334a Tuesday, February 14, 2017

fingerprints: a use-dependent, outwardly rectifying current sensitive to the biochemical and mutational analyses. Whether the GTP-induced conforma- 2þ KV1.3 blockers PAP-1 and ShK-186, an inwardly rectifying Ba -sensitive tional change of MxB is relevant to other antiviral activities or a vestige of evo- 2þ Kir2.1 current, and a Ca -activated, TRAM-34-sensitive KCa3.1 current. lution for Mx proteins remains to be clarified. Both KV1.3 and KCa3.1 blockers inhibited pro-inflammatory cytokine produc- 1638-Plat tion and iNOS and COX2 expression demonstrating that KV1.3 and KCa3.1 play important roles in microglia activation. Following differentiation with LPS or a An Atomic Model for a Complete Myosin Molecule within a Native Thick Filament combination of LPS and IFN-g microglia exhibited high KV1.3 current den- Kenneth A. Taylor1, Zhongjun Hu1, Dianne Taylor1, Michael K. Reedy2, sities (~50 pA/pF at 40 mV) and virtually no KCa3.1 and Kir currents, while mi- Robert J. Edwards2. croglia differentiated with IL-4 exhibited large Kir2.1 currents (~ 10 pA/pF at 1Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, 120 mV). KCa3.1 currents were generally low but moderately increased 2 following stimulation with IFN-g or ATP (~10 pS/pF). This differential Kþ USA, Cell Biology, Duke University Medical Center, Durham, NC, USA. We have obtained a 4.3A˚ resolution 3-D image of the relaxed thick filament channel expression pattern suggests that KV1.3 and KCa3.1 inhibitors could be used to inhibit detrimental neuroinflammatory microglia functions. This from Lethocerus indicus using IHRSR. The image resolution is variable being work was supported by NS098328 to H.W. and AG043788 to I.M. highest for the region where the myosin rods are packed and lowest for the blocked head within the interacting heads motif (IHM). Although the resolution Platform: Electron Microscopy is low for de novo atomic model building, by using coiled-coil constraints within the model building software Rosetta, we obtained a complete atomic 1636-Plat model for the Lethocerus flight muscle myosin rod, its least understood domain. Energetics of Sheath Contraction in Contractile Injection Systems For the rod, we started with a homology model of the Skip 1 crystal structure Alec D. Fraser, Michel Plattner, Petr G. Leiman. (PDB 4XA1) and built outward to the N- and C-termini. The myosin rods BMB, UTMB, Galveston, TX, USA. assemble into ‘‘curved molecular crystalline layers’’ (ribbons) and not as heli- The process of protein and DNA translocation across lipid membranes is cen- cal subfilaments. The number of plausible salt bridges between coiled-coils tral to the function of any organism. A large class of large multicomponent or- within ribbons is 30% greater than the number between ribbons. For the ganelles, such as bacteriophage tails, Type VI Secretion System, R-type IHM, we used flexible fitting starting with an homology model of the Lethoce- pyocins, Serratia antifeeding prophage, and others, translocate their substrates rus myosin sequence and the scallop transition state structure, PDB 1DFL. using a rigid tube/contractile sheath mechanism. Functionally and structurally, Notable features of the IHM atomic model include the following: (1) Hydro- these ‘contractile injection systems’ resemble a stretched spring (or sheath) phobic content in the free head RLC interaction with the myosin rods. This wound around a non-contractile tube. The system is locked in a high-energy is the best-ordered region of the IHM. (2) At the free head S1/S2 junction, metastable state by a baseplate structure that plays an important role in sheath 11 residues of the heavy chain a-helix are unwound. Density connecting the assembly and contraction triggering. Upon interaction of the baseplate with a displaced parts of the heavy chain a-helix is clearly resolved. (3) The heavy target cell membrane, the sheath contracts and drives the tube through the chain a-helices bound by the free head ELC and RLC could not be connected cell envelope. A full atomic model of the sheath and tube in the extended without unwinding 10 residues of the heavy chain a-helix. The blocked head and contracted state is available for R-type pyocin, one of the simplest repre- density is the lowest resolution region of the reconstruction and could be fit sentatives of contractile injection systems. The structure suggests that the without the heavy chain helical alterations seen in the free head. Supported contraction is accomplished by rigid body rearrangement of sheath subunits by NIH and AHA. and that the transformation is driven by the energy stored in the extended 1639-Plat conformation of the sheath during assembly. Other data show that the contrac- The Bacterial Flagellar Filament Re-Examined at High Resolution tion starts at the baseplate and propagates through the sheath as a wave. The Fengbin Wang1, Andrew Burrage2, Daniel Kearns2, Edward H. Egelman1. individual subunits’ trajectories are however unknown. We will discuss how 1University of Virginia, Charlottesville, VA, USA, 2Indiana University, we can derive these trajectories from energetics considerations. These finding Bloomington, IN, USA. are important for understanding the substrates that can be translocated by con- The bacterial flagellar filament has been an important model system in struc- tractile injection systems. tural biology. Current models are based upon the notion that subunits can switch between two stable states, one forming left-handed 11-start protofila- 1637-Plat ments and the other forming right-handed ones (Calladine, 1975; Kamiya et ˚ Cryoem Structure of the Full Length Dynamin-Like MxB at 4.6 A al., 1980). Pioneering work on the structure of two flagellin mutants came Resolution from the Namba lab, studying the Salmonella filament (Maki-Yonekura et 1 2 3 4 Frances Joan D. Alvarez , Shaoda He , Juan R. Perilla , Sooin Jang , al., 2010; Samatey et al., 2001). We have been using cryo-EM to study the 5 4 3 2 Peter E. Prevelige Jr. , Alan Engelman , Klaus Schulten , Sjors Scheres , structure of a gram-positive bacterium, Bacillus subtilis. We have now recon- 1 Peijun Zhang . structed five mutants, all at a better resolution than obtained for Salmonella. For 1University of Pittsburgh, Pittsburgh, PA, USA, 2MRC Laboratory of ˚ 3 one mutant, we have reached ~ 3.6 A, which allows for a full atomic model to Molecular Biology, Cambridge, United Kingdom, University of Illinois at be built with a high-degree of confidence. These results are being combined Urbana-Champaign, Urbana, IL, USA, 4Dana-Farber Cancer Institute, 5 into a new model for how structural polymorphism is dictated by the switching Boston, MA, USA, University of Alabama at Birmingham, Birmingham, between two states, only possible with the recent advances allowing for near- AL, USA. atomic resolution reconstructions of these filaments. Human dynamin-like, interferon-induced myxovirus resistance 2 or Mx2/MxB Calladine, C.R. (1975). Construction of bacterial flagella. Nature 255, 121-124. is a potent inhibitor of HIV-1 infection and a potential agent for a new thera- Kamiya, R., Asakura, S., and Yamaguchi, S. (1980). Formation of helical fila- peutic approach for the treatment of HIV/AIDS. MxB binds the HIV-1 CA ments by copolymerization of two types of ’straight‘ flagellins. Nature 286, core and blocks nuclear accumulation and chromosomal integration of the viral 628-630. DNA. The anti-HIV-1 activity and the CA binding require the unique N-termi- Maki-Yonekura, S., Yonekura, K., and Namba, K. (2010). Conformational nal domain of MxB, which has eluded structural determination due to the dif- change of flagellin for polymorphic supercoiling of the flagellar filament. Nat- ficulty of expressing the full length protein. In an effort to structurally StructMolBiol 17, 417-422. characterize this domain, we purified a GTPase active MBP fusion of the full Samatey, F.A., Imada, K., Nagashima, S., Vonderviszt, F., Kumasaka, T., Ya- length WT MxB in mammalian cells. We show that MxB is purified as large mamoto, M., and Namba, K. (2001). Structure of the bacterial flagellar proto- discrete oligomers and, in low salt conditions, further self-assembles into filament and implications for a switch for supercoiling. Nature 410, 331-337. helical oligomers. We further show that GTP binding results in the de- polymerization of the helical oligomers, while the subsequent GTP hydrolysis 1640-Plat allows the re-formation of said tubes. Using cryo-electron microscopy (EM) Using Cryoem to Understand How Phages Evade Bacterial CRISPR and helical reconstruction, we present the density map of the helical oligomer Defense System of MxB at 4.6 angstrom resolution, which revealed structural insights into the Saikat Chowdhury1, MaryClare F. Rollins2, Joshua Carter2, Ryan Jackson2, oligomerization interfaces and nucleotide-induced conformational changes of Lyn‘Al Nosaka1, Blake Wiedenheft2, Gabriel C. Lander1. MxB. Interestingly, only a partial density of the N-terminal domain is visible 1Int Struct and Comp Biol, The Scripps Research Institute, La Jolla, CA, on the map, suggesting an intrinsic flexibility or disorder of this domain. While USA, 2Immunology and Infectious Diseases, Montana State University, many studies point to the dispensability of the oligomerization and GTPase Bozeman, MT, USA. activity of MxB in its anti-HIV-1 activity, the oligomeric structure of MxB Bacteria and archaea have evolved different CRISPR based adaptive immune reported here provides the structural context to interpret the results from system for defense against phage infections. Pseudomonas aeruginosa has

BPJ 7825_7838 Tuesday, February 14, 2017 335a type I-F CRISPR-cas defense system, where the cas-crRNA surveillance com- 1643-Plat plex (csy-complex) binds to foreign DNA and recruits the cas-2/3 nuclease for Bacteriophage T7 DNA Translocation during Infection is Bolstered by the degradation of the foreign DNA. Bacteriophages have evolved mechanisms of Host ATP Synthase Complex evading this CRISPR defense system by encoding different anti-CRISPR Dustin R. Morado1, Chunyan Wang1,BoHu1, Ian Molineux2, Jun Liu1. (ACR) proteins. ACR1F and ACR2F are two such ACRs that bind to specific 1Pathology and Laboratory Medicine, University of Texas Health Science regions of the csy-complex and suppress the immune system. We present here a Center at Houston, Houston, TX, USA, 2Molecular Genetics and 3.4A˚ cryo-EM reconstruction of the ACR-bound csy-complex. This structure Microbiology, The University of Texas at Austin, Austin, TX, USA. not only shows the overall organization of the type I-F csy-complex, but Translocation of bacteriophage T7 dsDNA into its host cell is an integral step in also reveals how ACRs interact with this complex and prevent the binding of infection initiation that is energy dependent and is catalyzed principally by phage DNA. transcription per host and viral RNA polymerases. Here we show utilizing high-throughput cryo-electron tomography, a hexameric ring complex exists 1641-Plat below the T7 trans-envelope channel in the host-cell cytoplasm during infec- Unravelling the Mechanism of Membrane Attack Complex Pore Closure tion. Using sub-tomogram averaging and extensive classification we isolated Courtney Boyd1, Marina Serna1, Scott Gardner1, Agnel Praveen-Joseph2, T7 in the brief stage of genome translocation in coordination with the ring. Maya Topf2, B. Paul Morgan3, Doryen Bubeck1. This host-cell nanomachine is ~30nm in diameter, ~8nm in height with a 1Life Sciences, Imperial College London, London, United Kingdom, ~10nm channel. The complex was not observed in ATP synthase-deficient 2Institute of Structural and Molecular Biology, Department of Biological Escherichia coli mutants, suggesting that the complex functions in an Sciences, Birkbeck College, London, United Kingdom, 3Systems Immunity energy-dependent manner. T7 grows in these mutant strains, and thus DNA Research Institute, Division of Infection and Immunity, School of Medicine, ejection is not dependent on the complex. The ring is not host-specific; it has Cardiff University, Cardiff, United Kingdom. also been observed in an infected rough strain of Salmonella typhimurium, The membrane attack complex (MAC) is a large, multiprotein human immune which supports the growth of T7. These results, only obtainable by in situ struc- pore that is a major player in the complement terminal pathway. This complex tural analysis, provide details on the essential process of DNA translocation and macromolecular machine is composed 5 different proteins in different oligo- reveal the ability of T7 to recruit host-cell machinery to aid in the initiation of meric states, with a total of 24 proteins making up the final pore. MAC lyses infection. invading pathogens by puncturing of bacterial membranes, while sub-lytic concentrations of MAC deposited on host cells trigger inflammation and sig- Platform: Membrane Pumps, Transporters, and nalling cascades. Despite over half a century of study, the structure of the Exchangers II MAC was only recently elucidated, whereby it was revealed that the complex forms an irregular b-barrel with a ‘split-washer’ shape, distinct from other 1644-Plat pore-forming proteins studied to date. The asymmetric nature of the MAC Describing the Thermodynamics of the Secondary Transporter BetP by suggests an additional role in membrane distortion and poses the question Coupling Spectroscopic Measurements to Molecular Dynamics Simulations how such a pore could seal. Here we present a high-resolution cryo-electron 1 2 3 ˚ Vanessa Leone , Izabela Waclawska , Burkhard Endeward , microscopy structure of the full transmembrane MAC at 5.5A, enabling Thomas Prisner3, Christine Ziegler4, Lucy R. Forrest1. structural modelling and identification of domain re-arrangements that are 1NINDS-NIH, Bethesda, MD, USA, 2Max Planck Institute of Biophysics, necessary to facilitate MAC formation. We also report the sub-nanometer re- Frankfurt am Main, Germany, 3Johann Wolfgang Goethe University constructions of 3 distinct MAC intermediate conformational states, suggest- Frankfurt, Frankfurt, Germany, 4Institute of Biophysics and Physical ing a ‘bottom-up’ zippering mechanism in which strands of the b-barrel Biochemistry, Regensburg, Germany. initiate closure at the membrane. Irregularities within the wall of the pore The sodium-coupled glycine betaine symporter BetP regulates the osmotic indicated that the b-strands forming the contiguous b-barrel are not uniform. pressure in the microbe Corynebacterium glutamicum,asoilbacterium Incomplete membrane penetration and formation of transitional ‘toroidal’ used extensively in biotechnology. Crystal structures of BetP reveal it to pores highlight how the MAC may be able to sense different lipid environ- be a homotrimer, and computational and biochemical studies have shown ments and induce various signaling outcomes in conjunction with disruption that each protomer is able to transport betaine. X-ray structures of BetP of the lipid bilayer. are available for a number of different conformations, including outward- open, inward-open and occluded states; some are loaded with the substrate 1642-Plat and others are the apo form. Spectroscopic data collected using Pulsed Functional Study of the Ryanodine Receptor Type 1 using Cryo-Electron Electron Paramagnetic Resonance (PELDOR) on BetP in detergents and Microscopy in proteoliposomes provide information in the form of distance distribu- 1 2 3 3 Amedee des Georges , Oliver B. Clarke , Ran Zalk , Qi Yuan , tions about the conformation of the transporter outside of the crystal, and 4 4 4 Kendall J. Condon , Robert A. Grassucci , Wayne A. Hendrickson , its dependence on substrates. However, it is not trivial to connect the spec- 3 4 Andrew R. Marks , Joachim Frank . troscopic and structural data to obtain a description of the events being 1ASRC, CUNY, New York, NY, USA, 2Biochemistry and Molecular 3 measured at the microscopic level. Here, we coupled molecular simulations Biophysics, Columbia university, New York, NY, USA, Physiology and of structures of the BetP monomer with distance distributions from the Cellular Biophysics, Columbia University, New York, NY, USA, 4 PELDOR measurements. This allowed us to identify the states that are Biochemistry and Molecular Biophysics, Columbia University, New York, representative of different substrate/ion conditions. The approach employed NY, USA. here, known as EBMetaD (Marinelli & Faraldo-Go´mez, Biophys J, 2015), Cryo-electron microscopy (cryo-EM) is revolutionizing the structural biology enforces the system to sample only within the distance distribution, of membrane proteins. It has allowed the structural study of long sought after applying the minimum bias required to reproduce the experimental distri- targets, such as the type 1 ryanodine receptor (RyR1). But another very impor- bution. Comparison of different simulations permits us to identify the state tant advantage is that it allows to study the complex dynamics associated with or states in the transport cycle that are most prevalent under each of the membrane protein function. 2þ experimental conditions. The results of our computational studies may bet- The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca ) ter delineate the conformations that are thermodynamically accessible release channel required for skeletal muscle contraction. We used single- along the BetP transport cycle, and therefore provide important insights particle cryo-EM to study the dynamics of RyR1 in multiple functional states, into its mechanism. revealing the conformational changes key to channel gating and ligand- dependent activation. The binding sites for the channel activators Ca2þ, ATP 1645-Plat and caffeine were identified and the conformational changes associated with ANaD/HD Exchanger Regulated by an S4 Voltage-Sensing Domain and a their binding observed independently. They induce by themselves a priming Cyclic Nucleotide-Binding Domain of the cytoplasmic assembly without pore dilation. In contrast, in the presence Reinhard Seifert, Florian Windler, Wolfgang Bo¨nigk, of all three activating ligands, open and closed states of the pore were obtained Heinz-Gerd Ko¨rschen, U. Benjamin Kaupp. from the same sample, enabling analysis of conformational changes associated MNS, Caesar Research Center, Bonn, Germany. with gating. The analysis of multiple functional states was greatly facilitated by Sperm-specific electroneutral NHE proteins (sNHE or SLC9C1) feature a the use of holey-gold grids, which allowed cryo-EM reconstructions to reach unique tripartite structure including an exchanger homology domain, a proto- high resolution with much fewer number of particles than with conventional typical channel-like voltage-sensing domain (VSD), and a cyclic nucleotide- holey carbon grids. binding domain (CNBD). Because SLC9C1 proteins have not been functionally

BPJ 7825_7838 336a Tuesday, February 14, 2017 expressed, their physiological properties are largely unknown. Here, we char- in our molecular dynamic simulations, the introduction of a known inhibi- acterize the activation properties of SLC9C1 from Strongylocentrotus purpur- tory crosslink between residues A399 and A432 prevents rearrangement of atus (SpSLC9C1). SpSLC9C1 activity is gated by the VSD during membrane the side chain of F357. In further support for the proposed role of F357 in hyperpolarization and modulated by direct binding of cAMP. We recorded the CLC exchange cycle, we show using an uptake assay and ITC measure- SpSLC9C1 activity and the underlying gating currents by single cell electro- ments that the F357A mutation drastically impairs transport of CLC-ec1 physiology and voltage clamp fluorimetry. Both, the voltage dependence of with minimal effects on the Cl-/Hþ stoichiometry. Our results shed mecha- activation and that of the corresponding gating currents are shifted towards nistic light into the molecular basis of coupled Cl-/Hþ exchange in the CLCs more permissive membrane voltages by binding of cAMP to the CNBD. Our by identifying a novel conformational rearrangement essential for transport results indicate that the VSD is the pivot that gates the exchanger domain of both Cl- and Hþ. and itself is regulated by cAMP binding to the CNBD. Thus, SpSLC9C1 rep- resents a phylogenetic hybrid between ion antiporters, voltage-gated channels, 1648-Plat and cyclic nucleotide-gated channels. Insights into the Gating Mechanism of Excitatory Amino Acid Transporters-Associated Anion Channel 1646-Plat Delany Torres-Salazar1, Horacio Poblete2, Aneysis Gonzalez-Suarez3, Molecular Mechanism of Electrogenic Sodium/Proton Antiport Ariela Vergara-Jaque2, Jennie Garcia-Olivares1, Jeffrey Comer2, Oliver Beckstein1, David L. Dotson1, Mathieu Coincon2, Yandong Huang3, Susan G. Amara1. Chiara Lee4, Povilas Uzdavinys2, Emmanuel Nji2, Shoko Yashiro4, 1Laboratory of Molecular and Cellular Neurobiology, National Institute of Wei Chen3, Alexander D. Cameron5, Jana Shen3, David Drew2. Mental Health, Bethesda, MD, USA, 2Department of Anatomy and 1Physics, Arizona State University, Tempe, AZ, USA, 2Biochemistry and Physiology, Kansas State University, Manhattan, KS, USA, Biophysics, Stockholm University, Stockholm, Sweden, 3Pharmaceutical 3Interdepartmental Neuroscience Program, Yale University School of Sciences, University of Maryland School of Pharmacy, Baltimore, MD, USA, Medicine, New Haven, CT, USA. 4Division of Molecular Biosciences, Imperial College, London, United Excitatory Amino Acid Transporters (EAATs) control excitatory synaptic Kingdom, 5School of Life Sciences, University of Warwick, Coventry, transmission in the brain. They complete this task through a secondary United Kingdom. active transport mechanism. In addition, EAATs operate as substrate- Sodium/proton antiporters are integral membrane proteins that are vital for gated anion channels. These two mechanisms are known to be thermody- cell homeostasis. In humans, they maintain cellular pH and their dysfunction namically uncoupled. However, emerging evidence demonstrates a tight is linked to a variety of complex diseases, including cancer, cardiovascular structural coupling between them. We have recently identified a conserved pathophysiology, and autism. Naþ/Hþ antiporters are secondary active positively charged residue (R388, hEAAT1) in transmembrane domain 7 transporters that utilize the electrochemical gradient of one ionic species (TM7), which is crucial for such a coupling. Substituting R388 with a nega- to drive the energetically uphill transmembrane transport of the other spe- tively charged amino acid drives the carrier into a constitutive open channel cies. They operate by the alternating access mechanism whereby the protein state, while it limits the transport cycle. We here combine electrophysiolog- cycles between an outward facing and inward facing conformation to switch ical recordings and molecular dynamic simulations to characterize the the exposure of substrate binding sites between the extracellular and the mechanism that controls the switch between transport mode and channel intracellular environment. We used a combination of molecular dynamics mode. We have built a homology model of the mammalian isoform simulations with X-ray crystallography and functional measurements to EAAT1 using the crystal structure of the archaeal orthologue GltPh as a tem- adress key questions about the transport mechanism in the two electrogenic plate. Then using atomistic molecular simulations, we have identified resi- bacterial antiporters NhaA and NapA, which exchange 2 Hþ for 1 Naþ.In dues in TM2, TM5, TM7 and the hairpin loop 1 (HP1) which may be particular, the likely sodium and proton binding sites overlap, as predicted coordinating with R388, the anion channel opening. Our results show that by a previously proposed competitive binding mechanism. A large mutating E377 (HP1) to a positively charged residue, yields a large anion ‘‘elevator-like’’ conformational transition moves the Naþ binding site conductance and a significantly reduced transport activity (less than 1%), across the membrane. Based on simulations with varying protonation states similar to R388D/E. These results suggest that an interaction E377-R388 of conserved ionizable residues, absolute binding free energy calculations, is necessary to maintain an efficient transporter, and that affecting this inter- and explicit constant pH MD simulations as well as functional measure- action favors the equilibrium into an open channel and out of the transport ments of mutants, we put forward a detailed model for the transport mech- cycle. Interestingly, a double mutant swapping these charges (R388D- anism: Two protons are carried by a conserved aspartate and a conserved E377R) virtually recovered the wild type phenotype, corroborating this lysine residue. The Naþ binding site is formed by two conserved aspartate hypothesis. Our data provide additional insights into the mechanism that residues. The protonated lysine forms a salt bridge with the aspartate that facilitates opening and closing of the EAAT-associated anion channel and does not carry a proton. Binding of Naþ disrupts the salt bridge and facili- suggest that intramolecular interactionsbetweenR388andnearbyresidues tates the release of the proton from the lysine, thus maintaining the exper- are a crucial piece of this process. imentally observed competitive binding mechanism. The binding site itself is translocated in a rigid fashion across the membrane, consistent 1649-Plat with the alternating access mechanism. Molecular Insights on the Recognition of Substrates by the Promiscuous Efflux Pump AcrB 1647-Plat Ivana Malvacio1, Rosella Ombrato2, Paolo Ruggerone1, Attilio V. Vargiu1. Molecular Mechanism of Cl/HD Coupling in a CLC Antiporter 1Department of Physics, University of Cagliari, Cagliari, Italy, 2Angelini Yanyan Xu1, Malvin Vien2, Chungwen Liang1, Alessio Accardi2, Research Center, Angelini S.p.A., Rome, Italy. Simon Berne`che1. Efflux pumps of the Resistance Nodulation Division (RND) superfamily play 1Swiss Institute of Bioinformatics, Basel, Switzerland, 2Weill Cornell a major role in multi-drug resistance (MDR) to antibiotics in Gram- Medical College, New York, NY, USA. negatives. Understanding the molecular mechanism by which these pumps X-ray crystallography and mutagenesis data of the prokaryotic Cl-/Hþ anti- expel so many unrelated drugs is essential to develop antibiotics able to porter ClC-ec1 revealed the pathway for chloride ions and suggested that a escape efflux machineries, as well as efflux pumps inhibitors. The drug/ conserved glutamate, E148, is determinant for both chloride and proton proton antiporter AcrB, part of the major efflux pump AcrAB-TolC in transport. However, the mechanisms regulating the coupled transport of Escherichia coli, is the paradigm RND transporter. Unfortunately, only both substrates remain poorly understood. Using molecular dynamic free en- few compounds have been co-crystallized with AcrB, and thus computa- ergy simulations of ClC-ec1, we found that a conformational change of a tional approaches are essential in deciphering determinants of the interac- conserved phenylalanine residue lining the pore, F357, is a novel and essen- tions between diverse ligands and this promiscous protein. tial component of the transport cycle. Our simulations reveal that, in the Previously we used a truncated model of AcrB to characterize the binding of presence of an ion bound to the central site of the Cl- permeation pathway, several substrates and inhibitors. Here we performed docking calculations, mo- the side chains of F357 and E148 undergo a concerted conformational rear- lecular dynamics (MD) simulations and free energy calculations to investigate rangement that allows the aromatic ring of the phenylalanine to coordinate the interaction of 10 congeneric molecules with AcrB. Importantly, we use a the protonated E148 carboxylate group through a p;-dipole interaction. full model of the protein in the membrane in order to exploit interactions The reorientation of the F357 aromatic ring has two major functional conse- with all the possible binding sites. quences: 1) it favors the binding of a second Cl- to the pore, at the external Flexible ensemble-docking was performed using almost 40 non-redundant con- site; 2) it allows for the controlled diffusion of the proton attached to the formations of extracted from experiments and extensive MD simulations of E148 side chain along a path distinct from the Cl- pathway. Interestingly, AcrB and different protonation states for each compound. The top poses for

BPJ 7825_7838 Tuesday, February 14, 2017 337a each ligand (for a total of 200 AcrB-ligand structures) were then subjected to Platform: Cell Mechanics, Mechanosensing, and MD ms- long simulations and analyzed with respect to descriptors of their struc- ture, dynamics, and thermodynamics of binding. The outcome of this study, un- Motility II precedented for its extensiveness, will provide key information to medicinal chemists for the design of new compounds with improved efficiency. 1652-Plat Mechanical Response of Nesprin-2G to Cytoskeletal Forces Regulates Linc 1650-Plat Complex-Dependent Mechanotransduction Voltage-Gated Calcium Channels as Detecting Tools of Psychoactive Hengameh Shams1, Gant Luxton2, Mohammad R.K. Mofrad1. Drugs 1Bioengineering, University of California, Berkeley, Berkeley, CA, USA, Iwona Ruchala, Alan Harris, Louis J. De Felice, Jose M. Eltit. 2Genetics, Cell Biology, and Development, University OF Minnesota, Physiology and Biophysics, Virginia Commonwealth University, Richmond, Minneapolis, CA, USA. VA, USA. Nuclear geometry and positioning are affected by cytoskeletal forces, The dopamine, serotonin, and norepinephrine transporters are important deter- which plays an important role in cellular mechanotrasnduction. The LINC minants of monoaminergic neurotransmission. The pharmacological manipula- (LInkers of the Nucleoskeleton and Cytoskeleton) complex has been shown tion of these systems contributes not only to drug addiction, but also to the to bear mechanical load and mediate force transmission from the cytoskel- treatment of many mental diseases. The development of novel tools to expedite eton into the nucleus, however, molecular mechanisms of this phenomenon the identification of drug-effect profiles on monoamine transporters can benefit are not yet understood. Giant nesprin-2 (nesprin-2G) forms a direct linkage efforts to discover new psychotherapeutics. between the actin cytoskeleton and SUN proteins inside the perinuclear Monoamine transporters are natively expressed in the plasma membranes of space. These linkers are shown to be under constant tension especially presynaptic neurons, where they mediate the uptake of monoamine neurotrans- in the apical and equatorial planes of the nucleus and have 56 spectrin re- mitters. When expressed in heterologous expression systems, the uptake of sub- peats along their rod domain. A functional construct of nespring-2G con- strates is associated with an inward current that depolarizes the cell membrane. taining only four spectrin repeats, referred to as mini-nesprin-2G, was 2þ 2þ In addition, voltage-gated Ca channels modulate Ca permeability as a shown to rescue the nuclear movement defects observed in nesprin-2G- function of the membrane potential. We took advantage of these two elemental depleted fibroblasts. Since it is intrinsically difficult to measure forces and principles to develop a new assay to detect and characterize ligands of mono- track residue-level interactions across individual proteins using current amine transporters. experimental techniques, we used molecular dynamics simulations to study Using the Flp-In T-REx expression system, we established permanent mechanical response of nesprin molecules to cytoskeletal forces. Mini- cell lines expressing the human dopamine or serotonin transporters. We stud- nesprins are excellent candidates for this purpose as their small size allows 2þ ied the ability of these cells to produce Ca signals in response to mono- for long simulation periods necessary for capturing important molecular amine transporters’ substrates after transient transfection with different events. Our results provides a detailed understanding of how mechanical 2þ voltage-gated Ca channels. In addition we studied the dynamic range modes of mini-nesprin-2G are excited and contribute to its response to 2þ and sensitivity of two genetically encoded Ca sensors. Our results show various cytoskeletal forces around the nucleus. Also, we simulated force- that the co-expression of CaV1.2 and GCamp6s in monoamine transporter- induced conformational changes of the Actin Binding Domain (ABD) of 2þ expressing cells generates significant Ca signals in response to monoamine mini-nesprin-2G, which are most likely induced by the axial tension along transporter substrates. Moreover monoamine transporter blockers can inhibit the actin bundles at the apical plane of the nucleus. Furthermore, we exam- 2þ these Ca signals. Preliminary experiments measuring our optimized cell ined the effect of ABD conformation on its actin binding affinity by system in a Flex Station 3 plate reader suggest that the co-expression of a comparing the strength of interactions between distinct conformations of 2þ voltage-gated Ca channel, a monoamine transporter and a genetically en- the ABD domain of mini-nesprin-2G and actin. Lastly, we used force distri- 2þ coded Ca sensor constitute a rapid screening biosensor to identify active bution analysis to identify force-sensitive residues along the mini-nesprin- drugs at monoamine transporters. Supported by R01 DA033930 and R01 2G structure and tested the effect of mutating them to alanine on transferring AR067738. forces to the nucleus.

1651-Plat 1653-Plat Homology Modeling and Ligand Optimization for the Human Amino Acid Nuclear Constriction Segregates Mobile Nuclear Proteins Away from Exchanger Lat-1 (SLC7A5) Chromatin 1 2 Claire Colas , Avner Schlessinger . Charlotte R. Pfeifer, Jerome Irianto, Rachel R. Bennett, Yuntao Xia, 1Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New 2 Irena L. Ivanovska, Andrea J. Liu, Roger A. Greenberg, Dennis E. Discher. York, NY, USA, Pharmacological sciences, Icahn School of Medicine at University of Pennsylvania, Philadelphia, PA, USA. Mount Sinai, New York, NY, USA. As a cancer cell squeezes through adjacent tissue, penetrates a basement LAT-1 (SLC7A5) is a sodium-independent exchanger mainly located in the membrane, or enters the smallest blood capillaries, its nucleus can be highly blood brain barrier (BBB) where it mediates the transport of large neutral constricted, but any effects on chromatin density and other nuclear factors amino acids, thyroid hormones and prescription drugs. LAT-1 is also upregu- are poorly understood. Here, in cancer cell migration through rigid micro- lated in a variety of cancer types (e.g., prostate cancer), to provide amino acids pores and also in passive pulling into micropipettes, local compaction of that are used as nutrients and signaling molecules for growth. Thus, LAT-1 is chromatin is observed coincident in space and time with depletion of mobile an emerging drug target for substrates that can serve as prodrugs with optimal factors. Hetero/eu-chromatin has been previously estimated from molecular BBB or tumor permeability, or inhibitors that starve cancer cells. Here, we use mobility measurements to occupy a volume fraction f roughly two-thirds of computational modeling using homology modeling and ligand docking, fol- the nucleus, but based on the relative intensity of DNA and histones in lowed by experimental testing using synthetic chemistry and cellular uptake several cancer cell lines drawn into narrow constrictions, f can easily in- measurements, to develop homology models for LAT-1 and identify novel crease locally to nearly 100%. By contrast, mobile proteins in the nucleus, chemical tools for this transporter. We refine a homology model of LAT-1 including a dozen that function as DNA repair proteins (e.g. 53BP1) or nu- based on the arginine/agmatine transporter AdiC from E. Coli as modeling tem- cleases (e.g. Cas9, FokI), are seen to exhibit a much reduced density within plate and conduct SAR studies on amino acid derivatives to design compounds the constriction, approaching 0%. Such loss of mobile nuclear factors—com- with optimal Km/Vmax balance. Particularly, initial LAT-1 models and bind- pounded by the occasional rupture of the nuclear envelope—has important ing evaluation of putative compounds guided the synthesis of distinct com- functional consequences for the cell. Constricted migration indeed delays pounds, including tyrosine and phenylalanine derivatives, carboxylic acid DNA cleavage by a FokI-lacR fusion of a target locus integrated into chro- bioisosteres, and other unique scaffolds. These compounds were tested exper- mosome 1. imentally by your collaborators using cis-inhibition and trans-stimulation as- says. For example, we identified hydroxamic acids derivatives as LAT-1 1654-Plat substrates for the first time. Furthermore, iterative evaluation of these Microrheology of Astrocytes and Gliomas and Contribution of Inter- compounds by computational methods and experimental testing permitted to mediate Filaments to their Mechanics progressively refine our LAT-1 models, reaveling previously unknown drug- Alibert Charlotte1, Atef Asnacios2, Bruno Goud1, gable pockets on its surface. Our results improve our understanding of how in- Jean-Baptiste Manneville1. hibition and transport specificity is determined in LAT-1, as well as provide 1Institut Curie, Paris, France, 2Matie`re et Syste`mes complexes, Paris, France. novel chemical probes to further characterize the role of this transporter in can- Several studies show that cancerous tissues are stiffer than normal tissues. cer and neurological disorders. However, at the scale of the individual cell, tumor cells appear to be softer

BPJ 7825_7838 338a Tuesday, February 14, 2017 than normal cells. Here, we use two different microrheology techniques allow- 1657-Plat ing us to probe the mechanical properties of cells at two different scales: the Myofibril Organization in Cardiomyocytes is Guided by the Maximal scale of the whole cell using a single cell uniaxial rheometer, and the intracel- Tensile Stress Direction lular scale using a combination of micropatterning and optical tweezers. These Hongyan Yuan, Bahador Marzban. two techniques enable us to compare and correlate the internal visco-elastic Mechanical Engineering, University of Rhode Island, Kingston, RI, USA. properties with the mechanics of the entire cell. We focus our study on the The mechanisms underlying the spatial organization of self-assembled myofi- mechanics of astrocytes, the major glial cell type in the brain, and gliomas, brils in cardiac tissues remain incompletely understood. By modeling cells as brain tumors derived from astrocytes. We first discriminate astrocytes and elastic solids under active cytoskeletal contraction, we found a remarkable gliomas of different grades based on their mechanical properties. We show correlation between the predicted maximal principal stress directions and the that rat primary astrocytes are stiffer than glioma cells from both human in vitro myofibril orientations in individual cardiomyocytes. This implies that and rat grade III and grade IV cell lines, and that mechanical differences exist actomyosin fibers tend to assemble along the maximal tensile stress directions. between glioma cells grades. Next, since intermediate filaments (IFs) have By considering the dynamics of focal adhesion and myofibril formation in the been involved in the migration of glial cells and since the expression of IF model, we revealed that the different myofibril organizations in mature versus proteins is modified in gliomas, we evaluate the contribution of IFs to the immature cardiomyocytes are due to the different levels of force-dependent re- mechanics of gliomas and astrocytes. We show that the level of IF proteins modeling of focal adhesions. Further we found that the myofibril organizations correlate with the mechanical properties of the cells and that down- in microtissues are regulated by a combination of multiple factors including regulation of IF proteins lowers cell rigidity. Finally, using our intracellular cell/tissue shape, cell-substrate adhesions, and cell-cell adhesions. These find- technique, we are able to measure the force-deflection curve and the bending ings can guide the rational design in cardiac tissue engineering where recapit- rigidity of IF bundles in cellulo. ulating in vivo myofibril organizations is crucial to the contractile function of the heart. 1655-Plat Modelling the Flow of Dynamically Cross-Linked Biopolymer Networks 1658-Plat Arjan E. Boerma1, Patrick R. Onck1, Erik van der Giessen1, Microtubule Bundles in the Adult Cardiomyocyte Stefanos Papanikolaou2. Patrick Robison, Alexey I. Bogush, Benjamin L. Prosser. 1Micromechanics of Materials, University of Groningen, Groningen, Physiology, University of Pennsylvania, Philadelphia, PA, USA. Netherlands, 2Hopkins Extreme Materials Institute, Johns Hopkins The role of Microtubules (MTs) in mechanosignalling and mechanical University, Baltimore, MD, USA. properties has seen a recent surge of interest among the cardiac and skel- Networks of semi-flexible actin filaments in the cell cytoskeleton play a key etal muscle community. We recently reported that MTs buckle and bear role in biological functions such as cell motility and force transduction. load during the cardiac cycle. This has a significant effect on contractile Connections between actin filaments are formed and broken by cross- mechanics, suggesting that myocyte function is tunable by any regulatory linker molecules that thermally diffuse through the cell. The dynamic char- pathway that alters the mechanical behavior of MTs. Other work ongoing acter of these cross-links can be critical for the mechanical behavior of the in our lab indicates that a number of Microtubule Associated Proteins network. We present a coupled finite-element/Monte Carlo approach to (MAPs) are robustly upregulated in human heart disease. Multi-MT assem- model cross-linked filaments, and show that thermal diffusion of cross- blies or bundles associated with these and other MAPs have been observed links causes a transition from elastic to flowing behavior in response to me- and studied extensively in other contexts, including unicellular motile chanical loads. structures and neuronal axons, but have not been examined in cardiac myocytes. The number of MTs involved, their geometry and the relative 1656-Plat abundance of such structures compared to isolated MTs have significant Dictyostelium Mechanics Accurately Identifies New Targetable Drug implications for the mechanical properties of a myocyte. Using targeted Space for Pancreatic Cancer Delineated by Myosin IIS, Filamins, and overexpression of several MAPs in adult rat cardiomyocytes, we examine Alpha-Actinins, Collectively Comprimising the Mechanobiome the structure, magnitude and prevalence of MT bundles by electron micro- Alexandra Surcel1, Eric Schiffhauer1, Dustin Thomas1, Qingfeng Zhu2, scopy. We find that single MTs are the most prevalent MT elements in Robert Anders2, Douglas Robinson1. the unmanipulated myocyte, accompanied by comparatively rare and small 1 Cell Biology, Johns Hopkins University School of Medicine, Baltimore, groupings of 2-3 MTs with wide spacing. MTs are preferentially located 2 MD, USA, Pathology, Johns Hopkins University School of Medicine, at the interface between myofibril and mitochondria. Manipulation of MT Baltimore, MD, USA. binding partners can modestly increase the size and likelihood of MT bun- Pancreatic ductal adenocarcinoma (PDAC) is largely incurable, with a dles, and also dramatically reduces the inter-tubule spacing to be in-line 5-year survival rate of ~6% and a median survival of <6months.With with canonical bundle spacing. When we further characterized the functional 37,000 people dying annually in the US from pancreatic cancer, it is pre- consequences of MT bundling on cardiac contractility, we were surprised nd dicted to become the 2 leading cause of cancer deaths. PDAC cells to find that the promotion of bundling, at least with certain MAPs, does show extremely elevated rates of de novo and acquired resistance to tradi- not appear to impair contraction by reinforcing compliant microtubules, tional chemotherapies. Uncovering new drug spaces that target metastatic but rather uncouples the MTs from the process of contraction-associated hepatic disease, the primary mortality factor for patients, is essential. The buckling. ability of metastasizing cells to navigate through different microenviron- ments requires them to have altered mechanical profiles characterized by Platform: Protein-Small Molecule Interactions the cytoskeletal proteins that define the structural elements of the mechano- biome – the collection of proteins that sense and respond to mechanical 1659-Plat stimuli. Combined in Vitro and in Silico Approaches to the Assessment of Stimu- To identify new targetable drug spaces, we assessed the PDAC mechano- lant Properties of Novel Psychoactive Substances biome. We used our understanding of Dictyostelium mechanics and in silico Michelle A. Sahai1, Colin Davidson2, George Khelashvili3, analysis to identify the players and to accurately predict the mechanores- Vincenzo Barrese4, Neelakshi Dutta1, Harel Weinstein3, ponsiveness of specific isoforms of the actin crosslinkers alpha-actinin Jolanta Opacka-Juffry1. and filamin. We determined via western blot analysis and immunohistochem- 1Department of Life Sciences, University of Roehampton, London, United istry of patient-derived samples that key players involved in mechanosensa- Kingdom, 2Pharmacy and Biomedical Sciences, University of Central tion – myosin IIA, IIC, alpha-actin-4, and filamin B – show increased Lancashire, Preston, United Kingdom, 3Department of Physiology and expression in cancerous ductal epithelial over normal tissue, while non- Biophysics, Weill Cornell Medical College, New York, NY, USA, 4St mechanosensory paralogs show decreased expression. We have determined George’s University of London, London, United Kingdom. the contributions of each of isoform on PDAC mechanics across short and Novel psychoactive substances (NPS) are increasingly prevalent world-wide long-time scales and have assessed the impact of knockdowns and overex- although their pharmacological characteristics are largely unknown; those pression PDAC cell mechanics, migration, and invasion both 2D and 3D. with stimulant properties, due to interactions with the dopamine transporter Additionally, we have previously shown that targeting of myosin IIC by the (DAT), have addictive potential which their users may not realise. We evaluated small molecule mechanical modulator 4-HAP affects PDAC mechanics. the binding of 1-(1-benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB) to Mice treated with 4-HAP show a reduction in metastasis, suggesting rat striatal DAT by means of quantitative autoradiography with [125I]RTI-121, that direct targeting of the mechanobiome can be developed into new and the effects of 5-MAPB on electrically-evoked dopamine efflux by fast- treatments. cyclic voltammetry in rat brain slices. 5-MAPB displaced [125I]RTI-121 in a

BPJ 7825_7838 Tuesday, February 14, 2017 339a concentration-dependent manner, with significant effects at 10 and 30 mM. The 1662-Plat voltammetry data suggest that 5-MAPB reduces the rate of dopamine reuptake; Developing a Novel Class of CLC Chloride-Channel Inhibitors while the peak dopamine efflux was not increased, the area under the curve was Anna K. Koster1, Chase Wood2, Rhiannon Thomas-Tran1, augmented. 5-MAPB can also cause reverse dopamine transport consistent with Tanmay S. Chavan2, Jonas Almqvist3, Kee-Hyun Choi2,4, Justin Du Bois1, stimulant properties, more similar to amphetamine than cocaine. Molecular Merritt Maduke2. modelling and docking studies compared the binding site of DAT in complex 1Chemistry, Stanford University, Stanford, CA, USA, 2Molecular & Cellular with 5-MAPB to dopamine, amphetamine, 5-APB, MDMA, cocaine and RTI- Physiology, Stanford University, Stanford, CA, USA, 3Uppsala University, 121. This structural comparison reveals a binding mode for 5-MAPB found in Uppsala, Sweden, 4Korea Institute of Science and Technology, Seoul, Korea, the primary binding (S1) site, central to transmembrane domains 1, 3, 6 and 8, Republic of. which overlaps with the binding modes of dopamine, cocaine and its analogues. The chloride channel (CLC) family is a class of membrane proteins that regu- Atomistic molecular dynamics simulations further show that, when in complex lates the flux of chloride ions across cell membranes. Two of the nine mamma- with 5-MAPB, DAT can exhibit conformational transitions that spontaneously lian CLC isoforms, CLC-Ka and CLC-Kb, reside in the kidney where they are isomerize the transporter into inward-facing state, similarly to that observed in critical for maintaining proper water and salt balance and therefore are thera- dopamine-bound DAT. These novel insights, offered by the combination of peutic targets for treating hypertension and hyponatremia. CLCs are tradition- computational methods of biophysics with neurobiological procedures, provide ally difficult drug targets, as demonstrated by the low potency and general lack structural context for NPS at DAT and relate them with their functional proper- of isoform selectivity among existing classes of CLC inhibitors. In this work, ties at DAT as the molecular target of stimulants. we describe a novel small-molecule inhibitor that shows an unprecedented selectivity for CLC-Ka over CLC-Kb despite their 90% sequence similarity. 1660-Plat Through homology modeling, site-directed mutagenesis, and structure- Weighted Ensemble of Pathways for Ligand Unbinding from T4 Lysozyme activity relationship studies, we have identified and validated the inhibitor bind- Ariane Nunes-Alves1, Daniel M. Zuckerman2, Guilherme M. Arantes1. 1 ing site, as well as key molecular features that are stepping stones for designing Department of Biochemistry, Institute of Chemistry, University of Sa˜o CLC inhibitors with enhanced potency and isoform selectivity. Paulo, Sa˜o Paulo, Brazil, 2Department of Computational and Systems Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA. 1663-Plat T4 lysozyme L99A mutant is a protein often used as a model to study small Mapping Cholesterol Binding Sites on the Human Dopamine Transporter molecule binding to macromolecules. Experimental affinities and crystal Talia Zeppelin, Xavier Periole, Birgit Schiøtt. structures are available for its complex with several ligands. But pathways Chemistry, Aarhus University, Aarhus C, Denmark. for ligand entry and exit from the deeply buried and solvent inaccessible The human dopamine transporter (hDAT) is essential for regulating dopami- binding site have not been fully resolved. Protein conformational changes nergic neurotransmission by transporting dopamine from the synaptic cleft necessary to allow ligand excursion to the binding site have also been back into the presynaptic neuron. Dysregulation of hDAT is involved in debated. Here, we present molecular dynamics (MD) simulations that sam- several debilitating diseases such as Parkinson‘s disease, attention deficit hy- ple the pathways for benzene exit from the T4 lysozyme binding site and the peractive disease (ADHD) and Tourette‘s syndrome, among others. DAT is associated conformational changes. The weighted ensemble (WE) approach also the target of many illicit drugs and has been presumed to be involved wasusedtosearchforpossibleunbindingpathwaysandtoovercomethe in the development of addiction. It is therefore essential to further the under- limitations of MD simulations for sampling low probability regions in standing of the transporters architecture, transport mechanism and means of pre-defined progress coordinates. Interaction energies were obtained using regulation to aid in drug development. Recently, a range of DAT structures the CHARMM36 force field in combination with implicit solvation. Inde- has been published from the species Drosophilia Melanogaster (dDAT). All pendent WE simulations employing different progress coordinates revealed of which contain at least one conserved co-crystallized cholesterol molecule. four possible pathways for benzene exit. Displacements of alpha-helices It is believed that cholesterol acts in regulating hDAT activity, but it is unclear backbone and amino acid side chains on the order of 0.4 nm or less were whether the two cholesterol sites found on dDAT also exist on hDAT. Within enough to allow ligand exit. The side chains of Tyr88 and Leu133 were this contribution we use coarse-grained molecular dynamics simulations of a identified as the main gates for two of the pathways sampled. These results hDAT homology model embedded in a mixed POPC and cholesterol mem- complement previous proposals which identified only one or two transit brane. We will present the results, and furthermore compare any identified pathways. cholesterol binding sites to other membrane protein known to bind cholesterol. 1661-Plat Exploring New Pharmacological Perspectives of Fusicoccin, A Stabilizer of 1664-Plat 14-3-3 - Target Protein Complex Understanding the Binding Mechanism of Ryanodine to the Open- and Andrea Saponaro1, Alessandro Porro1, Antonio C. Sanjuan1, Closed States of the Ryanodine Receptor Pore Chiara Donadoni1, Marco Nardini1, Gerhard Thiel2, Anna Moroni1. Williams E. Miranda1, Van A. Ngo1, Laura L. Perissinotti1, 1Dept. Biosciences, University of Milan, Milan, Italy, 2Dept. Biology, S.R. Wayne Chen2, Sergei Y. Noskov1. TU-Darmstadt, Darmstadt, Germany. 1Biological Sciences, University of Calgary, Calgary, AB, Canada, 2Libin The development of small-molecules regulating protein - protein interaction Cardiovascular Institute, University of Calgary, Calgary, AB, Canada. has emerged as a powerful field in pharmacology. In this scenario, 14-3-3 Ryanodine (Ryd) is a poisonous plant alkaloid that specifically binds ryano- proteins represent a promising target, as they are an important class of dine receptors (RyRs) with distinguishable affinities. Some of its binding adapter proteins that regulate several hundred partners, many of them modes can sharply enhance the open probabilities, thus helping to demonstrate described as disease-relevant proteins such as p53, Raf1, a-Synuclein and the vital roles of RyRs as a family of intracellular calcium release channels in HERG channel. The fungal phytoxin Fusicoccin (FC) has shown to possess skeletal, cardiac and neuronal cells. Although electrophysiology and mutagen- stabilizing properties on the complex formed by 14-3-3 proteins with some esis experiments have shed some light on the binding mechanism of Ryd to of their targets, including ion channels and pumps. Thus, FC is a promising RyRs, a full atomistic insight of the receptor-ligand recognition process is still tool to control cellular processes regulated by 14-3-3 proteins. To date FC lacking. We used all-atom molecular dynamics (MD) simulations-based ap- action was thought to be limited to a specific subset of targets displaying proaches to study the binding of Ryd to the skeletal muscle isoform RyR1, the 14-3-3 binding site at their C-terminus (MODE III). The structure of which has been recently solved by Cryo-EM at 3.8-A˚ resolution for a closed the ternary 14-3-3 - target - FC complex shows that FC displays its stabiliz- structure and 4.1-A˚ for an open structure. We applied enhanced and bi- ing effect by developing an hydrophobic interaction with the C-terminal directional sampling methodologies to study not only energetics and thermo- non-polar residue of the target protein. In this work, by taking advantage dynamics, but also geometrical binding modes of single and multiple Ryds of our structural and functional study on the interaction between 14-3-3 along the pathways from the cytosol to cavity and passing the selectivity filter and the voltage-gated inward rectifier potassium channel KAT1 of Arabidop- to the lumen. Using the closed structure, our preliminary results suggest that sis thaliana, we expanded the palette of pharmacological targets of FC to a the pyrrolic ring of Ryd statistically favors R4892AGGG-F4921 residues of new kind of internal and C-terminal binding motifs lacking the hydrophobic RyR1‘s cavity, which explain the effects of the corresponding mutations in residue. By solving the structure of the ternary complex, we discovered a RyR2 (cardiac isoform) in experiments. This will be compared with the bind- new orientation of FC within the 14-3-3 - KAT1 complex, that accounts ing modes and thermodynamics of Ryds in the open structure of RyR1, thus for the unexpected stabilizing effect of the molecule. Thus, our structural providing some atomistic insights into how Ryd interacts with major residues and functional work opens new perspectives in the employment of FC as in the open and closed RyR1 that give rise to the enhancement of the open a stabilizing molecule for 14-3-3 - target complexes. probabilities.

BPJ 7825_7838 340a Tuesday, February 14, 2017

1665-Plat Workshop: Beyond Calcium: Imaging Voltage Mesoscopic Properties and Molecular Mechanisms of IAPP Amyloid Inhi- bition and Remodeling with Small Molecules and Other Ions Aleksandr Kakinen1, Bo Wang2, Xinwei Ge2, Raffaele Mezzenga3, Thomas P. Davis1,4, Feng Ding2, Pu Chun Ke1. 1667-Wkshp 1Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Fluorescent Visualization of Cellular Efflux Australia, 2Department of Physics and Astronomy, Clemson University, William R. Kobertz. Clemson, SC, USA, 3Department of Health Science & Technology, ETH Biochemistry and Molecular Pharmacology, UMASS Medical School, Zurich, Zurich, Switzerland, 4Department of Chemistry, Warwick University, Worcester, MA, USA. Coventry, United Kingdom. The fluorescent visualization of calcium ions entering the cytoplasm has reim- The amyloid aggregation of proteins is associated with a number of aged our basic understanding of the inner workings of calcium signaling in neurodegenerative diseases such like Alzheimer’s, Huntington’s and Parkin- cells, tissues and living organisms. In contrast, there is a dearth of tools to flu- son’s diseases, and also type-2 diabetes (T2D). Despite the physical and orescently visualize ions exiting cells. Part of the challenge stems from the fact structural properties of the aggregating proteins, the corresponding amy- that cellular egress is contrary to the pervasive intracellular-centric experi- loid aggregates feature some common characteristics including the forma- mental paradigm. Recently, we have been using glycan engineering to install tion of cross-b architecture and cytotoxicity to human cells, suggesting chemical handles into the cell’s glycocalyx that directly abuts the plasma mem- a common amyloid mechanism. Experimental studies have shown that brane in all cells. Subsequent chemical modfication of these unnatural sugars several naturally-occurring small molecules, such as epigallocatechin gallate ideally positions molecular probes within nanometers of the extracellular ves- (EGCG), have an inhibitory effect on the aggregation of a wide range of tibules of ion channels and transporters. My laboratory’s efforts to fluorescently those amyloid proteins, including islet amyloid polypeptide (IAPP, a.k.a. visualize ions exiting cells using this technology will be presented. amylin) in T2D, which is one of the most aggregation-prone peptides. Furthermore, EGCG was also reported having a novel function as amyloid 1668-Wkshp fibrils remodeling, indicating a promising therapeutic approach against am- Critical Evaluation of FRET-Based Biosensor Performance: Implications yloid diseases, and importance of understanding the molecular mechanism for Measuring Ion Concentrations of such anti-aggregation effect. In this study, we applied experiments and Amy E. Palmer. desecrated molecular dynamic (DMD) simulations to investigate the effects Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA. of EGCG on IAPP fibrillization. Our high- resolution transmission electron Fluorescent sensors are widely used to visualize, quantify, and reveal the dy- microscopy (TEM) images showed that EGCG inhibited IAPP aggregation namics of small molecules, secondary metabolites, metals, and ions. One of by rendering shorter and softer structures, while remodeled mature IAPP the great promises of such sensors is the ability to quantify cellular signals in fibrils by compromising fibril contour length and triggering splitting without precise locations with high temporal resolution, enabling the creation of real- altering the twisted fibril morphology. DMD simulations further revealed time dynamic maps of cellular signals. Yet this is coupled with the challenge that, upon binding with EGCG, IAPP monomers and oligomers tended of how to ensure that sensors are measuring what we think they are measuring, to form disordered off-pathway clusters instead of cross-b structure due developing robust approaches for quantification, and assessing whether sensors to competing interactions with amyloidogenic sequences. EGCG could are perturbing the underlying biology. This talk will highlight our efforts to also target fibril ends through capping and stacking the exposed sites and develop genetically encoded FRET-based sensors for quantitative mapping enable fibril remodeling. Our combined computational and experimental of zinc ions in cells. I will discuss approaches for defining whether sensors per- study offers a detailed mechanistic insight into the inhibitory effect of turb cellular ions, the importance of carefully defining dynamic range, strate- EGCG on IAPP fibrillization as well as fibril remodeling, suggesting a gies to compare sensor functionality in vitro, in cells, and in organelles, and promising approach for the mitigation of IAPP aggregation and future the specific challenges associated with quantifying ions in cellular organelles. T2D therapy. 1669-Wkshp Light Up the Brain with Genetically Encoded Sensors of Neural Activity 1666-Plat Lin Tian. Conformational Plasticity of the MAGE-A3 Protein as a Therapeutic University of California, Davis, Davis, CA, USA. Strategy in Multiple Myeloma A central challenge in neuroscience is to understand how neural circuits extract Roman Osman1, Hearn J. Cho2, Opher Gileadi3. 1 information from the environment and generate appropriate behaviors. To Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New address this challenge, one would like to measure the complex spatiotemporal York, NY, USA, 2Medicine, Hematology/Oncology, Icahn School of 3 neural activity on many different scales, from single synapses to microcircuits Medicine at Mount Sinai, New York, NY, USA, Structural Genomics to entire brains, in behaving animals. Recent breakthroughs in modern micro- Consortium, University of Oxford, Oxford, United Kingdom. scope and protein based fluorescence sensors including calcium, voltage and Type I MAGE proteins interact with the RING domain protein Kap1 through neurotransmitters, permit optically large-scale recording of neural activity their conserved MAGE Homology Domains (MHD) to form E3 ubiquitin with needed molecular and cell type specificity and spatiotemporal resolutions (Ub) ligases, which ubiquitinylate p53 targeting it for proteasomal degrada- in behaving animals. To further expand the kinds of neural activity that can be tion. RNAi experiments demonstrated that MAGE-A3 inhibits p53- measured with genetically encoded indicators, we applied the established dependent and independent mechanisms of apoptosis and confers resistance sensor design and optimization platform to the development of a set of specific, to chemotherapy-induced apoptosis in human myeloma cell lines. Since targetable and sensitive sensors for direct measurement neuromodulators of the MAGE expression correlates with progression of multiple myeloma biogenic amine family and activation of corresponding G-protein coupled re- (MM), preventing the interaction with Kap1 is a promising therapeutic inter- ceptors. We have characterized the expression, signal-to-noise ratio, dynamic vention against MM. The MHD of MAGE proteins are made of two winged range and kinetics of these sensors in dissociated neuronal culture, acute brain helix domains (WH) linked by a flexible b-hairpin. Structures of MAGE slice and in vivo in zebrafish and mouse. A broad application of these imaging proteins suggest that the WH domains need to undergo a conformational tools will enable neuroscientists to obtain a dynamic and comprehensive view change from a closed to an open form to interact with Kap1 and activate of synaptic transmission in action to decipher the codes for transferring infor- its Ub-ligase activity. Virtual screening on the closed form of MAGE-A3 mation across neural circuitry and systems. identified two compounds that recapitulate the RNAi experiments, suggest- ing that they may inhibit the interaction of MAGE-A3 with Kap1 leading to 1670-Wkshp the apoptosis of MM cells. Further experiments are being conducted to Functional Cortical Connectomics through Co-Expression of Genetically elucidate the nature of the observed effect. MD simulations of the com- Expressed Voltage and Calcium Indicators plexes of MAGE-A3 with the small molecules show binding modes in the Sam Barnes, Chenchen Song, Thomas Kno¨pfel. groove between the two WH domains. These may be responsible for inhib- Imperial College London, United Kingdom, London, United Kingdom. iting the conformational transition. Further simulations to estimate the effect The flow of information through networks of cortical neurons is thought to pro- of the small molecules on the conformational change as well as refinement vide the processing power that underpins cognition. Traditional structural of the initial leads to improve the affinity and selectivity of the compounds methods to map connectivity are blind to the functional dynamics of cortical are under way. circuits. Emerging approaches use genetically encoded activity indicators Supported by NIH R21 CA191898. such as GCaMP. However they are limited to reporting spiking activity and

BPJ 7825_7838 Tuesday, February 14, 2017 341a are blind to subthreshold excitatory and inhibitory events. The latter are, how- to a micrometer-sized bead, cantilever or a surface. We exploit the entropic ever, essential for the functional characterization of cortical circuits. This elasticity of single-stranded DNA to apply tension on a system mounted on problem could be overcome by a strategy based on the co-expression of a genet- our device [5]. Single-molecule FRET is used as a readout to study two dy- ically expressed, voltage sensitive fluorescent protein (VSFP) and calcium namic systems under different tensions: the transition behavior of a Holliday indicator (GECI) in the same neuron. Co-expression of VSFPs and GECIs in junction and the bending of a DNA promotor sequence induced by the specific cell-types facilitates direct visualization of sub and supra-threshold TATA-binding protein (TBP). activity, illuminating functional cortical circuits. This approach is possible References: in a new quadruple transgenic mouse: Rasgrf2-2A-dCre;CamK2a-tTA;Chi- Our results demonstrate the assembly power of DNA origami and our ability to VSFPBlfy1.2(TITL-GCaMP6f). This mouse line uses intersectional genetic fabricate materials and devices with control on the molecular level. targeting with (i) a GECI (GCaMP6f) targeted to the TIGRE locus (TITL) 1. P. W. K. Rothemund, Nature 440, 297-302 (2006) with expression driven in L2/3 excitatory neurons via Cre and tTA activities 2. N. C. Seeman, Annu. Rev. Biochem. 79, 12.1 (2010) (Rasgrf2 and CamK2A) and (ii) a VSFP (Chimeric-VSFPBlfy1.2) under a 3. R. Schreiber et al. Nature Nanotechnology 9, 74-78 (2014) tetracycline response element (pTRE) with expression driven by CamK2A to 4. J. Zheng et al. Nature 461, 74-77 (2009) excitatory neurons. The probability of GCaMP6f expression can be controlled 5. P. Nickels et al. Science, accepted by titrated trimethoprim application while VSFPs are broadly expressed in all excitatory cells. The co-expression of the two indicators combined with Workshop: Single-Particle CryoEM: A How-To 2-P microscopy gives simultaneous monitoring of both signals in the same neuron and across networks of neurons in vivo. We defined the spatio- Guide temporal profile of VSFP responses that co-occur with GCaMP transients in single neurons expressing both indicators in vitro and in vivo. We then create 1673-Wkshp event-triggered maps of co-active neural assemblies during visual stimulation. Using EM to Understand the Structure and Function of Molecular Thus we provide an approach to dissect functional cortical microcircuits in the Machines intact cortex. Bridget Carragher. New York Structural Biology Center, New York, NY, USA. Workshop: Next Generation Biophysics Enabled Cryo electron microscopy (cryoEM) has undergone an extraordinary period of rapid growth over the past several years. This growth resulted from the advent by Precision Chemical Biology Tools of technologies that make it possible to determine, at near-atomic resolution, the structure of many macromolecular complexes that previously could only 1671-Wkshp be imaged at low resolution. An introduction to the currently established Nanodiscs as a Precision Tool for Biophysics and Chemical Biology methods will be presented along with a discussion of some of the latest technol- Stephen G. Sligar. ogies that are still under active development. University of Illinois, Urbana, IL, USA. Membrane proteins are involved in numerous vital biological processes, 1674-Wkshp including transport, signal transduction and the enzymes in a variety of meta- Best Practices in Data Collection and DDD Movie Processing for Cryo-EM bolic pathways. Integral membrane proteins account for up to 30% of the hu- John Rubinstein. man proteome and make up more than half of all currently marketed Hospital for Sick Children Research Institute, Canada, Toronto, ON, Canada. therapeutic targets. Unfortunately, membrane proteins are inherently recalci- One peculiarity of the cryo-EM structure determination process is that the dif- trant to study using the normal toolkit available to scientists, and one is most ference between good data and bad data can often be detected only after the often left with the challenge of finding inhibitors, activators and specific anti- final 3D structure has been calculated. Direct detector device (DDD) movie bodies using a denatured or detergent solubilized aggregate. In many cases, acquisition introduces additional steps of image processing that can similarly since membrane proteins are inherently insoluble and prone to aggregation compromise the final 3-D map if not performed optimally. In this lecture I and oligomerization, the active state of interest is obscured. The Nanodisc plat- will explain aspects of microscope physics that must be considered to ensure form circumvents these challenges by providing a self-assembled system that that images contain as much information as possible. I will also describe renders typically insoluble, yet biologically and pharmacologically significant, methods for processing DDD movie data to allow optimal extraction of high- targets such as receptors, transporters, enzymes, and viral antigens soluble in resolution information about specimens. aqueous media. Because Nanodisc constructs provide a native-like bilayer environment that maintain a target’s functional activity, they are a versatile 1675-Wkshp tool in the study of membrane proteins such as ion channels, GPCRs, cyto- Optimization of a Cryo-EM Structural Model Representation chrome P450s, blood coagulation factors, various toxins and viral entities as Wah Chiu. well as a plethora of pharmaceutical targets. Nanodiscs also provide a nano- Baylor Col Med, Houston, TX, USA. meter scale vehicle for the in vivo delivery of amphipathic drugs, therapeutic Electron cryo-microscopy (cryoEM) has been used to resolve atomic reso- lipids, tethered nucleic acids, imaging agents and active protein complexes. lution biological structures. However, the resulting density maps and models In my presentation I will focus on recent uses of the Nanodisc technology in still lack assessment of accuracy and reproducibility. The densities of some seeking mechanistic understanding of critical cellular processes such as residues particularly, the negative charged residues have been found to be KRas4b signaling in cancer and Alzheimer’s disease intervention. consistently absent or have low values in near-atomic resolution cryoEM maps. We thus introduce a procedure to derive an atomic model that faith- 1672-Wkshp fully replicates the experimental map and then use this map to ensure data Molecular Tools and Nanodevices Built from DNA accuracy and reproducibility. We have applied this protocol to describe Tim Liedl. the 3.3 A˚ structure of the mature P22 bacteriophage capsid, a large and com- Physics, Ludwig Maximilian University of Munich, Munich, Germany. plex macromolecular assembly. This structure identifies previously unknown Over the last decades, DNA has proven to be an excellent choice for the de- molecular interactions between capsid subunits, crucial to maintaining par- signed self-assembly of functional two- and three-dimensional nanostructures ticle stability. and materials [1,2]. By offering attachment sites for active nano-components Acknowledgement: This research has been supported by NIH grants on DNA-based objects, our group has realized complex and nanometer- (R01GM079429, P01GM063210, T15LM007093) and R Welch Foundation precise assemblies of fluorophores, plasmonic nanoparticles and biomolecules (Q1242). [3]. The initial proposal catalyzing the rapid development of DNA nanotechnology 1676-Wkshp was to arrange periodic DNA frameworks to host guest molecules for crystal Structure Determination of Multi-Protein Complexes using Cryo-EM structure analysis. Ned Seeman reported a 3D DNA crystal based on the ‘‘ten- Lori A. Passmore, Christopher J. Russo. segrity triangle’’, where three DNA duplexes are interconnected in a self- MRC Laboratory of Molecular Biology, Cambridge, United Kingdom. restricting over-under, over-under, over-under fashion [4]. By adopting this Cryo-EM is a powerful approach for understanding the mechanisms of design principle, we present a tensegrity triangle design based on DNA origami multi-protein complexes that are often difficult to study by other methods. that crystallizes into three dimensional, micrometer-scale assemblies. Recent advances in the field have facilitated high-resolution structure deter- To explore dynamic aspects, we developed a nanoscopic DNA origami-based mination. Direct electron detectors have an improved signal-to-noise ratio single-molecule force spectroscopy device without any physical connection compared to film or CCD, and images can be acquired using a much lower

BPJ 7825_7838 342a Tuesday, February 14, 2017 electron dose than was previously required. These advances have been rev- Myriads of regulatory interactions have been identified at the level of transcrip- olutionary for biological cryo-EM specimens that are low contrast and radi- tion, translation, post-translational modification and metabolite feedback, but ation sensitive. Still, structure determination remains challenging for many our understanding which of them govern a given adaptation and actually con- proteins because cryo specimens move upon irradiation with the electron trol a given biological function is very limited. For metabolism this function is beam, resulting in blurred images with a limited resolution. Other practical the flux of small molecules that is coordinated through a variety of overlapping considerations also present challenges: Many complexes are only available regulation mechanism. Although the specific molecular implementation might in limited quantities and concentrations, and specimen preparation can be differ, the regulatory logic is usually highly similar in different microbes (1). In difficult and not easy to reproduce. We use a hybrid approach combining this lecture I will focus on the key role of metabolite-protein interactions in structural (cryo-EM, x-ray crystallography, NMR), biochemical, biophysical achieving this coordination in E. coli, both as input into transcriptional regula- and genetic techniques to gain insights into the molecular mechanisms of tion and by directly affecting enzyme activity, and outline how we delineate protein complexes that regulate gene expression. In this talk, I will discuss regulation events that actively control the coordination of metabolic fluxes some of our recent biological insights and technological advances. I will by combining various omics methods with computational analysis (2, 3). Out outline our approach to studying these multi-protein complexes using of the bewildering complexity of possible regulatory interactions, surprisingly cryo-EM in a step-by-step manner. few regulation events appear to be required for a given transition, typically involving less than a handful of active regulators. 1. Chubukov, Gerosa, Kocha- Workshop: Biological Networks from nowski & Sauer. Nat Rev Microbiol 12: 327 (2014) 2. Gerosa, Haverkorn, Christodoulou, Kochanowski, Schmidt, Noor & Sauer. Cell Systems Biology. Experiment to Modeling and Back 1: 270-282 (2015) 3. Link, Fuhrer, Gerosa, Zamboni & Sauer. Nature Methods 12: 1091-1079 (2015). 1677-Wkshp Using a Genome-Scale Model and Genomic Library to Discover the Func- 1679-Wkshp tions of Metabolic Genes Deciphering the Stochastic Kinetics of Gene Regulation Shu Pan1, Kiel Nikolakakis1, Edward Ruby2, Jennifer Reed1. Ido Golding. 1 2 University of Wisconsin-Mandison, Madison, WI, USA, University of Baylor College of Medicine, Houston, TX, USA. Hawaii, Manoa, Manoa, HI, USA. Gene activity is the prime mover in the living cell, driving a cell’s function at Vibrio fischeri is a bioluminescent marine bacterium that forms a symbiotic any given time. I will report on recent advances in our ability to describe the relationship in the light-emitting organ of the Hawaiian bobtail squid, stochastic kinetics of gene regulation, achieved through the combination of Euprymna scolopes. To better understand the metabolism of V. fischeri,we single-molecule fluorescence microscopy in individual cells, novel image anal- constructed a V. fischeri genome-scale metabolic model -and developed a novel ysis algorithms, and theoretical modeling. We apply our approach to explore approach for finding functions of metabolic genes using genomic libraries. Our gene regulation in a number of model organisms representing a gradation of V. fischeri model captures most V. fischeri biochemical reactions and their asso- complexity: E. coli bacteria, Drosophila embryos and mouse embryonic stem ciated genes based on the current genome annotation. While the model was cells. highly accurate at predicting carbon sources (86.2% accuracy) and gene essen- tiality (83.4% accuracy), the incorrect predictions by the model indicate that 1680-Wkshp there are metabolic functions missing from our model and the current genome Modeling Cytoplasmic Mechanics in Rapidly Moving Cells annotation. Julie Theriot. We developed a new integrated computational and experimental approach to Stanford University Sch Med, Stanford, CA, USA. find the genes responsible for these missing metabolic functions. This approach Directed crawling motility of animal cell types ranging from neurons to uses computational models to design selection strategies for these missing re- macrophages requires the coordinated force-generating activity of multiple actions. These selection strategies specify a medium condition and a recipient mechanical elements. Much molecular detail is known about the constitu- strain whose growth in this media is dependent on the metabolic function being ents of some mechanical submachines such as the polymerizing actin sought. Genomic libraries (in this case derived from V. fischeri) are then trans- network and the adhesion complexes, but it is not yet clear how these ele- formed into the recipient strain and growth selections are used to identify the ments all work together to generate coherent, directed motion at the level genes responsible for the missing metabolic function. This approach identified of the whole cell. In order to understand cellular mechanisms of large- 5 genes responsible for 4 missing metabolic functions (including enzymes and scale coordination, our work focuses on two extremely fast-moving cell transporters) in V. fischeri. Additional experiments involving knockout mutant types, the fish epidermal basal keratocyte responsible for the rapid closure complementation, mutant growth phenotyping, and enzyme assays further sup- of wounds in fish skin, and the human neutrophil that hunts down and kills ported that the genes identified from the genomic library are responsible for microbial invaders. Under normal culture conditions, motile keratocytes as- these metabolic activities in V. fischeri. This integrated computational and sume an unusually simple, stereotypical, fan-like shape with a very large, experimental approach can be generally applied to a broad range of microbes thin lamellipodium densely packed with actin filaments, and can move to improve their genome annotations and our understanding of their metabolic persistently with nearly constant shape and speed. In contrast, neutrophils capabilities. change shape dramatically during normal motion and exhibit significant intracellular fluid flows as well as complex cytoskeletal dynamics. We use 1678-Wkshp a variety of quantitative methods based on live cell videomicroscopy to Coordination of Metabolism Through Metabolite-Protein Interactions track the dynamic behavior of cytoskeletal elements and organelles as these Uwe Sauer. cells move and respond to signals and obstacles in their environments ETH Zurich, Switzerland, Zurich,€ Switzerland. including chemoattractants, electric fields, and physical variations such as The omics revolution has vastly expanded our ability to monitor intracellular changes in substrate adhesivity and microfabricated barriers. In parallel, molecular events. This technological advance has typically not been matched we develop mathematical models that describe simplified physical frame- with conceptual advances in our ability to interpret and understand such data works for intracellular mechanics and dynamics in ways that can make or to systematically generate hypotheses from them. The iterative systems quantitative predictions of experimental outcomes, which can then be biology cycle of experiment and computational analysis is a great promise, used iteratively to refine the mathematical models. Despite their very but turns in practise typically only one or twice per project. A particular chal- different biological roles and behaviors, we find that keratocytes and neutro- lenge for all cell types, from bacteria to humans, are the multiple, overlapping phils appear to share fundamental mechanisms of self-organization and regulatory mechanisms that coordinate adaptation to changing environments. movement coordination.

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Posters ture elements, placing insulin near the catalytic site. Concordantly, insulin un- dergoes conformational change; the insulin chain-B central a-helix becomes less ordered, a key step in the unfolding of insulin for degradation. The pres- Protein Structure and Conformation III ence of insulin moves IDE-N and IDE-C closer (the angle between them de- creases ~5) and causes these two domains to undergo a grinding motion as 1681-Pos Board B1 suggested by experimental data. Furthermore, greater flexibility for the door, Probing Substrate Sequestration in Carrier Proteins using Vibrational H-loop, and the hinge and interface between IDE-N and IDE-C is evident Spectroscopy Labels when insulin is present. These observations suggest that the presence of insulin Caroline A. McKeon, Samuel C. Epstein. induces both global and local conformational changes in IDE to unfold insulin Haverford College, Haverford, PA, USA. for cleavage. Principal component analysis shows negatively-correlated Carrier proteins are considered ‘‘lynchpin’’ enzymes of biosynthetic path- opening and closing motions of dimerized IDE monomers, suggesting that ways. The E. coli acyl carrier protein (ACP) is composed of multiple alpha the capture of substrate in one monomer may allosterically induce the other helices that form a hydrophobic, solvent-protected pocket that sometimes pro- to open. Our simulation and principle component analysis offer new insight vides a cavernous hiding spot to protect substrates bound to the ACP’s phos- into substrate unfolding inside the IDE catalytic chamber and its allosteric phopantetheine (Ppant) arm. The action of ‘‘chain sequestration’’ is thought to regulation. be important for driving the biosynthetic process. In this study, selected amino 1684-Pos Board B4 acid residues inside the ACP sequestration channel were targeted as possible Seca Functions In Vivo as a 1M6N-Like Dimer to Promote Protein incorporation sites for para-substituted aromatic nitrile or alkyne vibrational Transport spectroscopic probes. Using stop codon suppression, non-canonical amino Tithi Banerjee. acids were incorporated into the ACP via a co-transformed aminoacyl Wesleyan University, Middletown, CT, USA. tRNA synthetase. In addition, artificial substrates containing unique vibra- SecA ATPase motor protein plays a central role in bacterial protein transport by tional labeling groups like nitriles were covalently ligated to the ACP’s Ppant binding substrate proteins and the SecY channel complex and utilizing its arm. The vibrational frequencies of these labeling groups, measured via IR ab- ATPase activity to drive protein translocation across the plasma membrane. sorption or Raman scattering, report on mainly the solvent exposure of the CN SecA has been shown to exist in a dynamic monomer-dimer equilibrium modu- group, and the lineshape reports the distribution of environments around the lated by translocation ligands, and multiple structural forms of the dimer have label on either the protein or the growing substrate. IR and Raman results been crystallized. Whether SecA functions as a monomer or dimer in vivo, and that report on the sequestration of substrates of different length, as well as the structural form of the dimer remain highly controversial and unresolved methodological advances that enable this spectroscopic approach, will be questions. We addressed this matter by engineering r-benzoylphenylalanine discussed. along dimer interfaces corresponding to the five different SecA x-ray structures 1682-Pos Board B2 and assessing their in vivo photo-crosslinking pattern. A 1M6N-like dimer was Identification of Fluoxetine-Serotonin Transporter Interactions using the dominant if not exclusive dimer found in vivo, whether SecA was cytosolic Crosslinking-Mass Spectrometry (CX-MS) or in lipid or SecYEG-bound states. SecA bound to a stable translocation inter- Elizabeth Castellano. mediate was crosslinked in vivo to a second SecA protomer at its 1M6N inter- Duquesne University, Pittsburgh, PA, USA. face, indicating that this specific dimer promotes active protein translocation. The serotonin transporter (SERT) is a member of the neurotransmitter sodium Taken as a whole our study supports more recent thinking in the field that sug- symporter family of transporters (NSSs). SERT controls the magnitude and gests that SecA alternates between a dimer and monomer as part of the protein duration of serotonergic neurotransmission by facilitating the reuptake of sero- translocation cycle and creates a platform to directly test these and related ideas tonin back into the pre-synaptic neuron and is thus a target for antidepressants utilizing more direct biochemical and biophysical approaches at both the and selective serotonin re-uptake inhibitors (SSRIs) commonly prescribed to ensemble as well as single molecule level. treat depression. To understand drug binding to SERT, a photo-activatable fluoxetine analog was synthesized and crosslinked to human SERT expressed 1685-Pos Board B5 in tetracycline-inducible HEK293 cells. After purification, trypsinized peptides Comparison of Polarizable Continuum & Quantum Mechanics/Molecular were analyzed by ESI-Q-TOF and MS/MS studies identified sites of covalent Mechanics Models with Series of Tripeptides (AXA) attachment. Given that fluoxetine partitions in bilayers and might interact Allen Walker1, Lillian Zhu2, Esmael Jafari Haddadian3, Ahmed Lakhani1. 1Science, Calumet College of St. Joseph, Whiting, IN, USA, 2Northwood non-specifically with membrane proteins, control studies with photo- 3 activatable lipids were also conducted to map lipid accessible sites. The hSERT High School, Irvine, CA, USA, Biological Sciences, The University of expression system contains N-terminal tags for purification, so additional CX- Chicago, Chicago, IL, USA. MS studies were conducted using baculovirus overexpression of rat SERT con- In biomolecule simulations solvation is a key factor in representing experi- taining analogous tags, but at its C-terminus to control for artefacts attributable mental conditions. Recent studies use models like Quantum Mechanics and to the sites of the chimeric tags. Altogether, these studies identify non-specific Molecular Mechanics (QM/MM) and Polarizable Continuum Model (PCM) and specific sites of interactions between fluoxetine and SERT. to attempt to simulate solvent effects on spectra. For proteins and peptides vibrational analyses primarily focuses on amide I (due to its isolation and 1683-Pos Board B3 strong dipole moment) to determine information about the secondary structure Insights into Substrate Unfolding and Allosteric Regulation of Insulin (a-helix, b-sheet, PPII). In this study we compare the QM/MM and PCM Degrading Enzyme models with a focus on amide I at ~1650 cm1 (mainly amide C=O stretch) Felicia H. Jiang1, Wei-Jen Tang2, Esmael J. Haddadian3. and also extended to lower energy amide II (~ 1550 cm1) in order to gain 1Department of Statistics, University of Chicago, Chicago, IL, USA, 2Ben insight into the effectiveness of either method. We modeled a series of tripep- May Department for Cancer Research, University of Chicago, Chicago, IL, tides (AXA: where X=A, E, S, W) and capped at N- and C- termini to yield 3 USA, Biological Sciences Collegiate Division, University of Chicago, Ac-Ala-Xxx-Ala-NH2. For QM/MM studies, we first sampled the conforma- Chicago, IL, USA. tions with an explicit solvent using Charmm 36 force field. The simulations Insulin degrading enzyme (IDE) is involved in the clearance of insulin, amy- were executed for 20 ns using periodic boundary conditions, with constant lin, and glucagon, peptides involved in controlling blood glucose level. IDE pressure and temperature (NPT ensemble) at 300 K and 1 atm. We then ob- also degrades amyloid-b, a peptide vital for Alzheimer‘s disease progression. tained the most preferable conformation (which is PPII-like) and extracted IDE has two homologous N- and C-terminal domains, IDE-N and IDE-C. For the geometry for both PCM and QM/MM level calculation to generate IR IDE to effectively degrade its substrates, both domains must come together for and VCD spectra. Furthermore, PCM Model was computed with an ideal the catalytic cleft to assume fully closed conformation. Experimental data sug- PPII-like conformation (78,149,180) to compare with preferable conforma- gests that IDE undergoes at least two conformational switches in its catalytic tion obtained from MD simulation. The geometry was constrained (in terms cycle: one, the transition between the closed- and open-conformations, and of 4, c torsional angles) and prepared for optimization. With PCM model two, the ‘‘swinging door’’ motion at the catalytic cleft. However, the exact we use an implicit solvent while in QM/MM we use an explicit solvation shell mechanism of these two conformational switches is unknown. We investigated of 10A˚ . All calculations were fully optimized across all coordinates except (4, this mechanism using microsecond-long molecular dynamics simulations of c, u, which were fixed) at the DFT BPW91/6-31G** level of theory. By IDE structures with and without substrate. We find that the N-terminus of observing amide I and amide II, accurate inferences about the different types the insulin molecule chain-A forms a cross-b-sheet with IDE secondary struc- of theory were made.

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1686-Pos Board B6 the pockets between the dimer subunits of the HBV capsid protein. This bind- Identification of Key Structural Features Determining the Binding of ing alters the orientation of the subunits in the capsid assembly intermediates Alpha Globin and Endothelial Nitric Oxide Synthase and can inhibit the virus growth by either accelerating capsid assembly or by TC Stevenson Keller IV1, Nicole K. Swope2, Brant E. Isakson1, misdirecting it. However, the mechanisms of how CAMs affect assembly is Linda Columbus2. presently not well understood, preventing a rational enrichment of their ther- 1Molecular Physiology and Biological Physics, University of Virginia, apeutic potential. Here, we have used molecular dynamics simulations to Charlottesville, VA, USA, 2Chemistry, University of Virginia, study HBV capsid assembly with and without CAMs, focusing on early dimer, Charlottesville, VA, USA. tetramer, and hexamer intermediates. Based on principal component analysis Nitric oxide (NO), an endogenously produced gaseous molecule, is the most of tetramer and hexamer simulations combined with free-energy calculations, potent way of regulating blood pressure in the resistance vasculature. NO is we identified the structural bottlenecks for capsid nucleation and assembly. produced in endothelial cells by endothelial nitric oxide synthase (eNOS), Additionally, tetramer intermediates were simulated with different CAMs in and diffuses to the surrounding smooth muscle cells causing relaxation and the binding pocket, showing that they alter the proteins’ conformational dy- vasodilation. eNOS maintains blood pressure homeostasis through tightly regu- namics; free-energy perturbation simulations were also used to determine lated protein interactions and modifications. One of these regulatory pathways the affinities of CAMs for both open and closed hexamer intermediates. is a direct interaction with the alpha subunit of hemoglobin (alpha globin). Finally, structures of the tetramer from simulation were selected for docking, Alpha globin is expressed in endothelial cells and acts as both an NO scavenger resulting in one compound that was shown to accelerated HBV assembly and and an inhibitor of NO production via an interaction with eNOS. A peptide inhibit the virus growth without high toxicity liver cells. The activity of this mimicking a region of the alpha globin sequence (HbaX) directly interacts compound was further improved by virtual screening and experimental with eNOS in vitro, and lowers blood pressure when administered in vivo. testing. To improve the pharmacokinetic properties of this peptide, HbaX was cyclized and the secondary structure and binding affinity to eNOS were compared be- 1689-Pos Board B9 tween variants and linear peptides. The linker length and sequence of the cyclic Characterization of the Full-Length, Human Beclin-1 Purified from Escherichia Coli peptide were varied and compared to the nM KD of the HbaX-eNOS interac- tion. Secondary structure analysis of linear and cyclic peptides was determined Matthew Ranaghan1, Colin Garvie1, Doug Daniels2, Beth Levine3, with solution NMR techniques. These studies on the physical parameters of the Jose Perez1. 1Center for the Development of Therapeutics, Broad Institute of MIT and HbaX/eNOS interaction are the first step in investigating the viability of the 2 peptide for pharmaceutical use. Harvard, Cambridge, MA, USA, Department of Chemistry, University of Dayton, Dayton, OH, USA, 3Department of Internal Medicine and Howard 1687-Pos Board B7 Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX, Molecular Level Insight into a Unique Surface Protein Glycosylation USA. Pathway: Structure of the Actinomyces Oris LCP Enzyme that Mediates We report here the first synthesis and functional characterization of the full- Surface Protein Glycosylation length, human homolog of Beclin-1 that was generated as a recombinant pro- Brendan R. Amer1, Michael R. Sawaya2,3, Brandon Liauw1, Janine Fu1, tein from Escherichia coli. Beclin-1 is essential for initiating macroautophagy Hung Ton-That4, Robert T. Clubb5,6. (herein called autophagy); a highly conserved survival mechanism that recycles 1Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA, damaged cellular components (e.g., proteins, organelles) or pathogens by en- USA, 2DOE-Institute for Genomics and Proteomics, UCLA, Los Angeles, casing them in a bilayer vesicle (termed autophagosome) that fuses with a lyso- CA, USA, 3Howard Hughes Medical Institute, UCLA, Los Angeles, CA, some to enable degradation of the vesicular contents. Mutations in or altered USA, 4Department of Microbiology & Molecular Genetics, University of expression profiles of Beclin-1 are well documented to be involved in various Texas Health Science Center, Houston, TX, USA, 5Chemistry and cancers and neurodegenerative diseases. Moreover, several viruses (including Biochemistry, UCLA, Los Angeles, CA, USA, 6Molecular Biology Institute, HIV and HSV-1) specifically target Beclin-1 as a means to evade host defense UCLA, Los Angeles, CA, USA. mechanisms. We demonstrate that our recombinant Beclin-1 protein exists as a The peptidoglycan that surrounds Gram-positive bacteria is affixed with a range soluble homodimeric species in solution, undergoes reversible monomer-dimer of macromolecules that enable the microbe to effectively interact with its envi- equilibrium, binds lipid membranes, and interacts with HIV Nef and the anti- ronment. As surface displayed macromolecules frequently play key roles in apoptotic Bcl-2 proteins. bacterial pathogenesis, inhibitors that prevent their display could function as powerful antibiotics. Here we report studies of LytR-CpsA-Psr (LCP) proteins 1690-Pos Board B10 that attach teichoic acid polymers and capsular polysaccharides to the cell wall. Different Covalent Immobilization Strategies Influencing the Fibronec- LCP enzymes are important architects of the cell surface and potential drug tar- tin‘S Conformation and Bioactivity gets as the teichoic acid polymers they display on peptidoglycan are major con- Stephanie Vanslambrouck, Pascale Chevallier, Andree-Anne Guay-Begin, stituents of the cell surface, comprising up to 50% of its overall mass. Recently, Gaetan Laroche. Ton-That and colleagues discovered a new glycosylation pathway in the oral Laboratoire d‘inge´nierie de surface, Laval University, Quebec, QC, Canada. pathogen Actinomyces oris that employs an LCP enzyme. This LCP protein Fibronectin is one of the most investigated proteins due to its cell adhesion has a novel function in which it glycosylates the AcaA protein, which is then properties and its contribution to important physiological processes. However, displayed on the surface. This unique activity by A. oris LCP plays an important its conformation and, hence, its bioactivity strongly depend on the hydrophilic/ role in cell surface display and bacterial viability. A 2.7A˚ crystal structure of hydrophobic character of the surface, but also the immobilization strategies. the A. oris LCP enzyme reveals that the enzyme utilizes key conserved residues Therefore, the original approach of this work is to investigate the effect of in the active site to recognize its lipid-glycopolymer substrate. Additionally, the both parameters. For that purpose, the grafting of fibronectin onto silicon wa- structure reveals that the LCP enzyme co-purifies with E. coli isoprenoids, fers was performed by using four different crosslinkers with various hydrophi- defining one part of the enzyme active site where the enzyme binds its lipid- licity to link fibronectin either through its amino groups (lysine), or sulfhydryl glycopolymer substrate. Ongoing work includes development of an in vitro functionalities (cysteine). The grafting of all the crosslinkers and the fibronectin biochemical assay for LCP enzyme activity and structure-mechanism studies onto the surfaces was evidenced by X-ray photoelectron spectroscopy, and dif- of secondary substrate (peptide/peptidoglycan precursor) binding. The struc- ferences in hydrophilicity were highlighted with the contact angle measure- ture suggests mechanistic conservation between display of wall teichoic acids ments. Moreover, profiles based on atomic force microscopy images and surface protein glycosylation. exhibited different protein conformations. Indeed, hydrophilic spacer arms led to fibronectin spreading on the surface, which further exhibited a more elon- 1688-Pos Board B8 gated shape when grafted by cysteyl residues compare to lysyl ones. These dif- Rational Development of Hbv Capsid Inhibitors Aided by Molecular ferences in fibronectin conformation, related to its bioactivity, were also Dynamics supported by enzyme-linked immunosorbent assay (ELISA) tests. In fact, the Maksym Korablyov1, Anna Pavlova1, Christophe Chipot2, availability of the cell adhesion domains, using monoclonal anti-RGD anti- James C. Gumbart1. bodies, was also evidenced to be dependent on the crosslinkers. Furthermore, 1Georgia Institute of Technology, Atlanta, GA, USA, 2University of Illinois, they had an influence on the grafted fibronectin amounts, as shown by ELISA Urbana-Champaign, Urbana, IL, USA. tests with polyclonal anti-fibronectin antibodies. In conclusion, this work Chronic infection by the Hepatitis B virus (HBV) has no cure and is a major clearly demonstrated that hydrophilic crosslinkers allowed the grafting of a cause of liver damage and liver cancer. According to recent studies, the virus higher amount of fibronectin onto the surfaces as opposed to hydrophobic growth can be inhibited by capsid assembly inhibitors (CAMs), which bind in ones. Moreover, cell binding sequences were more available when fibronectin

BPJ 7839_7842 Tuesday, February 14, 2017 345a was grafted via its amino groups rather than its sulfhydryl groups, consequently In terms of function, it is widely accepted that only this tetrameric assembly the fibronectin bioactivity would be improved in this case. alpha2beta2 shows cooperative binding of oxygen molecules; in other words, the average affinity for oxygen is modulated from low to high depending on 1691-Pos Board B11 the average saturation level of the alpha2beta2 assembly. In contrast, isolated Structural Dynamics in the Myosin 7A Single a-Helix Domain subunits bind oxygen in a monotonic manner showing only high affinity. More- Matthew Batchelor, Marcin Wolny, Peter J. Knight, Emanuele Paci, over, this tetrameric assembly, once it is formed, dissociates only into dimers of Arnout P. Kalverda, Michelle Peckham. the form alphabeta, but reversion to pure assemblies of either subunit under Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom. equilibrium conditions is negligible. Therefore, the preferential formation of Myosin 7a (M7a) is an ‘unconventional’ myosin, highly expressed in the eye the alphabeta dimer is a crucial step and may hold clues about the emergence and ear. Mutations in M7a are a common cause of deafness and blindness of functional cooperativity. We have also investigated the role of chemical (Usher syndrome 1B). M7a consists of a motor domain, a neck region modifications inserted in the alphabeta interface and studied the impact on composed of five IQ motifs, a charge-rich segment originally predicted to the dimer assembly and the protein function as well. form a coiled coil, and a number of tail domains. Our aim here was to deter- mine if a polypeptide consisting of the M7a charge-rich segment (80 residues) 1694-Pos Board B14 forms a stable single a-helix (SAH) in solution, as we predicted from its Building a Better Phospholamban: Characterizing and Optimizing the sequence (Knight et al., J. Biol. Chem., 2005, 280, 34702). Circular dichroism Structural Dynamics of Domain Ib PLN Mutants to Tune SERCA Function spectroscopy demonstrated that this polypeptide is highly helical (>90% he- 1 1 2 3 Kim N. Ha , Mara Exline , Sarah E. Nelson , Gianluigi Veglia . lix at 10 C), and that it unfolds with a broad, non-cooperative thermal un- 1Chemistry and Biochemistry, St. Catherine University, St. Paul, MN, USA, folding transition, as expected for a SAH. Analytical ultracentrifugation 2Biochemistry, University of Minnesota, Minneapolis, MN, USA, showed that the polypeptide is monomeric. Rotary shadowing electron micro- 3Chemistry and Biochemistry, University of Minnesota, Minneapolis, MN, scopy, small angle X-ray scattering and size exclusion chromatography all USA. showed that it forms an elongated structure. Nuclear magnetic resonance The interplay between the sarco(endo)plasmic reticulum Ca2þ-ATPase (NMR) experiments corroborated this picture of an elongated, highly helical (SERCA) and its endogenous cardiac inhibitor phospholamban (PLN) lends structure and provided extra structural detail with regard to the residue side a critical role in Ca2þ signaling in cardiac myocytes. Dysfunctions in their in- chains. In common with other known SAH domains, the charge-rich segment teractions can lead to reduced cardiac contractility, and several mutations of is dominated by Glu (E), Lys (K) and Arg (R) residues. Formation of salt PLN have been linked to hereditary cardiomyopathy. The latest of these muta- bridges between the side chains of E-R and E-K pairs and their dynamic tions, R25C, was discovered in 2015 and is shown to lead to arrhythmias with behaviour are likely a major contributor to their stability. We are now eventual development of heart failure. The R25C mutation highlights the func- exploring salt bridge pairing preferences along the sequence with NMR ex- tional relevance of domain Ib of PLN in regulating its inhibitory potency and periments to determine how salt-bridge formation contributes to SAH mitigating allosteric signaling through the juxtamembrane region of the pro- stability. tein. In these studies, we seek to characterize the function, structure and dy- namics of domain Ib PLN mutants using a combination of structure 1692-Pos Board B12 prediction tools, site directed mutagenesis, enzyme assays, and solution state Protein Energy Network Models to Classify and Predict Functionally NMR. The ultimate goal of these studies is to use them to better inform rational Linked Interfaces of Proteins from Functionally Uncorrelated Interfaces structure-based design of therapeutic PLN species that could be used to treat Isha D. Mehta1, Brian W. Beck2. heart failure. The project is a working collaboration between St. Catherine 1Biology, Texas Womans University, Denton, TX, USA, 2Texas Advanced University, the University of Minnesota, and the Minnesota NMR Center, Computing Center, University of Texas, Austin, TX, USA. and provides a model of conducting collaborative undergraduate research in Proteins perform various cellular activities, often by interacting with other pro- partnership between a PUI, a major research institution, and an instrument teins. The structural stability and interaction specificity of such participating center. oligomers is likely representative of the function they carry out. The goal of our research is to characterize the organization of spatial and energetic features 1695-Pos Board B15 of interacting amino acid residues that occur during formation of Functionally Charge Neutralization, Not Salt Bridges, Stabilizes Alpha Helices in Linked Interfaces of Proteins (FLIPs) that are distinct from Functionally uncor- Repeating Blocks of Acidic and Basic Residues related Contacts (FunCs). Here, proteins are represented as networks of inter- Ammon E. Posey1, Tyler S. Harmon2, Rohit V. Pappu1. acting amino acid residues, where residues are the network nodes and their 1Department of Biomedical Engineering & Center for Biological Systems interacting energies (both bonded and non-bonded interactions) define the Engineering, Washington University in St. Louis, St. Louis, MO, USA, edges. Residue Interaction Networks (RINs, a network representation of protein 2Department of Physics, Washington University in St. Louis, St. Louis, MO, structure based on residue proximity) have been shown to have network prop- USA. erties that are distinct for active sites and residues important for protein folding. Proteins from a variety of organisms and protein families contain long single In our prior work, we could distinguish known FLIPs from FunCs using RINs alpha helices formed from repeating blocks of acidic and basic residues. The with ~70% accuracy and found FLIPs were generally much more organized consensus sequence for these types of helical regions is an E4K4 repeat, a than FunCs. In this work, we expand this concept to energetic organization fea- sequence that confounds IDP prediction algorithms, structural prediction algo- tures that can aid in a FLIP vs FunCs classification. This refinement of the rithms and many atomistic simulations alike. The prevailing hypothesis is that network model to directly use energy was found to improve the accuracy of these sequences form alpha helices through salt bridges between i and iþ4 res- two category classification thus also aiding in prediction of residues capable idue positions. However, a survey of the literature reveals that alternative of forming FLIPs. mechanisms have not been thoroughly investigated. We hypothesized that sequence context may favor altered charged states that stabilize helical con- 1693-Pos Board B13 formations. To test this hypothesis, we used a novel simulation approach in Assembly of a Multimeric Allosteric Protein from its Isolated Components which the neutralized forms of the ionizable residues were allowed to substi- Characterized by Isothermal Titration Calorimetry (ITC) tute for the corresponding charged forms in order to maximize agreement be- Antonio Tsuneshige. tween conformations in simulations and experiment. These simulations Frontier Bioscience, Hosei University, Tokyo, Japan. suggest that alpha helix formation in E4K4 repeats is the result of charge The structural aspects of the formation of human hemoglobin, a tetrameric allo- neutralization of glutamate residues instead of salt bridges. Careful potentio- steric protein of the form alpha2beta2, from its component subunits has been metric measurements of an E4K4 repeat peptide provided clear evidence of studied by isothermal titration calorimetry. In isolation, both alpha and beta large pKa shifts in a significant number of glutamate residues, supporting subunits form assemblies; alpha subunits form preferentially dimers of the the computational prediction. The distinction between the salt bridge vs. form alpha2, whereas beta subunits assemble tetramers of the form beta4. charge neutralization mechanisms might have important biological implica- Thus the overall reaction for the formation of the alpha2beta2 tetramer is rather tions since these two mechanisms result in vastly different pH-sensitivity of complex, since its formation involves the prior formation of a dimer of the form the alpha helical structures involved. These findings suggest that the effect alphabeta, a sine qua non step that precedes the formation of the tetrameric of sequence context on the charge state of ionizable residues must be taken form alpha2beta2. The formation of the alphabeta dimer implies necessarily into account when seeking to make bioinformatics- and molecular the dissociation of assemblies of isolated alpha and beta subunits into their evolution-based predictions of sequence-structure-function relationships. respective monomers. Furthermore, these findings emphasize the need for further development of

BPJ 7839_7842 346a Tuesday, February 14, 2017 constant-pH simulations that can be applied to sequences with a high content 1699-Pos Board B19 of ionizable residues. Characterization of a Novel FRET Probe in a Crowded Environment using Time-Resolved Anisotropy 1696-Pos Board B16 Hannah Leopold1, Megan Currie1, Jacob Schwarz1, Arnold J. Boersma2, Multi-State Modeling of Biomolecules by Satisfaction of Spatial Restraints Erin D. Sheets1, Ahmed A. Heikal1. from Single-Particle Electron Microscopy Images 1University of Minnesota Duluth, Duluth, MN, USA, 2University of Ilan E. Chemmama, Charles H. Greenberg, Andrej Sali. Groningen, Groningen, Netherlands. Department of Bioengineering and Therapeutic Sciences, U. of C. San Macromolecular crowding affects numerous biophysical properties of proteins Francisco, San Francisco, CA, USA. including molecular conformation, reaction kinetics and cellular localization. Single proteins and macromolecular complexes can exist in multiple confor- As a result, there is a need to quantify crowding in a heterogeneous environ- mational and compositional states. Accurate modeling of these ensembles of ment both in vivo and in vitro. Recently, a novel genetically encoded FRET states is key to understanding and modulating the molecular function. probe (CFP-linker-YFP) was developed as a sensor to quantitatively measure Single-particle electron microscopy (EM) can produce a wealth of structural crowding in vivo and was characterized with steady-state fluorescence (Nat information. Here, we describe an integrative method for computing a Meth [2015] 12:227). In this contribution, we investigated the excited state dy- multi-state model of a biomolecular system, based primarily on input particle namics of the FRET probe in Ficoll-70 crowded environments using time- images from EM. The approach also leverages prior knowledge about the ste- resolved fluorescence anisotropy. To differentiate between viscosity and reochemistry of molecules, image noise, and potentially other sources of infor- crowding effects, similar studies were carried out in glycerol-enriched buffer mation. We validate the approach by using a synthetic benchmark, comparing (PBS, pH 7.5). Time-resolved measurements were conducted as a function of it to our previous em2d method, and applying it to a real test case of both the excitation and emission wavelengths as a means to probe the tumbling g-secretase. motion of the donor and acceptor separately. Our results indicate that the 1697-Pos Board B17 anisotropy of the acceptor decays as a biexponential with a fast rotational Identification and Characterization of a Repressed Troponin I-Like time, which is attributed to the energy transfer efficiency between the donor Epitope Structure in the C-Terminal Region of Troponin T and acceptor. In addition, this fast, FRET-based component is sensitive to Taylor Heilig, J.-P. Jin. Ficoll-induced crowding in contrast with glycerol-enriched buffer. These re- Wayne State University, Detroit, MI, USA. sults indicate that time-resolved anisotropy can be combined with this novel The troponin complex plays a central role in striated muscle contraction. FRET probe for quantitative, non-invasive analysis of site-specific crowding. Among the three subunits of troponin, troponin I (TnI) and troponin T Now that a more comprehensive picture of how the FRET probe is functioning (TnT) are encoded by genes originated from a TnI-like ancestor (Chong & at the molecular level in vitro, further structural modifications of the probe have Jin, J. Mol. Evol. 68:448-60, 2009). Here we report a study of an intriguing been carried out to increase FRET efficiency. observation that the restrictive N-terminal truncation of cardiac TnT restores an epitope structure that is recognized by a monoclonal antibody (mAb) 1700-Pos Board B20 TnI-1 originally generated against a C-terminal epitope of TnI. We tested Crytochrome and Nuclear Receptors - Expanding the Role of a Canonical the hypothesis that this restored epitope of cardiac TnT represents an evolu- Circadian Repressor tionarily repressed allosteric structure. Removal of the evolutionarily added Colby Sandate. N-terminal segment of cardiac TnT removes the repression and restores a The Scripps Research Institute, San Diego, CA, USA. TnI-like structure in TnT, which may confer a conditional TnI-like function. Many organisms have evolved circadian rhythms as a molecular adaptation to Using genetically engineered TnT and fragments, we performed enzyme- the Earth’s daily light and dark cycle. Cryptochrome (CRY) is well established linked immunosorbent assay (ELISA) to assess the binding affinity of mAb as the key member of the repressive arm of the mammalian molecular clock. TnI-1 for the restoration and localization of the TnI-like epitope. While Outside of the sleep/wake cycle, the biological clock regulates many core pro- mAb TnI-1 strongly recognizes TnI with no detectable binding to intact car- cesses, such as metabolism. However, many of the biochemical mechanisms diac TnT, N-terminal truncated cardiac TnT and its T2 fragment showed sig- governing these processes have yet to be elucidated - particularly in regards nificant cross reactions. ELISA titrations further showed that this repressed to CRY repressive activity. Here, we present evidence that CRY also functions TnI-like epitope structure is also present in the slow and fast skeletal muscle as a regulator of some nuclear receptors (NRs) - transcription factors that con- isoforms of TnT and restorable in their T2 fragments. Since the TnI-1 epitope trol the expression of myriad genes involved in metabolism, development and structure in TnI is related to a calcium-regulated tropomyosin-binding site inflammation, among others. Using an array of biochemical techniques and (Zhang et al., FEBS J. 278:3348-59, 2011), the TnI-like structure restored in electron microscopy, we aim to characterize the interaction between the NR the C-terminal region of TnT may represent a conditional tropomyosin- Hepatocyte Nuclear Factor 4a (HNF4a) and CRY2 in order to shed light on binding site. Further characterizations of the function of this TnI-like tropomy- CRY repressive activity in the clock and beyond. osin-binding activity will provide novel insights into the structure-function relationship of TnT and the physiological significance of N-terminal modifica- 1701-Pos Board B21 tions of TnT. Quo Vadis, Biomacromolecular Structure Quality Radka Svobodova Varekova1, Vladimir Horsky1,2, David Sehnal1, 1698-Pos Board B18 Veronika Bendova3, Lukas Pravda1, Jaroslav Koca1. NMR Studies of Conformational Selection of hnRNP H on RNA Splicing 1Central European Institute of Technology, Brno, Czech Republic, 2Faculty Liang-Yuan Chiu, Srinivas Penumutchu, Blanton S. Tolbert. of Informatics, Masaryk University, Brno, Czech Republic, 3Faculty of Chemistry, Case Western Reserve University, Cleveland, OH, USA. Science, Masaryk University, Brno, Czech Republic. Modularity provides proteins versatility in regulating many cellular processes. Biomacromolecular structural data is one of the most interesting and impor- The RNA Recognition Motif (RRM) is a common protein family that adopts tant results of modern life sciences. However, all that glitters is not gold, since modularity to facilitate its biological activity. hnRNP H contains three RRMs this treasure throve is inevitably plagued by errors and discrepancies. The that play important roles in RNA splicing by binding G-rich enhancer element2. issue of structure data reliability accompanies the whole process of structure However, the mechanism three RRMs use to recognize RNA is still unknown. discovery. It has stimulated the research community to concentrate more on Here, based on the analysis of paramagnetic relaxation enhancement (PRE), the data quality improvement - e.g., development of much more sophisticated data indicates that hnRNP H RRM12 adopts a closed conformation in the data deposition workflows, highly sensitive validation tools, etc. A major issue absence of RNA. Second, dynamics experiments, T1, T2 and NOE also reveals is, how these validation efforts influence the real quality of structural data? that RRM1 and RRM2 domain share similar motion, which suggests that And in general, how is structure quality changing over time and which factors compact (closed) form is major population in solution. Moreover, in closed affect it? form, one of two bindings site was buried in compact structure. Based on above Missing answers to these key questions motivated us to focus on an analysis of observation, we propose a hypothesis that there is chemical equilibrium be- the current state of data quality and validation trends. Among others, we paid a tween open form and close form. Once hnRNP H recognizes its G-rich binding special attention to ligand validation criteria as they are the most sensitive and sequencing, it will release the blocking RRM from closed form and reestablish interesting part of validation outputs. Our research has been based on valida- chemical equilibrium toward to open form through conformational selection in tion data from our validation database ValidatorDB (http://ncbr.muni.cz/ presence of RNA3. Integrated methods like NMR paramagnetic relaxation ValidatorDB) and PDB validation reports. Furthermore, we have compiled enhancement, dynamics experiments and SAXS would be utilized to address all these validation information into the ValTrendsDB (http://ncbr.muni.cz/ this hypothesis. ValTrendsDB). This rich database enables the whole scientific community

BPJ 7839_7842 Tuesday, February 14, 2017 347a to see a wide range of ongoing trends related to the structure quality. As the a GEF is poorly understood. In fact, rates of GDP dissociation, formation of the matter of fact, researchers can ask questions: Does the quality improve with Gai:Ric-8A complex and dissociation of Ric-8A resulting in the Gai:GTP active better resolution of the structure? Does the quality decrease with the size of state have not been well established. Previously, stopped-flow fluorescence ex- the structure? Which particular quality criteria are improving in newer struc- periments have been used effectively to measure GEF activity on small tures and which are stagnating? Careful analysis of the assembled dataset re- GTPases. This method provides the opportunity to study the Gai:Ric-8A system veals some surprising facts. While certain discovered trends are very positive under pre-steady state conditions, which to our knowledge has not been per- (e.g., clashscore markedly decreases with the year of structure publication), formed previously. By looking at the microscopic rate constants we perform others are alarming (e.g., ligand quality stagnates with the year of structure new measurements of GEF activity by monitoring GDP release rather than publication). GTP binding. Use of pre-steady state kinetics may also enable identification of previously uncharacterized intermediary complexes such as Gai:GDP:Ric- 1702-Pos Board B22 8A. Recent data suggest that Gai myristoylation leads to altered rates of GEF Acetyl-Group Sensing through Modulation of Conformational Dynamics activity. This work will compare the kinetics of Ric-8A mediated GEF activity in an Arylalkylamine N-Acetyltransferase on both myristoylated and non-myristoylated Gai. Adam A. Aboalroub, Ashleigh Bachman, Ioannis Gelis. Chemistry, University of South Florida, Tampa, FL, USA. 1705-Pos Board B25 Arylalkylamine acetyltransferases (AANAT) belong to the ubiquitous super- Using Single Molecule FRET to Examine the Impact of Enzymatic Degly- family of GNAT acetyl transferases that catalyze the transfer of acetyl group cosylation on the Structure of Immunoglobulin G Antibodies to a diverse set of acceptor substrates using primarily acetyl-CoA as donors. Alan J. Mlotkowski, Brandon Roman, Mark S. Piraino, Kinetic analyses on GNAT family members have revealed that catalysis oc- Cathrine A. Southern. curs through an ordered sequential mechanism where acetyl-CoA binds first, Chemistry, DePaul University, Chicago, IL, USA. followed by the substrate binding. After the chemical step, the acylated sub- Immunoglobulin G (IgG) antibodies have sugar molecules bound in the frag- strate is released first, followed by CoA. Despite of the wealth of available ment crystallizable (Fc) region. The removal of these sugar molecules dimin- structural data on GNATs, the molecular events that impose a sequential ishes the ability of IgG molecules to bind to receptor molecules, leading to a mechanism remain elusive. Using NMR spectroscopy, in combination with diminished immune response. Some studies explain the abrogated receptor ITC and kinetic experiments, we show that the position of the acetyl group binding interaction as a result of a collapse of the Fc region, which causes during catalysis regulates protein conformational dynamics. Part of the sub- the receptor binding site to be occluded. Other studies indicate that an increase strate binding site switches from a stable, ordered state in the presence of in the flexibility of the Fc region is a more plausible explanation. We have used acetyl-CoA to a conformational labile ensemble of structures in the presence single molecule Fo¨rster Resonance Energy Transfer (smFRET) to study glyco- of CoA which may allow for the ordered binding of substrates and release sylated and enzymatically deglycosylated IgG molecules to further examine of products. In addition, scanning through available GNAT structures, a this question. By examining freely diffusing IgG antibodies labeled with dye conserved salt-bridge connecting the cofactor and substrate binding sites molecules, we have obtained smFRET efficiency histograms of the Fc region was identified, that when broken disrupts the positive binding cooperativity conformations present for both glycosylated and enzymatically deglycosylated of the substrate. IgG. We have used accessible volume calculations to validate the histograms obtained for the glycosylated structures. We have also obtained mass spectrom- 1703-Pos Board B23 etry data confirming the complete enzymatic removal of the sugar molecules. Interdomain Conformational Changes in Visual Arrestin upon Binding to This allows differences in the FRET histograms to be attributed to the removal Different Forms of Rhodopsin of the sugar molecules. Our results support the notion that the flexibility of the Wei-Lin Ou, Ned Van Eps, Takefumi Morizumi, Aidin R. Balo, Fc region increases upon deglycosylation. Oliver P. Ernst. 1706-Pos Board B26 Biochemistry, University of Toronto, Toronto, ON, Canada. Monitoring Macromolecular Crowding via FRET using Wavelength- Visual arrestin (arrestin-1) and its C-terminally truncated splice variant p44 Dependent Fluorescence Lifetime Measurements reside in photoreceptor cells, regulating the desensitization of rhodopsin by Jacob Schwarz1, Megan Currie1, Hannah Leopold1, Arnold J. Boersma2, blocking G protein signalling. The binding of full-length arrestin-1 is highly Ahmed A. Heikal1, Erin D. Sheets1. specific to phosphorylated light-activated rhodopsin (P-Rh*); however, p44 1Chemistry and Biochemistry, University of Minnesota Duluth, Duluth, MN, is capable of binding to different forms of rhodopsin, including unphosphory- USA, 2Biochemistry, University of Groningen, Groningen, Netherlands. lated light-activated rhodopsin, dark phosphorylated rhodopsin, and phosphor- Macromolecular crowding influences many intracellular processes such as ylated opsin. Several crystal structures have shown that the two lobes of protein folding, biochemical reactions and diffusion. Recently, a rationally activated arrestin undergo a rotation of 20 as compared to arrestin in the basal designed FRET probe was developed to quantify macromolecular crowding state, while there are still questions remaining: whether this twist conformation in vitro (Boersma et al. Nat. Meth. 12:227, 2015). The probe consists of a of arrestin is induced by active rhodopsin and whether arrestin-1 exist other FRET pair (mCerulean and mCitrine) that is connected with a flexible linker. conformations when it binds to different functional forms of rhodopsin. To Here, we characterized the same FRET probe in crowded solutions using fluo- probe the conformational changes within arrestin-1 and its splice variant p44 rescence lifetime measurements as a function of the concentration of a crowd- upon complex formation, a pair of disulfide-linked imidazoline nitroxide spin ing agent (Ficoll-70) as well as the excitation/detection wavelengths towards label (V1) was introduced to residues 105 and 285 on monomeric arrestin-1 selective monitoring of the donor and acceptor. We also carried out similar mutant and the interspin distances between spin labels were measured by dou- measurements on the intact probe in a homogenous environment (glycerol- ble electron-electron resonance (DEER) spectroscopy. The distance measure- enriched buffer) as a means to differentiate between viscosity and crowding ef- ments indicate that the two lobes of p44 have more flexibility than arrestin-1 fects on energy transfer. Importantly, we used the cleaved version of the probe, and p44 is likely to have at least two different receptor-binding conformations, where the donor and acceptor are no longer linked, to distinguish between of which only one is known from X-ray crystallography. Moreover, our data changes in the fluorescence lifetime due to FRET and refractive index of the also suggest that the rotated conformation of arrestin-1 can only be stabilized crowded solutions. These studies complement the previous steady-state spec- by the active state of rhodopsin. troscopy measurement on this novel probe. Importantly, our results provide a reference point for future studies using the quantitative, non-invasive fluores- 1704-Pos Board B24 cence lifetime imaging microscopy (FLIM) of this genetically encoded Effects of Gai Myristoylation on RIC-8A GEF Activity FRET probe in living cells for the ultimate site-specific crowding studies in Levi J. McClelland, Stephen R. Sprang. living cells. Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT, USA. 1707-Pos Board B27 Resistance to inhibitors of cholinesterase (Ric-8) proteins have been shown to The Thermodynamics of Divalent Metals Binding to Taurine/a-Ketogluta- play a role in neurotransmitter release and embryogenesis. Further, Ric-8A acts rate-Dependent Oxygenase (TAUD) as a chaperone for Gai in vivo and demonstrates Guanine nucleotide Exchange Mingjie Li. Factor (GEF) activity for Gai in vitro by releasing GDP from Gai:GDP to form Chemistry, Mississippi State University, Starkville, MS, USA. Gai:Ric-8A which then dissociates to Gai:GTP and free Ric-8A in the presence Taurine/a-ketoglutarate (aKG) dioxygenase (TauD) is a nonheme iron(II) and of GTP. To date, the specific reaction mechanism by which Ric-8A functions as aKG dependent metalloenzyme, which catalyzes the hydroxylation of taurine

BPJ 7839_7842 348a Tuesday, February 14, 2017 leading to its decomposition into aminoacetaldehyde and sulfite, where sulfite in-house programs often in combination with dedicated software tools. Devel- is a key sulfur containing metabolite in E. coli. The nonheme iron(II) center in opment of such solutions is generally error-prone and time-consuming. Hence TauD is formed from two histidine side chain residues and a glutamic acid the question is, can we do any better? Do we really need all these single purpose coordinating to one face of the octahedral coordination geometry. This com- programs? Or can we extract biologically important sites in an easy user- mon metal binding motif has been termed the 2-His-1-carboxylate facial triad defined and customizable way? and is found in a number of nonheme manganese, iron, and cobalt containing We have developed PatternQuery (PQ - http://ncbr.muni.cz/PatternQuery)-an proteins. Here we have focused our efforts to measure the thermodynamic online service for mining structural databases such as Protein Data Bank. It en- driving forces that lead to formation of these bioinorganic centers in biology, ables description of a relationship between atoms, residues, and other structural by studying divalent metal ion coordination to TauD using isothermal titration elements using a simple, yet robust, query language. Each query specifies the calorimetry. Titrations of metal complexes into the metal-free (apo) TauD and composition, topology, connectivity, and 3D structure of a pattern. This allows the corresponding chelation experiments were performed under anaerobic to relate the primary, secondary, and tertiary structure information simulta- environment. neously. Smaller datasets of hundreds of structures can be processed within sec- The thermodynamic terms associated with cobalt(II), iron(II), and mangane- onds in interactively. The whole Protein Data Bank or its subset can be se(II) binding to apoTauD were deconvoluted from complex experiments, processed under an hour. All the results are made available for download where the pH and buffer independent binding constant (K) were measured and presented in a clear graphical form for online inspection. to be 2.9 109, 2.4 107, and 9.8 105, respectively. (The corresponding DG values were calculated to be 12.8 kcal/mol, 10.1 kcal/mol, and 1710-Pos Board B30 8.2 kcal/mol, respectively.) Interestingly the measured enthalpy changes Interconnection between Parallel Assembly Pathways in Large Ribosome for these binding events (DH) are 17.8 kcal/mol, 12.8 kcal/mol, and Subunit Biogenesis 12.2 kcal/mol, respectively. These data are fully consistent with the Riley C. Gentry, Eda Koculi. Irving-Williams series, which suggest there is increasing affinity for transition University of Central Florida, Orlando, FL, USA. metal ions from left to right across the periodic table. However, it seems this The ribosome is the macromolecular machine responsible for protein produc- the increasing affinity is derived from increasing favorability of both the tion in every living cell. While structural studies have provided information related DH and DS terms. on the fully assembled bacterial ribosome, knowledge of bacterial ribosome as- 1708-Pos Board B28 sembly remains limited. This knowledge is invaluable to facilitate the design of novel antibiotics that target the ribosome assembly process. Ribosome assem- Discriminating Residue Substitutions between Two Single Proteins with a Sub-Nanopore bly involves rRNA folding, rRNA processing and post-transcriptional modifi- Zhuxin Dong. cation, r-protein associations, and both association and release of ribosomal Electrical Engineering, University of Notre Dame, Notre Dame, IN, USA. maturation factors. In vivo, all of these processes are highly coordinated, but The two variants of human histone, H3.2 and H3.3, are essentially the same the nature and extent of their coordination is not well understood. In order to chains of 136 amino acids (AAs), differing by only four residue substitu- study this coordination in vivo, we will exploit the phenotype created by ex- pressing the helicase inactive DbpA protein construct, R331A. DbpA is an Es- tions, yet the replacement of H3.2 with H3.3 in the chromatin structure is 1 cherichia coli (E. coli) DEAD-box RNA helicase believed to perform RNA supposed to promote organismal aging through aberrant gene regulation. To discriminate between them, measurements of force and concomitant cur- structural rearrangements near the peptidyl transferase center. E. coli cells ex- rent blockade were performed as these histones, denatured in sodium do- pressing R331A DbpA accumulate three large subunit intermediates. More decyl sulfate (SDS) and tethered to the tip of an AFM, were impelled importantly, previous investigation by our lab of the large ribosomal subunit systematically (one-at-a-time) through a sub-nanometer diameter pore, i.e. intermediates’ kinetics of conversion and the 5’ processing of the 23S rRNA a sub-nanopore. The force measurements revealed that, once the denatured demonstrated that these intermediates convert to the large subunit through par- allel assembly pathways. Currently, we are probing the RNA processing and protein translocated through the sub-nanopore, a disproportionately large force was required to pull it back through, indicating that the SDS was structural characteristics of the intermediates using chemical modification fol- cleaved from the protein allowing it to refold during the translocation. The lowed by next generation sequencing. Additionally, we are in the process of force measurements also exposed a dichotomy in the translocation kinetics: determining the protein composition of the intermediates with mass spectrom- either the molecule slid nearly frictionlessly through the pore or it slipped- etry. Combined, the RNA structure, processing, and protein composition of the and-stuck. When it slid frictionlessly, regular patterns were observed inter- particles from these three parallel assembly pathways will reveal how the matu- ration processes are interconnected during large subunit ribosome assembly mittently in the force and blockade current fluctuations that corresponded in vivo. to the distance between stretched residues. Furthermore, the amplitudes of the fluctuations in the current blockades were correlated with the occluded volume associated with the AAs in the pore waist.2 Finally, the difference Protein-Small Molecule Interactions I in the patterns in the blockade current fluctuations associated with the two histones consistently peaked near position 91 in the sequence, where AA 1711-Pos Board B31 substitution (M->G) occurs, corresponding to volume difference of only Kinetics and Pathways of Extremely Long Ligand Release Events 0.085 nm3 in a read. Revealed by Wexplore and Conformation Space Networks 1Saade, E.; Pirozhkova, I.; Aimbetov, R.; Lipinski, M.; Ogryzko, V. Molecular Samuel D. Lotz, Alex Dickson. turnover, the H3.3 dilemma and organismal aging. Aging Cell 2015,14, Biochemistry and Molecular Biology, Michigan State University, East 322-333. Lansing, MI, USA. 2Kennedy, E.; Dong, Z.; Tennant, C.; Timp, G. Reading the primary structure It has been reported on a set of applications that the binding kinetics is of a protein with 0.07 nm3 resolution using a sub-nanometre-diameter pore. more predictive than the binding affinity of the efficacy of a drug molecule Nat. Nanotech. 2016, DOI: 10.1038/NNANO.2016.120 in vivo. Although the thermodynamics depends only on the endpoints of the binding pathway, the kinetics depends on details of the binding process, partic- 1709-Pos Board B29 ularly the ligand binding transition state. Unfortunately, the ligand release Effective on-Demand Mining of Structural Databases timescales of pharmaceutically relevant drug molecules can extend up to Lukas Pravda, David Sehnal, Radka Svobodova Varekova, Jaroslav Koca. thousands of seconds: far beyond the reach of conventional molecular sampling Central European Institute of Technology, Brno, Czech Republic. approaches. Using WExplore, an algorithm based on concurrent loosely- The majority of in silico experiments often heavily relies on a data collection. coupled trajectories without biasing forces, we have characterized the ligand Indeed, identification of biomolecular substructures (patterns) within biomole- release pathways of a number of systems, with residence times extending up cular databases, such as Protein Data Bank is a common procedure in structural to thousands of seconds. By measuring the trajectory flux into the unbound bioinformatics and related fields. We are seeking for well-defined molecular state, we directly compute ligand residence times without using a Markovian patterns such as binding or catalytic sites, transcription factors, protein struc- assumption that show excellent agreement with those determined experimen- tural, or sequence motifs, etc. These are in turn used to aid structural and func- tally. Throughout we obtain broad sampling of ligand exit pathways, involving tional characterization and comparison of proteins, analysis of newly distinct exit channels and significant coupled motions between the ligand and determined protein structures, identification of similar binding sites in off- the bound receptor. The broad set of ligand bound poses and exit pathways al- target proteins, discovery of new inhibitors, facilitation of protein-protein inter- lows us to determine general physical principles of ligand binding, including action and more. This is usually done using a plethora of one-time-only use the specific molecular interactions that govern binding kinetics.

BPJ 7839_7842 Tuesday, February 14, 2017 349a

1712-Pos Board B32 Gefitinib, Dasatinib, Sunitinib, Pazopanib) with its known targets with a sensi- Machine Learning Guided Ligand-Protein Simulation Approach Eluci- tivity of 50% and specificity of 56%. Four out of the five top predicted targets dates the Binding Mechanism of Abscisic Acid for Sorafenib were shown to interact with it using in vitro experiments. Our Saurabh Shukla1, Moeen Meigooni1, Chuankai Zhao1, Diwakar Shukla1,2. method is broadly applicable for the prediction of protein-small molecule inter- 1Department of Chemical and Biomolecular Engineering, University of actions with several novel applications to biological research and drug Illinois at Urbana-Champaign, Urbana, IL, USA, 2Department of Plant development. Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA. Rising temperatures and climate change are threatening agricultural productiv- 1715-Pos Board B35 ity and have motivated efforts to elucidate the molecular mechanisms of A Synthetic Peptide from the N-Terminal of Hexokinase I Prevents the drought resistance in plants. Abscisic acid (ABA) is a key plant hormone Interaction Between VDAC1 and SOD1 G93A Mutant Recovering the that imparts drought resistance in plants. Crystal structures of receptors Viability of an ALS Cell Model (PYLs) involved in ABA perception have shed light on the interactions ABA Andrea Magri1,2, Ramona Belfiore3, Loredana Leggio1, Francesca Guarino3, makes in the binding pocket of the receptor but these structures provide limited Angela Messina1. knowledge about the dynamic mechanism of ABA binding. Mechanistic under- 1Biological G. & E. Sciences, University of Catania, Catania, Italy, standing of ABA recognition by plants could provide new avenues for develop- 2Biometec, University of Catania, Catania, Italy, 3BIOMETEC, University of ment of drought resistant agrochemicals. In this work, we have performed Catania, Catania, Italy. extensive atomistic molecular dynamics (MD) simulations using Markov state Superoxide Dismutase 1 mutants associate with 20-25% of familial Amyotro- models (MSMs) based adaptive sampling protocols to characterize ABA bind- phic Lateral Sclerosis (ALS) cases, producing toxic aggregates on mitochon- ing pathway for two ABA receptors in A. Thaliana, AtPYL5 and AtPYL10 re- dria, notably in spinal cord [1,2]. The Voltage Dependent Anion Channel ceptors belonging to different sub-classes of ABA receptors to identify the isoform 1 (VDAC1), the main pore in the outer mitochondrial membrane major bottlenecks in their binding pathways. Our results not only explain the [3] is a docking site for SOD1 G93A mutant in ALS mice and the physiolog- binding mechanism of ABA molecule but also provide atomistic information ical receptor of Hexokinase I (HK1) [4], which is poorly expressed in mouse about the intermediate states along the binding pathways. We have also iden- spinal cord [5, www.brain-map.org]. Our results demonstrate that HK1 com- tified a new non-productive pose, which we call the inverted state, where petes with SOD1 G93A for binding VDAC1, suggesting that in ALS spinal ABA gets trapped in the binding pocket while forming an unproductive com- cord available HK1-binding sites could be used by SOD1 mutants for docking plex. We validate our results with previously published NMR and Hydrogen/ mitochondria, producing thus organelle dysfunction [6]. We tested this Deuterium exchange experiments to show that receptors retain their flexibility model by studying the action of a HK1-N-end based peptide (NHK1). This even after ABA binding. Our findings shed light on the role of specific receptor NHK1 peptide specifically interacts with VDAC1, inhibits the SOD1 G93A residues in ABA binding, explore role of water in binding and characterize ma- binding to mitochondria and restores the viability of ALS model NSC34 cells jor barriers to ABA binding. Our results demonstrate the efficacy of MD sim- [6]. Altogether, our results suggest that NHK1 peptide could be developed ulations and MSM framework in elucidating the mechanism of ABA signaling. as a therapeutic tool in ALS, predicting an effective role also in other Knowledge of ABA binding mechanism will help in improving drought resis- proteinopathies. tance in plants by informing genetic manipulations and agrochemical discovery 1. Magrı` A et al, Biochim Biophys Acta (2016) 1857:789-98 efforts. 2. Vande Velde C et al, PNAS (2008) 105:4022-7 3. De Pinto V et al, Biochim Biophys Acta (1989) 987:1-7 1713-Pos Board B33 4. Tomasello MF et al (2013) PlosOne 8, e81522 Hydration Effects on Binding Equilibria: Role of Desolvation Energy 5. Pastorino JG & Hoek JB J Bioenerg Biomembr (2008) 40:171-82 Daryl K. Eggers. 6. Magrı` A et al, SciRep. 2016, in press. Chemistry, San Jose State University, San Jose, CA, USA. This study tests a new thermodynamic framework for aqueous binding equi- libria that features an explicit consideration for the change in hydration that oc- 1716-Pos Board B36 curs when two solvated surfaces come into contact. As an outcome of this Multi-Conformer Hierarchical Virtual Screening Workflow to Identify approach, the standard state free energy of binding is defined by the summation Potential K-Ras Inhibitors of two terms, the traditional term (-RTlnK) plus a desolvation free energy term Amit K. Gupta1, Priyanka Prakash1, John A. Putkey2, Alemayehu A. Gorfe1. 1Integrative Biology and Pharmacology, UT Health houston, Houston, TX, that is weighted by the number of complexes formed at equilibrium. The gov- 2 erning equation suggests that the equilibrium ratio (K) is not a constant; this USA, Biochemistry and Molecular Biology, UT Health Houston, Houston, equation is supported firmly by results from isothermal titration calorimetry us- TX, USA. ing the chelation of calcium(II) by EDTA as a model binding reaction. In addi- K-Ras is a small GTPase that plays a critical role in the regulation of a variety tion, we demonstrate that secondary solutes can shift the equilibrium by of signaling pathways involved in cell growth and proliferation. Somatic muta- altering the average free energy of bulk water; molar solutions of urea, sucrose, tions on K-Ras are associated with many different cancers, accounting for and trehalose result in significant changes in the equilibrium ratio without about 85% of all Ras-associated cancers or 15-20% of all human cancers. K- altering the standard state free energy, as defined by our working equation. Ras is a very dynamic allosteric enzyme and our previous studies revealed This investigation provides a fresh approach for characterizing concentrated, that K-Ras harbors four allosteric ligand-binding pockets. This suggested that nonideal solutions, as relevant for understanding the driving forces behind targeting K-Ras directly is a viable strategy to abrogate its abnormal functions. molecular interactions in a cell or tissue. Future investigations will target other In the current study, we conducted extensive multi conformer hierarchical vir- examples of concentration-dependent equilibria that appear to follow the pro- tual screening to identify potential hits targeting each of these four pockets. In posed equations. the first step we used clustering, structure-based pocket analysis and knowledge-based filters on conformers obtained from all-atom MD simulations 1714-Pos Board B34 of oncogenic mutant K-Ras to select representative structures for docking. The A Structure Based Framework to Identify Novel Targets of FDA Approved representative structures were further analyzed in terms of their physico- Kinase Inhibitors chemical properties to gain an insight into the unique features of each pocket. Hammad Naveed. Based on this the large purchasable chemical space of the ZINC database was Computer Science, Toyota Technological Institute at Chicago, Chicago, IL, tailored to generate complementary pocket-specific chemical libraries. We per- USA. formed ensemble docking of these tailored ligand libraries against each pocket The promiscuity of drugs towards protein binding sites and vice versa hinders with the standard precision module of the Glide docking software, and used hi- our understanding of the metabolism of healthy and diseased states. The iden- erarchical post-docking analysis (PDA) to identify plausible K-Ras binders. In tification of all protein targets is therefore important to understand a drug’s PDA the docking outputs of ligands were grouped based on their common res- (side) effects, and to reuse existing drugs. Current FDA approved kinase inhib- idues interaction pattern (A) and chemical scaffold diversity (B) at the each itors have significant side-effects, many of which might be due to the off-targets binding site. A primary list of highest scoring hits per pocket was made from of these drugs. We have developed a novel computational approach for drug the ligands in the B subgroup that belonged to subgroup A, followed by visual target prediction for large-scale discovery of new targets for existing drugs. inspection of the binding pose. This yielded a list of 761 potential hits. 217 of For a given drug, we construct a probabilistic pocket ensemble that captures these were procured for experimental testing and about 100 were tested for their the promiscuous structural features of drug binding sites. We were able to pre- ability to bind to GDP-bound K-Ras using N15-labeled heteronuclear single dict the interaction of six FDA approved kinase inhibitors (Sorafenib, Imatinib, quantum coherence (HSQC) NMR. Of the 100 tested, 11 showed significant

BPJ 7839_7842 350a Tuesday, February 14, 2017 chemical shift perturbations at several residues, suggesting potential binding. ous observations that the affinity of RGD for aIIbb3 is greater than AGDV and We will discuss these results and their implications for future efforts in K- support our hypothesis that the RGD motifs promote the interaction of aIIbb3 Ras drug discovery. with immobilized fibrinogen and fibrin. 1719-Pos Board B39 1717-Pos Board B37 Brushed Polyethylene Glycol and Phosphorylcholine as Promising Database of Ca Protein-Ligand Binding Gibbs Energies, Enthalpies, Entropies, Volumes, and Crystal Structures Grafting Agents against Protein Binding _ _ _ Bo Wang1, Thomas Blin2, Aleksandr K€akinen2, Xinwei Ge1, Vaida Linkuviene, Asta Zubriene, Vaida Paketuryte, Alexey Smirnov, 2 2 2 Vytautas Petrauskas, Daumantas Matulis. Emily H. Pilkington , John F. Quinn , Michael R. Whittaker , Thomas P. Davis2, Pu Chun Ke2, Feng Ding1. Institute of Biotechnology, Vilnius University, Vilnius, Lithuania. 1 2 We have designed, synthesized and determined the binding thermodynamics Dept. of Phys. & Astro., Clemson University, Clemson, SC, USA, ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, of over 700 aromatic sulfonamides to the family of 12 human carbonic anhy- Monash Institute of Pharmace, Monash University, Parkville, Australia. drase (CA) isoforms. The proteins were cloned and expressed in bacterial and human cell cultures and affinity-purified in large quantity sufficient for ITC The grafting of linear polyethylene glycol (PEG) is a common strategy in and crystallography. The binding affinities were determined by the thermal ascribing the stealth effect to nanostucture against protein adsorption for shift assay (FTSA, also termed ThermoFluor or differential scanning fluorim- biomedical applications. However, PEG may still evoke side effects in vivo etry, DSF), a high-throughput method. The enthalpies and entropies of bind- that compromises the circulation and efficacy of PEGylated drugs or nanocar- rier. Recently, we demonstrated the synthesis of brushed phosphorylcholine ing were determined by ITC, a medium throughput method, for a selection of (PC) as biomimetic alternative to linear PEG for the grafting of superparamag- compounds and CA isoforms. A correlation map between the compound chemical structure and the binding DG and DH was drawn. The map showed netic iron oxide nanoparticles (IONP). Compared to brushed PEG (bPEG), which structural features of the compounds generated the highest increments brushed PC (bPC) rendered even better suspendability, stability, biocompati- in exergonicity and exothermicity of compound binding. Furthermore, only bility and cellular distribution. To further examine the structures of bPEG some structural features were most useful in generating compounds that and bPC and directly test their antifouling properties against proteins, discrete would selectively bind to cancer-expressing CA isoforms, but would not molecular dynamics (DMD) simulations were performed. We found that brushed polymers were more rigid than the linear PEG while bPEG and bind to essential for life human CA isoforms. Volumes and compressibilities bPC ligands displayed distinct globular and cylindrical morphologies. Grafting of binding were determined by PressureFluor and densitometry for several compounds. Over 60 X-ray crystal structures showed the position of com- either bPEG or bPC onto IONPs led to different characteristics of the grafting pounds bound in the enzyme active center. ITC was essential technique layer, namely ligand coverage, height, and conformational strains both later- that enabled the dissection of unknown contributions from linked reactions ally and vertically. Upon mixing both bPEG and bPC onto the same IONP sur- such as buffer protonation to the binding reaction. Only after the subtraction face while maintaining the same grafting density, a conformational relaxation of the bPEG was observed in DMD simulations, as corroborated by fluores- of pH-dependent buffer contribution to the enthalpy of binding, the intrinsic cence quenching of Cy5 attached to bPEGs in the experiment. Both bPEG- Gibbs energies and enthalpies of binding were obtained. All methods that determine the binding reaction, such as FTSA, ITC, SPR, thermophoresis, and bPC-grafted NPs displayed antifouling against human serum albumin and enzymatic inhibition methods would provide only the observed thermo- (HSA), with an increased grafting density giving rise to enhanced protein dynamics of binding that is pH and buffer-dependent. It was important to avoidance. Our results suggest that, with a stronger repulsion to HSA and calculate the true (intrinsic) parameters and use them in the structure- the capability to a higher grafting density due to its cylindrical shape, bPC thermodynamics correlation maps. is more advantage than both linear and brushed PEG for grafting NPs with minimal protein binding. These new structural and energetic insights offer a general guidance for NP synthesis and anti-fouling applications employing 1718-Pos Board B38 branched polymers. Comparison of the RGD- and AGDV-Containing Peptide Interactions with the Platelet Integrin Alphaiibbeta3 Rustem I. Litvinov1,2, Olga Kononova3,4, Dmitry S. Blokhin2, 1720-Pos Board B40 Vladimir V. Klochkov2, Valeri Barsegov3,4, Joel S. Bennett1, Insights into a Low Promiscuous Aminoglycoside Modifying Enzyme, John W. Weisel1. Aminoglycoside N3 Acetyltransferase-Via 1University of Pennsylvania, Philadelphia, PA, USA, 2Kazan Federal Fnu Prashasti. University, Kazan, Russian Federation, 3University of Massachusetts, Genome Science and Technology, University of Tennessee, Knoxville, TN, Lowell, MA, USA, 4Moscow Institute of Physics and Technology, Moscow USA. Region, Russian Federation. Aminoglycoside modifying enzymes (AGMEs) are plasmid-encoded enzymes The C-terminal AGDV-containing dodecapeptide (gC-12) sequence of the found in resistant bacteria. AGMEs covalently modify their substrates and thus fibrinogen g-chain mediates binding of soluble fibrinogen to the activated in- render the drug ineffective for the bacteria. Aminoglycoside (AG) antibiotics tegrin aIIbb3, leading to platelet aggregation. However, the RGD-containing are bactericidal agents used to treat various bacterial diseases like tuberculosis peptide sequences located in two places in the fibrinogen Aa-chain inhibit and meningitis. However, the clinical effectiveness of these drugs has been this process and contribute to aIIbb3 binding when fibrinogen is immobilized drastically affected by the emergence of AGMEs. More than 50 different and when it is converted to fibrin. Although the interaction of aIIbb3 with AGMEs are known, having variable levels of substrate promiscuity. However, various RGD-containing and gC-12 peptides has been studied extensively, no correlation has been confirmatively observed between the sequence or struc- there is still not a comprehensive characterization of their binding to the active ture of an AGME and its substrate profile. We aim to understand the molecular open and inactive closed conformations of aIIbb3. Here, we combined exper- principles underlying this ligand selectivity by deciphering the thermodynamic, imental and computational approaches to compare kinetics, thermodynamics structural and dynamic properties of enzyme-ligand complexes. Kinetic, ther- and structural details of cycloRGDFK (cRGDFK) and gC-12 binding to modynamic and structural properties of the aminoglycoside N3 acetyltransfer- aIIbb3. Using an optical trap-based single-molecule technique, we measured ase VIa (AAC-VIa) are described. Despite having significant sequence the probability of peptide binding to aIIbb3 as a function of aIIbb3-peptide similarity to highly promiscuous acetyltransferase, AAC-VIa can modify interaction time and extracted first-order binding/unbinding rates and binding only 5 aminoglycosides, with a ~4-fold difference in the kcat values. Thermo- affinity constants (Kb), which indicate that cRGDFK binds to aIIbb3 tighter dynamic studies determined the binding of ligands to be enthalpically driven 14 2 14 2 than gC-12 (Kb=0.4610 cm and Kb=0.310 cm , respectively). Next, and entropically unfavorable. Unlike other AGMEs, the formation of binary we performed docking modeling and implicit solvent MD simulations on and ternary complexes was accompanied by a net deprotonation of the enzyme, GPUs using the NMR-determined solution structures of the peptides, and crys- ligand or both. Also, the values for heat capacity change (DCp) were within the tallographically resolved structures of the aIIbb3 conformers. The results range of protein-carbohydrate interactions. Another significant difference was showed that cRGDFK and gC-12 bound to the aIIbb3 at the physiological observed in the structure of AAC-VIa and other AGMEs in solution. Analytical binding site with similar strength for the open and closed aIIbb3 conforma- ultracentrifugation (AUC) studies showed that AAC-VIa exists in a monomer- tions. The Gibbs free energy (DG) of the aIIbb3-peptide complexes, calculated dimer equilibrium, with more dimeric form appearing with increasing concen- using Umbrella Sampling technique, indicated that the overall thermodynamic trations of the enzyme. Binding of ligands drive the enzyme to a more stability of the complex is higher for cRGDFK than for gC-12 (DG=16.1 kcal/ monomeric form. Also, dimer formation is achieved mainly through polar in- mol and DG=13.5 kcal/mol, respectively). These results account for our previ- teractions. Crystal structures of different complexes of the enzyme showed

BPJ 7839_7842 Tuesday, February 14, 2017 351a that structures of apo-and ligand-bound forms were identical which suggests (HDX) will be used to map the drug-protein interface. We will present the that, unlike other AGMEs, more rigid structure of AAC-VIa may limit the mass spectrometry data for the solvent exposed peptides after digestion with active site to accommodate only few selected aminoglycosides, hence low sub- pepsin when PfGR is complexed with methylene blue, which will allow us strate promiscuity. to narrow down the active site. 1721-Pos Board B41 1724-Pos Board B44 The Role of Solvent Effect on the Ligand Binding Properties of the Ther- Ligand-Adopting Mechanism of the P53-Binding Pocket of the Oncopro- mostable Variants of Aminoglycoside Nucleotidyltransferase 4(ANT4) tein MdmX Seda Kocaman. Zheng Su1, Lingyun Qin1, Rong Chen1, Fei Yang1, Jingjing Zhou1, Biochemistry, Cellular and Molecular Biology, University of Tennessee at Huili Liu2. Knoxville, Knoxville, TN, USA. 1Biotechnology, Hubei University of Technology, Wuhan, China, 2State Key The aminoglycoside nucleotidyltransferase 4’ (ANT) is a homodimeric Laboratory of Magnetic Resonance and Atomic and Molecular Physics, enzyme that detoxifies a large number of aminoglycoside antibiotics by nucle- Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, otidylating at the C4’-OH site. Two thermostable variants for this enzyme Wuhan, China. show only a single amino acid changes in their primary amino acid sequences The intrinsic conformational dynamics of proteins play pivotal roles in deter- (T130K, D80Y). It is not known how single residue replacements, which are mining the ligand-binding properties. Although the molecular dynamics simu- distant from active site and monomer-monomer interface, result in various de- lation enables the understanding of protein-ligand interactions, the mechanism grees of changes on thermostability of the enzyme. Thermodynamic parame- underlying how a dynamic protein binding pocket adopts a ligand remains ters of the binary enzyme-aminoglycoside complexes, however, show highly elusive. The cancer-inducing interaction of Mdm2 (mouse double minus 2) significant differences. The data, acquired in H2O and D2O in this work by and its homologue MdmX with p53 is an attractive system not only for anti- isothermal titration calorimetry also demonstrate that solvent reorganization cancer drug discovery, but also for exploring the molecular mechanisms of upon ligand binding show large differences between the two variants. The protein-ligand interactions, as Mdm2 inhibitors exhibit weak affinities for heat capacity change (DCp) also show antibiotic-dependent differences be- MdmX. Here, we report the effects of the conformational dynamics of the tween the two variants. Thus, data shown in this work suggest that thermody- p53-binding domain of MdmX (N-MdmX) on the recognition of the Mdm2 in- namics of ligand-protein interactions and solvent effects may be among hibitor nutlin-3a by shaping the dynamic surface of N-MdmX through the molecular parameters that separate thermophilic proteins from simply mimicking the pre-defined nutlin-3a binding pocket of Mdm2. The interaction those that are thermostable but otherwise identical to the mesophilic of the ligand with N-MdmX via an induced-fitting mechanism traps the dy- counterparts. namic conformation of MdmX into an unfavorable state, resulting in a low ligand-binding affinity. Using a protein engineering strategy, we demonstrated 1722-Pos Board B42 that the global flexibility of the binding pocket is a pre-request for improving an In Vitro Development of an FRET Based Inhibitory Assay for the Coiled- unoptimized ligand. Our results provide a reverse-engineering rationale for Coiled Interaction between MBD2CC and P66ACR1 lead optimization and an understanding of protein recognitions in native bio- Victor C. Ekehchiadi. logical systems. Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK, USA. 1725-Pos Board B45 The Nucleosome Remodeling and Deacetylase (NuRD) complex plays a crit- Biochemical and Biophysical Characterization of a Small Molecule Inter- ical role in the methylation dependent gene silencing, and a small coiled-coil action with the Ras-Related Protein Cdc42 interaction is critical for it’s function. The coiled-coil interaction between the Djamali Muhoza, Alix Montoya-Beltrand, Paul D. Adams. p66a and MBD2 proteins is responsible for the recruitment of a chromatin Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. modeling protein (CHD4), which is responsible for the formation and func- Deleterious Protein-Protein Interactions (PPIs) involving Ras-related proteins tion of the NuRD complex. Developing selective inhibitors would help us un- have been known to play significant roles in events leading to cellular prolif- derstand this role and possibly lead to a novel therapeutic for sickle cell. We eration, inhibition of cell death, and cell transformation. Therefore, it is impor- have shown that a small peptide inhibitor can block the recruitment of CHD4, tant to characterize molecular features of Ras protein interactions that dictate a by inhibiting the p66 -MBD2 interaction. This inhibition prevents the their function as ‘timing switches’ in signal transduction pathways. This is a silencing of Y-globin and activation of b-globin, there by preventing critical step towards targeting the inhibition of cell transformation as well b-type globin disorder such as sickle cell anemia. Hence we would like to as controlling abnormal signaling activity of tumor-causing Ras variants. establish a FRET based assay for in vitro screening and characterization of Cell division cycle 42 (Cdc42) is a member of the Ras family of proteins. Pre- potential inhibitors. This assay is based on appropriately pairing of the fluo- vious structural studies from this laboratory have outlined differences in rophores to optimize the energy transfer, for whom we used eGFP and conformational dynamics in an important region of Cdc42 that interacts TAMRA. with various effectors, known as the Switch 1 region, using a single-point 1723-Pos Board B43 mutant variant of the wild type protein. These differences resulted in an in- The Binding of Methylene Blue to Plasmodium Falciparum Glutathione crease in GTP hydrolysis in the presence of an effector protein that inhibits Reductase GTP hydrolysis when bound to wild type Cdc42. These findings suggest Judith H. Prieto. that induced conformational changes in key effector-binding regions of Ras- Chemistry Department, Western Connecticut State University, Danbury, CT, related proteins may serve to alter effector protein interactions. As such, the USA. targeting of a small molecule towards an important effector-binding region Plasmodium falciparum is the cause of human malaria and is one of two ma- of Cdc42 could facilitate changes in local conformation as well as dynamics laria parasites known to have drug resistance. Since there are no preventative without affecting the overall stability of the protein. We are currently using vaccinations against malaria, the control of this disease is heavily dependent biochemical and biophysical approaches to characterize the influence of a upon the use of antimalarial drugs. Antimalarial drugs, such as methylene small molecule (ZCL278) on the interaction between Cdc42 and an effector blue, are effective therapies against human malaria. At a specific concentration, protein that inhibits Cdc42-stimulated GTP hydrolysis. Molecular modeling methylene blue has been shown to be a selective inhibitor of the parasite’s studies suggest that this small molecule binds to the Switch 1 region of glutathione reductase (PfGR). Glutathione reductase is an important target Cdc42 and ‘‘Mimics’’ an interaction with an effector protein. We present a when studying malaria drug resistance because it is a flavoenzyme that regen- preliminary characterization of the Cdc42-ZCL278 interaction. We also erates glutathione, which is an essential protein for antioxidant defense against discuss the influence this small molecule has on Cdc42’s interaction with an cell damage. Methylene blue is also a substrate that is reduced by gluthathione effector protein that inhibits Cdc42-stimulated GTPase activity in the presence reductase to produce leucoMB. This is then spontaneously oxidized by molec- of ZCL278. ular oxygen to form methylene blue again. During this process, reactive oxygen species, such as hydrogen peroxide and superoxide form. These act as recycling 1726-Pos Board B46 catalysts against infectious organisms. Due to PfGR’s central position in redox Shark-Derived Single Domain Antibodies Targeting Apolipoprotein E control, it is ranked number one as an antimalarial drug target. The goal of this Shane E. Gordon1, Michael Foley1,2, Matthew A. Perugini1. research is to study the interface between methylene blue and the putative pro- 1Department of Biochemistry and Genetics, La Trobe Institute for Molecular tein target, glutathione reductase, in order to understand the drug action mech- Science, Melbourne, Australia, 2AdAlta Pty. Ltd., 15/2 Park Drive, anism. PfGR was expressed and purified and hydrogen-deuterium exchange Bundoora, VIC, Australia.

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Human apolipoprotein E (ApoE) exists as three common isoforms that differ 1729-Pos Board B49 by single amino acid substitutions. ApoE4 (Arg112/Arg158) is the strongest Defining and Exploring Allosteric Mutations genetic risk factor for late-onset Alzheimer’s disease (LOAD)—a significant Igor N. Berezovsky. cause of mortality and disability worldwide. At the protein level, previous Bioinformatics Institute, Singapore, Singapore. studies suggest that the conformational landscape of ApoE4 is different to Published in 1963, the ‘‘Allosteric proteins and cellular control system’’ that of the wild-type ApoE3 (Cys112/Arg158). To date, however, effective marked a birth of the new area of research, which is nowadays called allosteric ligands capable of selectively targeting the ‘‘ApoE4-conformation’’ remain regulation. Recently established omnipresence of the allosteric control in all elusive. In the present study we sought to use phage display technology to types of proteins motivated numerous efforts on detecting regulatory exosites isolate several single domain antibody fragments (i.e. VNAR), initially and design of effector molecules [1]. By the analogy with allosteric ligands, derived from the wobbegong shark, that are reactive against human ApoE protein mutations that modulate protein activity by affecting its dynamics isoforms. To this end we have expressed, purified, and characterized recom- rather than directly changing catalytic and binding sites can be considered as binant ApoE3 and ApoE4 for use as bait in phage display. After 3-5 rounds allosteric. We resort to recently developed structure-based statistical mechani- of panning enrichment against ApoE isoforms was observed. From these cal model of allostery [2] in order to quantify the effects of allosteric signaling rounds of panning several unique clones were identified and subsequently caused by mutations. The model consists of three major components: (i) the subcloned, expressed, and purified to near homogeneity. We are currently modeling of dynamics in unperturbed (i.e. unligated/wild-type) and in per- in the process of more fully characterising the binding propensities of these turbed (i.e. ligated, mutate, modified, etc.) protein forms using normal modes; clones. (ii) a per-residue allosteric potential is used for the energy evaluation of the ef- fects of structural changes caused by protein dynamics; (iii) the evaluation of a per-residue allosteric free energy, that is the work exerted on the residue of in- 1727-Pos Board B47 terest as a result of allosteric signaling caused by a perturbation of the residue’s Molecular Dynamics and Docking Studies on Neurotensin Receptor structural environment. We use above model to introduce the definition of allo- Rejwan Ali1, Yasmeen Beasley1, Tetyana Skazko1, Mostafa Sadoqi1, steric mutation, which allows one to explore causality and energetics of the Mihaly Mezei2. regulation of protein activity caused by mutations of amino acids in the sites 1 2 Physics, St John‘s University, Queens, NY, USA, Chemical & Structural distant to the functional ones. Biology, Ichan School of Medicine at Mount Sinai, New York, NY, USA. 1. Guarnera, E., and Berezovsky, I. N. (2016) Allosteric sites: remote control in In recent times, a significant portion of molecular simulation works have been regulation of protein activity, Current opinion in structural biology 37, 1-8. focused on G-protein Coupled Receptors (GPCR). Given the fact that drug 2. Guarnera, E., and Berezovsky, I. N. (2016) Structure-Based Statistical Me- discovery paradigm has identified GPCR as important class as target mem- chanical Model Accounts for the Causality and Energetics of Allosteric brane protein, molecular modelling of GPCR has itself become a unique Communication, PLoS Comput Biol 12, e1004678. area in the broad field of bio-molecular simulations. Atomistic simulation studies of GPCR provide rich information about ligand binding and the active conformation states related to onset of G-protein activation and downstream 1730-Pos Board B50 signaling. Starting with rhodopsin, the first GPCR to be reported for crystal Transcriptional Regulation by Locking and Unlocking the Intrinsic Disor- structure, growing number of solved structures have opened up scopes for der in NfkB molecular simulation studies that potentially can assist novel design of Wei Chen, Clarisse G. Ricci, J. Andrew McCammon, Elizabeth A. Komives. high efficacy drugs molecules. In this presentation, we report our initial Department of Chemistry and Biochemistry, University of California, San data on ongoing molecular docking and dynamics studies of the agonist- Diego, La Jolla, CA, USA. bound neurotension receptor. AMBER and GAFF force fields for organic Intrinsic disorder plays a crucial role in the NFkB/IkB/DNA genetic regulatory molecules have been used to simulate the ligand/GPCR system under peri- network. Past studies on intrinsic disorder in the NFkB-IkB interaction have odic boundary conditions. Three initial independent 50 ns molecular dy- been focusing on IkB, the inhibitor of NFkB, while the dynamics of NFkB itself namics as well as docking studies show that natural agonist bound to the remains little understood. Previous coarse-grained energy landscape models outer helical region has an unstable binding conformation in contrast to the and hydrogen-deuterium exchange mass spectrometry (HDXMS) experiments agonist bound in the site embedded in intra-cellular helical region. Molecular showed intrinsic disorder in the N-terminal DNA-binding domain (NTD) of docking using blind targeting of the X-ray protein structure also support our NFkB which becomes ‘‘locked’’ upon binding of DNA or IkB. Here we present findings from initial molecular dynamics results. As neurotensin receptor is all-atom simulations with conventional molecular dynamics (cMD) and abundant both in brain and gastronomical regions, we are interested in active Gaussian accelerated MD (GaMD) to investigate the NFkB NTD dynamics. conformations of the receptor in physiological and acidic pH conditions. Our simulations showed that, starting from the DNA-bound conformation as Delineation of energy and entropy in different constituent components in the crystal structure, the free NFkB without binding to anything moves to in different pH medium by MM/PBSA and MM/GBSA techniques can pro- an ensemble of ‘‘open’’ states which are never seen in crystal structures. Com- vide information of neurotensin binding mode in different parts of human parison with HDXMS was also carried out to explore the local unfolding in the body. NTD. Our results not only implied how transcription is turned on by locking the intrinsic disorder in NFkB, but also provided insights into how IkB accelerates NFkB dissociation from DNA by ‘‘unlocking’’ the intrinsic disorder to turn off Protein Dynamics and Allostery III transcription as quickly as needed.

1728-Pos Board B48 1731-Pos Board B51 A Web-Based Interface for the Identification of Allosteric Ligand Binding Construing the Dynamic Complexity at a Plausible IKK2-Nemo Interface Sites Jamie M. Schiffer, Thuy Tien Nguyen, Ozlem Demir, Garret Chan, Nabina Paudyal, Alemayehu A. Gorfe. Rommie E. Amaro, Gourisankar Ghosh. Integrative Bio and Pharma, UT Health at Houston, Houston, TX, USA. Chemistry, UC San Diego, San Diego, CA, USA. The development of efficient tools for binding site identification in MD-derived The Nuclear factor kB (NF-kB) pathway is central to the regulation of gene ensembles with potentially open druggable pockets is an important step for expression in cells, leading to downstream effects like the inflammatory computational drug design. Ligand binding specificity analysis (LIBSA) and response, proliferation, and differentiation. In the canonical pathway, phos- probe-based molecular dynamics (pMD) simulation are some of the protocols phorylation of the inhibitor of NF-kB (IkB) occurs after the inhibitor of kB ki- that utilize MD-ensembles to access the propensity of a site to bind a ligand. nase (IKK) complex is activated, which results in the relocation of NF-kB to the These tools require expert skills to be properly executed. A python wrapper nucleus. The IKK complex is thus the central regulatory complex in the NF-kB has been developed to streamline pre-existing algorithms of LIBSA and auto- pathway. This complex is composed of the catalytic subunits IKK1 and IKK2, mate its disparate protocols. The wrapper helps in the preparation of files, the later being the active kinase in the canonical pathway, as well as the regu- execution of individual programs and generation of appropriate results. The latory subunit NEMO. Here, through computational modeling, anisotropic goal of the python wrapper is to integrate available algorithms in a systematic network models, and molecular dynamics simulations, we reveal the dynamic way and build a python based web interface for submission of jobs and moni- motions surrounding a plausible NEMO interface in the scaffold dimerization toring the results. I will present a design of web interface that provides an ideal domain. This conformational ensemble illuminates possible pockets for drug platform for making complex binding site identification protocols readily avail- discovery efforts and aligns computation with experiment to understand how able for non-expert users. NEMO and IKK2 interact.

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1732-Pos Board B52 to functions must be extracted from the time-series data. One of the multivar- What Makes Enzymes Work? Exploring Protein Dynamics in P-T-X iate analysis methods, principal component analysis (PCA), is widely used Qi Huang1, Jocelyn M. Rodgers2, Russell J. Hemley3, Toshiko Ichiye1. for analysing static properties of systems. On the other hand, relaxation 1Chemistry, Georgetown University, Washington, DC, USA, 2Geophysical, mode analysis (RMA) has been used to elucidate dynamic properties in a Carnege Institute for Science, Washington, DC, USA, 3Civil and random spin system [1], a homo-polymer system [2], and hetero-polymer sys- Environmental Engineering, George Washington University, Washington, tems [3, 4, 5]. This method, on the basis of statistical physics, finds slow DC, USA. relaxation modes in systems with relaxational dynamics. In its previous Although both stability and flexibility are necessary for enzymes to function, application to a protein system [4], however, the long-time behavior was general quantitative measures of flexibility have yet to emerge so that design not described well. Two-step RMA has recently been developed in a of functional enzymes is difficult. Clarifying what type of flexibility is impor- homo-polymer system in order to improve description of long-time behavior tant may help to resolve this question. For instance, collective motions are [6]. We applied this method to a hetero-polymer system, hen egg-white lyso- important, but are the atomic fluctuations that give rise to the collective mo- zyme (HEWL) protein. We found that two-step RMA not only describes tions more fundamentally important? Exploring ‘‘Nature’s laboratory’’ has long-time behavior well, but also extracts slow structural transitions of a pro- become a powerful method, namely, by examining enzymes from ‘‘extremo- tein more effectively. philic’’ organisms that live under extreme conditions. Extremophiles must [1] H. Takano, S. Miyashita, J. Phys. Soc. Jpn. 64, 3688 (1995). maintain enzyme activity under extreme conditions to grow, and often the ad- [2] H. Hirao, S. Koseki, H. Takano, J. Phys. Soc. Jpn. 66, 3399 (1997). aptations necessary for stability and flexibility conflict. While enzymes from [3] A. Mitsutake, H. Iijima, H. Takano, J. Chem. Phys.135, 164102 (2011). extremophiles who live under extremes of temperature (T) and even chemical [4] T. Nagai, A. Mitsutake, and H. Takano, J. Phys. Soc. Jpn. 82, 023803 composition (X) have been well-studied, enzymes from piezophiles who live (2013). at high pressure (P) have not been, in part because high pressure is a difficult [5] A. Mitsutake, H. Takano, J. Chem. Phys. 143, 124111 (2015). variable in the field and in the laboratory. In addition, pressure apparently af- [6] S. Natori, H. Takano, submitted J. Phys. Soc. Jpn. fects proteins differently than temperature, and new high-pressure instrumen- tation makes experimental studies of high pressure effects on enzymes possible. Here, flexibility measures are assessed in homologous enzymes 1735-Pos Board B55 from mesophiles and piezophiles under different P-T-X conditions using Importance of Protein Vibration Directionality on Function 1 1 1 2,3 molecular dynamics simulations in light of results from high pressure Katherine A. Niessen , Mengyang Xu , Yanting Deng , Edward H. Snell , 1 experiments. Andrea G. Markelz . 1Physics, SUNY University at Buffalo, Buffalo, NY, USA, 2Hauptman- 3 1733-Pos Board B53 Woodward Medical Research Institute, Buffalo, NY, USA, Structural Extreme Biophysics: Enzymes under Pressure Biology, SUNY University at Buffalo, Buffalo, NY, USA. Qi Huang1, Jocelyn M. Rodgers2, Russell J. Hemley3, Toshiko Ichiye1. Global protein vibrations have long been associated with protein functionality. 1Georgetown University, Washington, DC, USA, 2Georgetown University, Long-range intramolecular vibrations have been measured in proteins using Carnegie Institution of Washington, Washington, DC, USA, 3George anisotropic absorption in the terahertz frequency range [1,2]. These measure- Washington University, Washington, DC, USA. ments directly correspond to the directionality of vibrations and show large The discoveries of ‘‘extremophilic’’ microbes that thrive under extremes of changes in the directionality of the vibrational displacements for free chicken temperature, pressure, pH, etc. raise many questions including the adaptations egg white lysozyme (CEWL) and inhibitor bound CEWL. Normal mode made in their constituent molecules such as enzymes in order to function under ensemble analysis (NMEA) and quasiharmonic analysis (QHA) calculations conditions where their counterparts in mesophiles fail. Of extreme conditions, of the free and tri-acetylglucosamine (3NAG) bound CEWL dynamics were the effects of high pressure have been relatively unexplored, both because of performed and also show large changes in vibration directionality with bind- the difficulty in producing high pressure in the lab and because of the difficulty ing. This in spite of the calculated energy distribution showing very little in collecting samples of ‘‘piezophiles’’, microbes adapted for high pressure en- change, in agreement with neutron scattering measurements. We investigate vironments. Here, factors are examined that may lead to the increased activity the importance of the protein directionality by extending the calculations to under pressure in dihydrofolate reductase from the piezophilic Moritella pro- a double deletion mutant (DD CEWL) with an activity rate 1.4 times that of funda compared to the homologous enzyme from the mesophilic Escherichia WT [3]. The deletions are far from the active site, indicating that the increased coli. Molecular dynamics simulations are performed at various temperatures activity arises from changes in the dynamics. The calculated anisotropic and pressures to examine how pressure affects the flexibility of the enzymes spectra show large changes with mutation, whereas the energy distribution from these two microbes, since both stability and flexibility are necessary for show practically no change from the WT. Similarities in the bound WT, enzyme activity. The results suggest that collective motions on the 10 ns time- free DD, and bound DD CEWL NMEA spectra indicate the mutation may scale are responsible for the flexibility necessary for ‘‘corresponding states’’ be changing the directionality of the vibrations toward more efficient motions. activity at the growth conditions of the parent organism. The results also sug- Projections of the low frequency vibrations, from QHA, on the functional gest that while the lower stability of many enzymes from deep-sea microbes displacement show higher overlap of residues around Glu35 and Asp52 in may be an adaptation for greater flexibility at low temperatures, it may also the mutant system. These residues are known to be critical in CEWL function- enable the enzymes to withstand high pressure. Determination of the adapta- ality. The results reveal that the mutation may be steering the protein toward tions of enzymes for extreme conditions can lead to a greater understanding more functional dynamics without significantly affecting the energy distribu- of enzyme structure-function relationships. In addition, understanding these tion. [1] K.A. Niessen, et al. (2015) DOI: 10.1007/s12551-015-0168-4 adaptations can be used in biotechnology so that enzymes can be bioengineered [2] G. Acbas, et al. (2014) DOI: 10.1038/ncomms4076 to function under specific conditions. Moreover, determining the limiting con- [3] S. Mine, et al. (1999) DOI: 10.1006/jmbi.1999.2572. Supported by NSF ditions where enzyme activity can be maintained is one of the factors in deter- (DBI 1556359, MCB 1616529) and DOE (DE-SC0016317). mining the ‘‘limits of life’’, which could guide the search for life in extreme environments such as beneath the oceanic and continental surface or even 1736-Pos Board B56 extraterrestrially. Conformational Evolution of Three Regulator of G-Protein Signaling Proteins (RGS4, RGS8, RGS19) in Microsecond-Scale Simulations 1734-Pos Board B54 Hossein Mohammadiarani, Harish Vashisth. Improved Relaxation Mode Analysis of a Hen Egg-White Lysozyme Chemical Engineering, University of New Hampshire, Durham, NH, USA. Protein Regulators of G protein signaling (RGS) proteins modulate GPCR signaling Naoyuki Karasawa1, Ayori Mitsutake1,2, Hiroshi Takano1. by binding to Ga-subunits of heterotrimeric G proteins and accelerating hy- 1Keio University, Yokohama, Japan, 2JST PRESTO, Chiyoda, Japan. drolysis of GTP. Therefore, RGS proteins are becoming increasingly impor- Proteins have various functions within living organisms and play important tant therapeutic targets to be directly or allosterically inhibited from binding roles in life phenomena. Their functions are related to not only structures to Ga-subunits. While structures of several known RGS proteins are highly but also structural fluctuations around them. Molecular dynamics (MD) simu- similar and largely contain a-helical motifs, some thiadiazolidinone lation is an effective tool to investigate protein dynamics by tracing motions (TDZD) compounds that target cysteine residues have shown different levels of all atoms in a system. However, structural fluctuations of proteins include of specificities and potencies for closely related proteins thereby suggesting various time-scale dynamics and are complex. Therefore, the motions related intrinsic differences in dynamics of these proteins. In this work, we have

BPJ 7839_7842 354a Tuesday, February 14, 2017 studied the dynamics of three different RGS proteins (apo-RGS4, apo-RGS8, Malaria is one of the most prevalent and fatal infectious diseases of the world and apo-RGS19) using microsecond-scale classical molecular dynamics and is compounded by wide spread drug resistance, with some strains devel- (MD) simulations with CHARMM and AMBER force-fields. Analyses of oping resistance to current front-line anti-malarials. Plasmodium falciparum these trajectories reveal high fluctuations in a5anda6 helices and the loops M1 alanyl aminopeptidase (PFM1AAP) is involved in the terminal stages of connecting them. These fluctuations lead to perturbations in residues in the haemoglobin degradation and the generation of an amino acid pool to support RGS-Ga interface and in the vicinity of cysteines that are targets of allosteric parasitic growth and development. This represents a promising new anti- inhibitors. These findings have significant implications for understanding dif- malarial target where inhibition of this enzyme is lethal to the malarial parasite. ferences in potencies and specificities of inhibitory small-molecules. To start understanding ligand recognition and binding in PFM1AAP for future drug design efforts, we explored (un)binding pathways of substrates, inhibitors and a product in the enzyme using steered molecular dynamics (sMD) simula- 1737-Pos Board B57 tions. The results of our simulations further clarify the substrate entrance and Comparative Structural Dynamic Analysis of GTPases exit pathways to the buried active site of PFM1AAP and identify their respec- Hongyang Li, Xin-Qiu Yao, Barry Grant. tive physiochemical profiles. Our SMD simulations reveal several binding Computational Medicine and Bioinformatics, University of Michigan, Ann characteristics: (1) A substrate/inhibitor recognition mechanism, (2) active Arbor, MI, USA. migration into the entrance channel, (3) a water reservoir which facilitates cor- GTPases regulate a multitude of essential cellular processes ranging from rect substrate orientation, (4) a molecular gate controlling product egress. movement and division to differentiation and neuronal activity. These ubiqui- Furthermore, several critical residues have been identified that in concert, facil- tous enzymes operate by hydrolyzing GTP to GDP with associated conforma- itate these processes. These residues are now the focus of a mutagenesis study tional changes that modulate affinity for family specific binding partners. There to fortify the in silico predictions. We observe differences in (un)binding path- are three major GTPase superfamilies: Ras-like GTPases, heterotrimeric G pro- ways of substrates, inhibitors and product. A novel residue-based network anal- teins and protein-synthesizing GTPases. Although they contain similar ysis has been developed to highlight repetitive rather than sporadic events in the nucleotide-binding sites, the detailed mechanisms by which these structurally multiple sMD simulation trajectories of ligand (un)binding. Our work paves the and functionally diverse superfamilies operate remain unclear. Here we way toward designing novel potent PFM1AAP inhibitors for the treatment of compare and contrast the structural dynamic mechanisms of each superfamily malaria. using extensive molecular dynamics (MD) simulations and subsequent network analysis approaches. In particular, dissection of the cross-correlations of atomic displacements in both the GTP and GDP-bound states of Ras, transducin and 1740-Pos Board B60 elongation factor EF-Tu reveals analogous dynamic features. This includes Dynamic Allostery in Regulated Entry of Newcastle Disease Virus into similar dynamic communities and subdomain structures (termed lobes). For Host Cells all three proteins the GTP-bound state has stronger couplings between equiva- Nalvi D. Duro, Sameer Varma. lent lobes. Network analysis further identifies common and family specific res- University of South Florida, Seminole, FL, USA. idues mediating the state specific coupling of distal functional sites. Mutational Newcastle disease virus (NDV) belongs to the family of paramyxoviruses that simulations demonstrate how disrupting these couplings leads to distal dynamic cause numerous fatal diseases in humans and farm animals. Here we focus on effects at the nucleotide binding site of each family. Collectively our studies mechanisms that underlie its regulated entry into host cells. To gain entry, NDV extend current understanding of GTPase allosteric mechanisms and highlight utilizes two of its membrane proteins: Hemagglutinin-Neuraminidase (HN) and previously unappreciated similarities across functionally diverse families. the fusion (F) protein. HN binds to sialic acids expressed on the host cell, and this binding stimulates HN to activate F, which, in turn, mediates virus-host membrane fusion. HN interacts with sialic acid and F through separate sites 1738-Pos Board B58 located on two different domains, however, no models explain this allosteric The Molecular Mechanism of Entropic Allostery of Hemoglobin coupling. In fact, the analogous mechanisms in other paramyxoviruses also Takashi Yonetani1, Kenji Kanaori2. remain undetermined. Starting with X-ray structures of HN‘s receptor binding 1 2 Biophys/Biochem, Univ. Pennsylvania, Philadelphia, PA, USA, Biomol domain (bound to sialic acid derivatives), we examine using molecular dy- Engineering, Kyoto Inst Tech, Kyoto, Japan. namics how sialic acid affects HN’s receptor binding domain (RBD) as well Protohemes in Hb (Hb) are reversible low-affinity binding sites for diatomic li- as HN’s RBD-RBD dimeric interface. We note first that sialic acid induces gands such as O2, CO, and NO. The ligand-affinity of Hb is regulated by con- only minor structural changes in individual RBDs - an observation similar to trolling two distal effects, i.e., distal H-bonding and geminate-recombination of proteins like GPCRs that are regulated by dynamic allostery. Despite inducing the ligand, which enhance retention of the ligand at the hemes to increase minor changes in individual RBDs, we find that sialic acid reorients the RBD- apparent ligand-affinity of Hb. Ligands, dissociating from hemes, encounter RBD interface, and that this reorientation contributes to HN stimulation. physical barriers of globin matrix (‘‘Caged’’ states), before either geminately Finally, we note that the induced RBD-RBD reorientation is unlike what we recombining to the hemes to regenerate the respective ligated hemes [Route observed in the case Nipah’s HN homolog (Dutta et al. Biophys. J. 2016), sug- A] or migrating through globin matrices, followed by bimolecular dissociation gesting that fusion stimulation mechanisms are not conserved across to form deoxy-Hb [Route B]. Therefore, only a fraction of the dissociating paramyxoviruses. ligands moves out of Hb (bi-molecular quantum yield [Q]). The remainder re- verts back to the hemes to form the ligated Hb (geminate quantum yield [1-Q]). This is resulted in increases in the apparent ligand-affinity of Hb in comparison 1741-Pos Board B61 The Allostery Landscape: Quantifying Thermodynamic Couplings in Bio- with those of free hemes (P50a [Q]/ [1-Q]). Binding of heterotropic effectors reduces the O -affinity of Hb (increases in P ). Effectors cause enhancement molecular Systems 2 50 1,2 1,2 1,2 of very high-frequency (>tera-Hz) thermal fluctuations of globin in both in Michel A. Cuendet , Harel Weinstein , Michael V. LeVine . 1Department of Physiology and Biophysics, Weill Cornell Medical College deoxy- and oxy-Hb (1), resulting in increases in apparent transparency/porosity 2 of globin matrix toward migrating ligands, enhancing bi-molecular dissociation of Cornell University, New York, NY, USA, HRH Prince Alwaleed Bin (Route B) and reducing geminate-rebinding (Route A) This results in substan- Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College of Cornell University, New York, NY, USA. tial decrease in the apparent O2-affinity (KT and KR) without detectable changes in static crystallographic structures of deoxy- and oxy-Hb as well as Allostery plays a fundament role in most biological processes. However, little the coordination/electronic structures of the hemes in T(deoxy)- and R(oxy)- theory is available to describe it outside of two-state models. Here we use a Hb (2). statistical mechanical approach to show that the allosteric coupling between References: [1] Laberge & Yonetani, Molecular dynamics simulations of he- two collective variables is not a single number, but instead a two- moglobin A in different states and bound to DPG, Biophys. J. 94 (2008) 2737; dimensional thermodynamic coupling function that is directly related to the [2] Yonetani & Kanaori, How does hemoglobin generate such diverse function- mutual information from information theory and the copula density function ality of physiological relevance?, BBA 1834 (2013) 1873. from probability theory. On this basis, we demonstrate how to quantify the contribution of specific energy terms to this thermodynamic coupling function, 1739-Pos Board B59 enabling a decomposition that reveals the mechanism of allostery. We illus- Steered Molecular Dynamic Simulations Reveal Critical Residues for (Un) trate the thermodynamic coupling function and its use by showing how allo- Binding of Substrates, Inhibitors and a Product of the Malarial PFM1AAP steric coupling in the alanine dipeptide molecule contributes to the overall Daniel S. Moore, John P. Dalton, Irina G. Tikhonova. shape of the F/J free energy surface, and by identifying the interactions Molecular Therapuitics, Queen‘s University, Belfast, United Kingdom. that are necessary for this coupling.

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1742-Pos Board B62 Membrane Protein Structures I How Does a Ligand Explore the Deep Channel of Neurolysin? A Confor- mational Dynamics Study with Wexplore and Elastic Network Modeling 1744-Pos Board B64 1 2 1,3 Arzu Uyar , Vardan Karamyan , Alex Dickson . Improving Expression in Yeast of A1 Adenosine Receptor using Chimeric 1Department of Biochemistry and Molecular Biology, Michigan State 2 Receptors - Design and Optimization of the Host/Protein Platform for University, East Lansing, MI, USA, Department of Pharmaceutical Therapeutic Development Sciences, Texas Tech University Health Sciences Center, Amarillo, TX, 3 Abhinav R. Jain, Anne S. Robinson. USA, Department of Computational Mathematics, Science and Engineering, Chemical and Biomolecular Engineering, Tulane University, New Orleans, Michigan State University, East Lansing, MI, USA. LA, USA. Neurolysin is a zinc metallopeptidase involved in neuropeptide degradation G Protein-Coupled Receptors (GPCRs) are seven a-helical transmembrane re- and modification by cleaving a number of bioactive peptides. In vivo, neuro- ceptor proteins that bind to specific extracellular ligands to initiate a down- lysin hydrolyzes the short neuropeptide neurotensin, to create inactive shorter stream signaling response. GPCRs comprise nearly 4% of all human genes, fragments. Neurolysin is of interest as a therapeutic target since a change in but are targets of approximately 25-40% of all drug discovery efforts. Aden- neurotensin level may cause a range of disorders such as pain perception, osine receptors (A1R, A2aR, A2bR and A3R) are a subfamily of GPCRs that schizophrenia, addiction, cardiovascular disorders, and Huntington and Par- recognizes adenosine, an important energy metabolite, have been implicated kinson diseases. Neurolysin has a prolate ellipsoid shape with a deep channel in numerous cardiovascular and neurodegenerative diseases, and therefore that runs almost the entire length of the molecule. Although other metallopep- are targets for drug development. Biophysical characterization and high reso- tidases show a hinge-like motion, available crystal structures of neurolysin do lution structure determination are routinely used for GPCR drug design and not reveal any significant conformational changes. Thus, elucidation of its in- discovery and require heterologous expression and purification. High-level ternal dynamics and how they are affected by ligand binding can aid in further GPCR expression has facilitated many structure determination, as for A2aR, design of potent ligands that either activate or inhibit neurolysin function. For which shows high yield (milligram protein per liter of culture) and has mul- this purpose, we combine two different computational techniques, namely tiple resolved crystal structures. Yet to date, heterologous expression of the WExplore and Elastic Network Modeling (ENM), to examine possible ligand other adenosine receptor family members has resulted in low yields of active binding sites and their effect on large-scale motions in neurolysin. WExplore receptor, suggesting additional strategies are needed to enable structural char- uses the weighted ensemble algorithm and generates full-atomistic conforma- acterization. Previous studies suggest the C-terminus of the A2aR plays a role tional ensembles that can span large free energy barriers. In this study, we use in the observed efficient expression and trafficking to the plasma membrane of WExplore to generate a network of binding sites in the neurolysin channel, the receptor. To understand the role of the C-terminus and to test its efficacy and predict their relative probabilities. ENM analysis, which is a fast in improving the expression of a subfamily member, we developed A1R chi- coarse-grained normal mode analysis that provides collective deformations meras with the A2aR C-terminus using homologous DNA recombination in of proteins, reveals the hidden hinge-like motion of neurolysin, where the yeast. The role of the C-terminus of the A2aR on expression, trafficking two domains rotate toward each other by narrowing the channel. The differ- and activity relative to wildtype receptors will be described. With our engi- ences observed during hinge-like motion in apo and ligand bound forms of neered chimeric receptors, we hope to improve expression of other recombi- neurolysin pinpoint specific binding regions that affect low-frequency motions nant GPCRs and to screen ligand libraries via receptor binding affinity and in an allosteric manner. This combined multiscale approach helps elucidate signaling activity. the coupling between ligand binding, hinge motion and neurolysin catalytic activity. 1745-Pos Board B65 A Practical Method to Prepare GLP-1 Receptor 1743-Pos Board B63 Zhongping Hu, Fan Yang, Nian Cheng, Zhengding Su. Variable Dynamics of IgG4 Monoclonal Antibody Controlled by Single Hubei University of Technology, Wuhan, China. Point Mutations GLP-1 receptor (GLP-1R) belongs to one of B-class G protein-coupled recep- 1 2 Ettayapuram Ramaprasad Azhagiya Singam , Shahid Uddin , tors (GPCRs) and is a therapeutic target for the treatment of type-2 diabetes. 3 1 Jose Casas-Finet , Donald J. Jacobs . The receptor is composed of a globular N-terminal extracellular domain 1UNC Charlotte, Charlotte, NC, USA, 2Formulation Sciences, MedImmune 3 (ECD), a seven-helical transmembrane domain (TMD) and a small intracellular Ltd, Cambridge, United Kingdom, BioPharmaceutical Development, domain (ICE). In this work we demonstrated a practical procedure to utilize mi- MedImmune LLC, Gaithersburg, MD, USA. crobial systems to prepare GLP-1R for structural biology study. The ECD of An immunoglobulin G (IgG) antibody consists of two heavy and two light GLP-1R was expressed in the form of GST and intein fusion protein using chains that bind together to form a Y-shaped structure. Although IgG anti- either yeast or E. coli protein expression systems. The TMD of GLP-1R was bodies are naturally bivalent, weak interaction within the CH3-CH3 domain prepared with an MFH fusion protein in E. coli. The ECD and TMD were interface together with a Fab-arm exchange allows the IgG4 antibody to released from their fusion proteins and then ligated via intein-mediated native become an effective monovalent half molecule. Recent experiments deter- ligation, which generated a biologically-active full-length GLP-1R protein. Our mined a number of single point mutations in the CH3-CH3 interfacial region work can not only provide high-quality sample for protein structure character- that significantly increase the monovalent form [1,2]. In this investigation, we ization, but also enable to isotopically label GLP-1R protein segmentally for explored the effect of single point mutations in the interface of the CH3-CH3 NMR study. domain on the structure and dynamics of the full IgG4 molecule using coarse- grained molecular dynamics (MD) simulation employing a Martini force field. We analyzed MD trajectories for the wild type IgG4 and the single 1746-Pos Board B66 point mutations F405R, T394D, Y349D and L368W. For the wild type and Predicting Membrane Protein Expression in Yeast from Sequence-Derived each mutant system, three 2 ms MD trajectories were generated using Features different initial conditions (in total 6 ms/system). In agreement with experi- Samuel J. Schulte, Shyam Saladi, William M. Clemons. mental results [2] the mutations F405R and T394D increase the half- California Institute of Technology, Pasadena, CA, USA. antibody population, while Y349D and L368W establish monomer-dimer Despite comprising one-quarter of most organisms’ proteome and serving as equilibrium. From principal component analysis it is determined that all the target of over half of all drugs, integral membrane proteins remain difficult four mutations increase mobility. However, the F405R and T394D mutations to characterize. Poor expression in heterologous systems often hinders IMP severely weaken the interaction between the two CH3 domains resulting in study, and large-scale efforts to express IMPs have proven time-consuming, dissociation. Free energy of binding for the CH3-CH3 domain is calculated costly, and capricious. As such, we recently used quantitative experimental using umbrella sampling along a representative dissociation pathway. These expression studies to train a machine learning model capable of predicting results highlight mechanistic causes for the loss in binding affinity in the membrane protein expression in Escherichia coli solely from sequence- mutated systems. derived features. Though our linear bacterial model generalizes well to eukary- [1] Shan, L., et. al. Generation and Characterization of an IgG4 Monomeric Fc otic membrane proteins expressed in E. coli, we observe poor prediction for Platform. PLoS One 11:e0160345 (2016). IMPs heterologously expressed in yeast, a host frequently chosen for its greater [2] Wilkinson, I.C., et. al. Monovalent IgG4 molecules: immunoglobulin similarity to higher eukaryotes. Thus, we report a new model capable of Fc mutations that result in a monomeric structure. MAbs 5:406-417 predicting IMP expression in Saccharomyces cerevisiae. To avoid overfitting (2013). resulting from the limited size of our training dataset, the number of

BPJ 7839_7842 356a Tuesday, February 14, 2017 sequence-derived features used to predict expression is reduced from the 89 onto one of the recombinant proteins enables in vivo biotinylation. Immobi- used for the E. coli model to just eight. Strikingly, in agreement with recent lized avidin allows for binding of the biotinylated protein for purification findings in the wet laboratory, the disorder of the C-terminus is identified as directly from cell lysate, either on beads for structure analysis or a surface the most predictive feature. We additionally incorporate new features, appropriate for binding measurements, such as with surface plasmon reso- including predicted N- and O-glycosylation and disulfide bond formation, nance of biolayer interferometry. The protein binding partner is purified using into our algorithm. We are working to verify the model across a wide variety a standard His-tag purification, and, in the case of membrane proteins, is re- of small- and large-scale expression datasets from the literature. We will share constituted into liposomes. This allows for investigations of membrane our predictor with the broader community to help accelerate membrane protein protein-protein interactions using proteoliposomes for the mobile component, biochemical and biophysical study. which can interact with immobilized soluble proteins. In addition to facili- tating the comparison of the structure and binding of the same sample, this 1747-Pos Board B67 set-up allows for a quick screening method of protein mutations and/or mod- Statistical Models Robustly Predict Membrane Protein Expression in ifications which alter or eliminate binding, before using those mutants for E. Coli structural studies. Several model protein-protein systems will be used to Shyam Saladi, Alexander E. Chu, William M. Clemons. develop this approach. The first system presented is the Neisserial Opa pro- California Institute of Technology, Pasadena, CA, USA. tein, an eight-stranded b-barrel membrane protein which interacts with recep- Membrane protein production is difficult; their biogenesis does not stop with tors on the surface of human cells. Binding affinities for the mutants and translation but also requires translocation and integration into a lipid bilayer. structural restraints measured with DEER experiments will be presented using These additional steps hamper their heterologous expression which signifi- this approach to demonstrate the application of the method. Additional cantly impedes biophysical and structural studies. Detailed and anecdotal protein-protein complexes will be investigated to determine the general appli- evidence in the literature suggests that a variety nucleotide and amino- cability of the method. acid sequence level determinants may potentially support or hinder their biogenesis, e.g. mRNA pausing elements, codon adaptation, transmembrane 1750-Pos Board B70 segment hydrophobicity, ‘‘positive inside rule.’’ In previous work, we Sans Studies of Membrane Protein Incorporation and Crystallization in demonstrated that a linear, preference-ranking Support Vector Machine the Lipidic Cubic Phase can capture heterologous membrane protein expression in E. coli.Here Thomas E. Cleveland, Paul Butler. we present work showing statistical models with greatly improved perfor- Center for Neutron Research, National Institute of Standards and mance across small- and large-scale laboratory experiments (e.g. expression Technology, Gaithersburg, MD, USA. tests that routinely precede structural studies) published in the literature. We Membrane proteins can be incorporated into lipidic mesophases, such as the also present progress towards a Bayesian model that can be refined as new lipidic cubic phase (LCP), in order to facilitate the growth of crystals for struc- expression data becomes available on-line and that can be extended across tural studies. However, although this technique has had great empirical suc- expression systems (e.g. yeast, insect cells), plasmids, and conditions. cesses in generating membrane protein crystals, many details of the process Furthermore, parameter values from this model will facilitate reliably char- are not well understood; for example, the mechanisms of initial incorporation acterizing the sequence features underpinning membrane protein expression of membrane proteins into the LCP, the localization of protein after incorpora- suggesting intriguing areas for further biophysical and computational tion, and the mechanisms of nucleation and crystal growth. It has long been hy- experiments. pothesized that protein monomers add to the growing crystal through a ‘‘lamellar portal:’’ a lamellar region of lipid connecting the bulk cubic phase 1748-Pos Board B68 to the crystal. While microbeam diffraction experiments in the vicinity of Establishing Rhodobacter Sphaeroides for Expression of Large Mitocon- LCP-embedded crystals have provided some experimental support for this hy- drial Membrane Complex YME1L for Cryo-EM Structural Studies pothesis, these experiments are performed only after the growth of macroscopic Cristina Puchades, Luke Rockland Wiseman, Gabriel Lander. crystals; few experiments have probed the distribution of incorporated protein The Scripps Reseach Institute, san diego, CA, USA. in LCP prior to crystallization, or the processes leading to initial nucleation and Mitochondrial function is reliant on a balanced mitochondrial proteome, main- crystal growth. tained by a set of intricately regulated quality control proteases, such as We have used Small-Angle Neutron Scattering (SANS) to study each step of YME1L. YME1L is AAAþ ATPase located in the inner membrane of mito- the cubic phase crystallization process using Bacteriorhodopsin (bR) as a model chondria that has been shown to be essential for cardiac function. However, system. By contrast-matching the non-protein components of the system, i.e. a lack of structural information limits molecular understanding of its activity detergents and lipids, the protein may be studied directly and in isolation, and regulation. We aim to fill this knowledge gap by employing an electron mi- greatly simplifying the interpretation of scattering data from these complex croscopy (EM)-based approach for structural studies of full length human multi-component systems. We will present the results of our contrast- YME1L. Structural studies require expression and purification of stable, matched SANS studies of bR in detergent solutions; immediately after cubic natively folded protein, which constitutes a major bottleneck in the field of phase incorporation; and finally, after precipitant addition. The implications membrane protein biology. Expression of full length YME1L is hindered in of these results regarding the mechanisms of bR incorporation, localization E. Coli by insufficient membrane surface for protein integration, and a lack in the LCP, and crystallization will be discussed. of appropriate folding machinery leading to formation of inclusion bodies. Furthermore, low endogenous expression levels and limited overexpression ca- 1751-Pos Board B71 pabilities limit purification of membrane complexes from mammalian cells. We Crystallogenesis of Membrane Proteins Mediated by Polymer-Bounded have expressed full-length FLAG-tagged human YME1L in Rhodobacter Lipid Nanodiscs sphaeroides, establishing an expression platform for this and other large, insol- Jana Broecker1, Bryan T. Eger1, Oliver P. Ernst2. uble, multi-subunit membrane complexes with key implications for mitochon- 1Biochemistry, University of Toronto, Toronto, ON, Canada, 2Biochemistry drial function. and Molecular Genetics, University of Toronto, Toronto, ON, Canada. For many membrane proteins, detergent-mediated solubilization compromises 1749-Pos Board B69 protein stability and functionality, often impairing biophysical and structural Investigation of Affinities and Structures of Membrane Protein-Protein analyses. Hence, membrane-protein structure determination is a continuing Interactions using Comparable Samples bottleneck in the field of protein crystallography. Here, as an alternative to ap- Jennifer Martin, Marissa Kieber, Linda Columbus. proaches mediated by conventional detergents, we report the crystallogenesis Chemistry, University of Virginia, Charlottesville, VA, USA. of a recombinantly produced membrane protein that never left a lipid bilayer When identifying protein-protein interactions, traditional methods require the environment. We used styrene-maleic acid (SMA) copolymers to solubilize use of a variety of different protein preparations or protein modifications for lipid-embedded proteins into SMA nanodiscs, purified these discs by affinity structural and quantitative binding studies. The downside to this approach is and size-exclusion chromatography, and transferred proteins into the lipidic cu- that the differently prepared proteins may have alterations in dynamics or con- bic phase (LCP) for in meso crystallization. The 2.0-A˚ structure of an a-helical formations which can affect function, and conflicting results can be obtained. 7-transmembrane microbial rhodopsin thus obtained is of high quality and To combat these challenges, we aim to develop a method for investigating virtually identical to the 2.2-A˚ structure obtained from traditional detergent- protein-protein interactions which uses the same sample preparation for based purification and subsequent LCP crystallization. The highlights of this both structure and binding investigations. Incorporating a small BAP tag work are: i) Crystallization of a membrane protein that never left a lipid bilayer

BPJ 7839_7842 Tuesday, February 14, 2017 357a environment; ii) Lipid nanodiscs enable transfer of membrane proteins between 1755-Pos Board B75 lipid bilayer systems; iii) SMA copolymers are compatible with lipidic cubic Structure of an Inward Proton Transporting Anabaena Sensory phases; and iv) Method expands the toolbox for membrane-protein Rhodopsin Mutant: Mechanistic Insights crystallography. Hartmut Luecke, Bamboo Dong, Lissete Sanchez Magraner. MB&B, UC Irvine, Irvine, CA, USA. 1752-Pos Board B72 Microbial rhodopsins are light-activated, seven-a-helical, retinylidene trans- Structure of the STRA6 Receptor for Retinol Uptake membrane proteins that have been identified in thousands of organisms Jonathan Kim. across archaea, bacteria, fungi, and algae. Although they share a high degree Physiology, Columbia University, New York, NY, USA. of sequence identity and thus similarity in structure, many unique functions Vitamin A homeostasis is key to cellular function and human physiology. The have been discovered and characterized among them. Some function as out- integral membrane receptor STRA6 mediates tissue uptake of vitamin A ward proton pumps, some as inward chloride pumps, whereas others function through recognition of the retinol-binding protein-retinol complex. STRA6 as light sensors or ion channels. Unique among the microbial rhodopsins acts as a transporter, but it has alternatively been suggested to function in trans- characterized thus far, Anabaena sensory rhodopsin (ASR) is a photochromic membrane signaling. Here we present the structure of STRA6 determined to sensor that interacts with a soluble 14-kDa cytoplasmic transducer that is en- 3.9A˚ resolution by single-particle cryo-electron microscopy. STRA6 is a dimer, coded on the same operon. The sensor itself stably interconverts between all- in which each protomer contributes nine transmembrane and a central horizon- trans-15-anti and 13-cis-15-syn retinal forms depending on the wavelength of tal intramembrane helix that is positioned at the core of the dimer interface. illumination, although only the former participates in a photocycle with a Surprisingly, the C-terminus of each protomer is tightly bound to calmodulin signaling M intermediate. A mutation in the cytoplasmic half-channel of in a noncanonical arrangement. The structure suggests alternate modes for the protein, replacing Asp217 with Glu (D217E), results in the creation of internalization of retinol, and provides a template to further investigate the a light-driven, single-photon, inward proton transporter. We present the 2.3 physiological role of STRA6. A˚ structure of dark-adapted D217E ASR, which reveals significant changes in the water network surrounding Glu217, as well as a shift in the carbon 1753-Pos Board B73 backbone near retinal-binding Lys210, illustrating a possible pathway lead- Biosynthetic Production of an isotopically Labelled Retinal in E. Coli ing to the protonation of Glu217 in the cytoplasmic half-channel, located ˚ Rachel Munro1, Meaghan Ward1, So Young Kim2, Keon Ah Lee2, 15 A from the Schiff base. Crystallographic evidence for the protonation Kwang-Hwan Jung2, Vladimir Ladizhansky1, Leonid Brown1. of nearby Glu36 is also discussed, which was described previously by Four- 1Department of Physics, University of Guelph, Guelph, ON, Canada, ier transform infrared spectroscopy analysis. Finally, two histidine residues 2Department of Life Science and Interdisciplinary Program of Integrated near the extracellular surface and their possible role in proton uptake are Biotechnology, Sogang University, Seoul, Korea, Republic of. discussed. Solid-state Nuclear Magnetic Resonance (ssNMR) is an emerging biophysical 1756-Pos Board B76 technique which has been useful in studying the structure of integral mem- Spectroscopic Investigation of the Protonpump CsR and its Leaky brane proteins such as microbial rhodopsins. However, this technique requires Derivatives the incorporation of isotopically-labelled atoms into the protein. This is usually Roman Fudim, Arend Vogt, Peter Hegemann. accomplished through over-expression of rhodopsins in E. coli, in minimal Humboldt University Berlin, Berlin, Germany. media wherein all carbon and nitrogen sources are isotopically labeled. The Microbial Rhodopsins are integral seven-transmembrane helix proteins, which isomerization of a covalently bound retinal is an integral part of both microbial covalently bind all-trans-Retinal as their light-sensitive chromophore. In nature and animal rhodopsin function. As such, the retinal binding pocket is of signif- they are utilized as sensors, enzymes, ion channels or ion pumps. Light-driven icant interest for ssNMR assignments. Unfortunately, the de novo organic syn- proton pumps compose one subfamily of light-driven ion pumps and transport thesis of an isotopically-labelled retinal is cost-prohibitive in large scale protons across the plasma membrane against an electrochemical gradient. expression. Previously, the biosynthesis of a retinal precursor, beta-carotene, Although the most prominent member of this group Bacteriorhodopsin (BR) has been introduced into many different organisms. This system can be was intensively studied for the past forty years, less was known about the elec- extended to the E. coli expression strains UT5600 and BL21. We have shown trophysiological characteristics of pumps in native membranes under that the novel biosynthetic production of an isotopically labelled retinal ligand controlled electrochemical conditions. The recent electrophysiological investi- concurrently with its apoprotein proteorhodopsin in E. coli presents a cost gation of the rhodopsin from the eukaryotic microalga Coccomyxa subellipsoi- effective alternative to de novo organic synthesis. By using alternately labeled dea (CsR) led to a more profound understanding of the pumping mechanism carbon sources (glycerol) we were able to verify the biosynthetic pathway for under electrochemical load. Replacement of Arg83 in CsR (Arg82 in BR) by retinal and assign several new carbon resonances for proteorhodopsin-bound Gln drastically reduced the power of the pump and caused outward directed retinal. pumping at neutral pH but inward directed passive currents at high load, which indicated a channel-like behavior. We were able to functionally express and pu- 1754-Pos Board B74 rify CsR-WT as well as R83Q from HEK293T cells and further characterize Exploring Complexes of Rhodopsin and Binding Partners in Virtual those using spectroscopic methods. We investigated the photochemistry of Reality CsR-WT and R83Q using laser-flash photolysis experiments. We identified a 1 1 2 2 Aidin R. Balo , Ned Van Eps , Aditya Dhoot , Merry Wang , strong pH dependency of the stationary absorption as well as on kinetic prop- 1,3 Oliver P. Ernst . erties in the CsR-R83Q mutant. These results are in agreement to findings for 1Biochemistry, University of Toronto, Toronto, ON, Canada, 2Bio/Nano 3 BR-R82Q mutant, where R82 is assumed to regulate the pKa of the group Group, Autodesk Research, Toronto, ON, Canada, Molecular Genetics, responsible for proton release, but these earlier results were not linked to the University of Toronto, Toronto, ON, Canada. occurrence of passive proton conductance. Finally through sequence alignment Protein structures are important for understanding biological processes on the with other microbial rhodopsins we were able to identify and partly charac- most fundamental level. However, communicating 3D protein structures us- terize more positions which show channel-like behavior, e.g. Y207, to get a ing 2D projections in publications or presentations does not deliver a com- more profound understanding of the relation between photochemistry and plete representation of the important structural components, especially to actual ion conductance. scientists working outside of structural biology, to students, and to a lay audience. 1757-Pos Board B77 We present our latest structural work characterizing complexes of rhodopsin, Construction of a GPR3 Homology Model Using Conformational the G protein-coupled photoreceptor in found in rod cells, and its binding part- Memories ners based on structures solved by crystallography or structural models derived Paula Morales, Dow P. Hurst, Patricia H. Reggio. from sparse structural data, such as DEER spectroscopy distance measure- Chemistry and Biochemistry, University of North Carolina at Greensboro, ments. In collaboration with Autodesk Research, we present in an accessible, Greensboro, NC, USA. immersive virtual reality environment to allow attendees to freely explore GPR3 is an orphan receptor which belongs to the Class A family of G-Protein the important, highlighted structural features to be discussed. The presentation Coupled Receptors. It shares high sequence similarity with GPR6, GPR12 (64 of pre-authored models generated in Autodesk’s web-based Molecule Viewer is and 61%), the lysophospholipid receptors S1P1 and LPA1 (34 and 33%), and accessed by entering a URL into a web browser on a smartphone. These struc- the cannabinoid receptors CB1 and CB2 (21 and 26%). GPR3 is predominantly tural models can be accessed and explored in virtual reality on any smartphone expressed in mammalian brain and oocytes and it is known as a Gas-coupled using provided cardboard virtual reality devices. receptor activated constitutively in cells.

BPJ 7839_7842 358a Tuesday, February 14, 2017

As demonstrated by different research groups, GPR3 represents a possible GxxxG-like motifs usually have a few dimerization modes with similar en- target for the treatment of different pathological conditions such as Alzheimer’s ergies. Proteins with Phe-rich sequences often show multiple helix association disease, oocyte maturation or neuropathic pain. However, the lack of potent and modes. The preferred modes of dimerization are frequently evolutionarily selective GPR3 ligands is delaying the exploitation of this promising therapeu- conserved (e.g. in cadherin, HLA histocompatibility antigen, receptor tyrosine tic target. In this context, we aim to develop a homology model which helps us kinase, and cytochrome P450 families), but they can also change even after a to elucidate the structural determinants governing ligand-receptor interactions. single mutation. For that purpose, we constructed a GPR3 model based on the crystallized S1P1 receptor structure (Hanson et al, Science 2012). Sequence divergences in trans- 1760-Pos Board B80 membrane helixes 1, 4, 6 and 7 have been explored using the Monte Carlo/ First Membrane-Bound, Atomic Level Structure of the Ternary Extrinsic simulated annealing program, Conformational Memories (Whitnell et al, J. Coagulation Complex Comput. Chem. 2007). Extracellular and intracellular loop geometries were Melanie Muller, Emad Tajkhorshid. calculated using Modeller v9.1. Energy minimizations of the resultant R and University of Illinois, Urbana, IL, USA. R* models were performed using the OPLS2005 all atom force field in Macro- Formation of the ternary extrinsic complex (Tissue Factor (TF): Factor VII model (Schrodinger, 2015). As in S1P1 receptor, the N-terminus of the GPR3 (FVII): Factor X (FX)) is is a critical step in blood clotting. Knowledge of spe- model displays a helical portion and short random coil region that covers the cific interactions important to the complex’s formation and to FX activation in top of the binding crevice. the complex, an event required for clot formation, could allow for development The development of the GPR3 active and inactive state models will enable the of targeted drugs for thrombosis and other coagulation disorders. Despite high rational design of novel GPR3 ligands which may serve as research tools for interest in characterizing the structure of the extrinsic complex, the challenging further understanding of the biological role of this orphan receptor. [Support: nature of the complex has prevented experimental studies from making prog- NIH KO5 DA021358 and NIH RO1 DA03934 (PHR)]. ress in this direction. The complex only forms on bilayers with specific anionic phospholipids, making experimental structure determination prohibitively 1758-Pos Board B78 difficult. We present the first atomic resolution structure of the ternary extrinsic Computational Analysis of OmpG Gating complex to include membrane interactions, developed using a novel methodol- Alan Perez-Rathke1, Christina Chisholm2, Min Chen3, Jie Liang1. ogy which combines ensemble protein-protein docking and extensive non- 1Bioengineering, University of Illinois at Chicago, Chicago, IL, USA, equilibrium molecular dynamics simulations. First, a membrane-bound model 2Chemistry, University of Massachusetts, Amherst, MA, USA, 3Chemistry, of FX was developed for the first time and simulated. This dynamic sampling of University of Massachussets, Amherst, MA, USA. the flexible FX protease allowed us to select 10 distinct poses for use in protein Outer membrane protein G (OmpG), a monomeric beta-barrel membrane pro- docking. The protein-protein docking program DOT2 was used to develop pro- tein, has the potential to be used as a nano-sensor device. To achieve this goal, tein only structures of FX bound to a structure of TF:FVII. Top docked struc- it is important to understand OmpG’s ion current conducting properties. Previ- tures were selected based on agreement with experimental information. ous studies have demonstrated pH-dependent gating, namely, current flow Specific geometrical aspects derived from the binding mode of the docked through the porin decreases as the pH decreases. While it is thought that structure were used to develop collective variables to drive, in biased all- loop regions have strong influence on the OmpG gating state, there is uncer- atom simulations, the process of complex formation in the presence of a mem- tainty on the mechanism that induces the functional switch as the pH is altered. brane. Using non-equilibrium simulations, binding between membrane-bound Based on the DiSGro method of protein loop modeling, we extensively sample structures of FX and TF:FVII was then slowly induced. The highly mobile the three-dimensional conformations of multiple interacting loop regions of membrane mimetic (HMMM) model, which replaces slow-moving lipid tails OmpG at various pH levels. Specifically, 100,000 wild type multi-loop sam- with detergent molecules, was used to allow rapid lipid dynamics and relaxa- ples are generated at both pH 5 and pH 7. From the 5,000 lowest energy con- tion during this deliberate complex formation. Our structure represents the first formations at each pH, we infer which ionizable residue interactions are model of the extrinsic complex to include the critical membrane interactions predictive of the gating state, as well as which interactions and residues are required for complex formation. consistently stabilizing or destabilizing towards a single state. Lastly, we investigate the role of solvent screening effects in relation to both the open 1761-Pos Board B81 and closed states. Determine the Substrate-Regulated TonB-Dependent Transporter and TonB Interactions 1759-Pos Board B79 Lishan Liu1, David Cafiso2. Proteome-Wide Modeling of Transmembrane Alpha-Helical Homodimers 1University of Virginia, Charlottesville, VA, USA, 2Chemistry, University of by TMDOCK Virginia, Charlottesville, VA, USA. Andrei L. Lomize, Irina D. Pogozheva. Gram negative bacteria utilize a family of specific, high affinity transport pro- College of Pharmacy, University of Michigan, Ann Arbor, MI, USA. teins to scavenge rare nutrients such as iron, vitamin B12 and certain carbohy- A novel energy-based method, TMDOCK, has been developed for ab initio drates from their environment. These active transport proteins are localized to modeling of 3D structures of parallel homodimers in membranes. Instead of the bacterial outer-membrane and obtain energy for transport from cytoplasm exploring the entire conformational space, the method employs a fast membrane proton motive force by coupling to the inner membrane protein template-driven global energy optimization procedure that combines the TonB. Although a number of high-resolution crystal structures have been ob- knowledge of geometrically feasible packing arrangements and local energy tained for TonB-dependent transport proteins (TBDT), the molecular mecha- minimization with a novel force field. Ranking and selection of optimal dimer- nism by which transport takes place still remains unknown. ization modes is based on the calculated free energy of a-helix association TonB is stoicheometrically limited with respect to the outer membrane (DGassoc) which includes van der Waals, hydrogen bonding and dipole- TDBT, and transport is thought to involve cycles of attachment and dissoci- dipole interactions between helices, side chain conformational entropy, and sol- ation of TonB from the outer-membrane transporter. Previous studies have vation energy in the anisotropic lipid environment. The fast version of the shown that the affinity of TonB for several transporters is enhanced by sub- TMDOCK method is available through the web server (http://membranome. strate binding. In both BtuB, the vitamin B12 transporter and FecA, the ferric org/tm_server.php). citrate transporter, binding of substrate induces conformational changes in TMDOCK successfully reproduced 26 experimental dimeric structures formed the energy-coupling segment termed Ton box. Upon substrate binding, Ton by transmembrane (TM) a-helices of 21 single-pass membrane proteins box of BtuB and FecA become less ordered and increase their exposure to (including 4 mutants) with Ca atom r.m.s.d. from 1.0 to 3.3 A˚ . Additional the periplasm. testing demonstrated consistency of calculated dimer structures with published Here, we studied the structure and dynamics of the FhuA Ton box in the pres- TOXCAT, mutagenesis, or other experimental data for more than 80 bitopic ence or absence of substrate utilizing both CW and pulsed EPR techniques in proteins. Subsequent application of the method to 6041 bitopic proteins from isolated native outer membrane. Unlike BtuB and FecA, the Ton box of six proteomes included in the Membranome database (membranome.org) FhuA is disordered with or without substrate. However, substrate binding shows that ~70% of the proteins tend to form stable dimers with DGassoc<-3 does change the position of Ton box relative to the barrel axis and appears to kcal/mol. Results of modeling indicate three different types of dimerization extend the Ton box further into the periplasm. The results suggest a mechanism behavior of TM a-helices. A unique dimerization mode is usually observed that could enhance TonB affinity for FhuA in the presence of substrate. for TM domains carrying a single GxxxG-like motif, interhelical disulfide Remarkably, each of these three homologous transporter, BtuB, FecA and bridges, or polar residues in the middle of a helix. Proteins with several FhuA appears to regulate TonB affinity by a different mechanism.

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Protein Assemblies II 1765-Pos Board B85 Improved EphA2 SAM:SHIP2 SAM Domain Association by Increasing the 1762-Pos Board B82 Solvation of the Proteins in Molecular Dynamics Simulation Constructing an in Silico Model of the Gram-Negative Cellular Envelope Zhenlu Li, Matthias Buck. Curtis Balusek, James C. Gumbart. Department of Physiology and Biophysics, Case Western Reserve University, Physics, Georgia Inst. of Technolgy, Atlanta, GA, USA. Cleveland, OH, USA. The cell envelope of Gram-negative bacteria varies from other living organisms Characterizing the features important for protein-protein interactions is an due to the presence of two lipidic (inner and outer) membranes and a cell wall important stepping stone for understanding the structure, dynamics and func- separating them. The periplasm, or region between the inner and outer mem- tion of complicated protein complexes. Theories of energy landscapes, confor- branes, is host to numerous proteins that facilitate cellular growth and regulation mation ensembles, binding sites/interfaces, as well as allosteric communication in the near absence of cellular energy. In order to construct an accurate model of networks in protein-protein interaction are being gradually established. Molec- the Gram-negative cell envelope, we have utilized numerous proteomics studies ular dynamics simulations have long been used and have been proven as a bene- of Escherichia coli and compiled a comprehensive list of associated proteins ficial tool in protein studies. However, computational studies of protein-protein ranked by abundance. The abundant proteins with crystal structures in each of association are still confronted with difficulties in sampling the whole energy the associated regions, inner membrane, periplasm, cell wall, and outer mem- landscape of a protein complex. The protein complex usually trapped into local brane, were then incorporated procedurally into an all-atom model of the minima with relaxation times that can be comparable or are often larger than Gram-negative cell envelope. Here we present the methods for constructing an computational power that is available. Moreover, an emerging viewpoint sug- accurate, large scale model of the cell envelope and further employ this model gests that especially for a soluble protein, the current all atom force field to determine novel properties of the Gram-negative cell envelope. possibly underestimates the solvation energy, and as a result, the protein- protein association will be overestimated. 1763-Pos Board B83 Our previous studies have unveiled the structure of EphA2 SAM: SHIP2 SAM Extending Upside, a Near-Atomic Level Model for Fast Protein Folding, complex [1], and showed that the complex samples multiple conformation states for Predicting Protein-Protein Interactions in a dynamic way [2], also extending to the observations of multiple dissociation Nabil F. Faruk1, John Jumper2, Benoit Roux3, Tobin R. Sosnick3. pathways [3]. In this study, we studied the association process of the SAM do- 1Biophysical Sciences, University of Chicago, Chicago, IL, USA, 2 3 mains of the EphA2 receptor and SHIP2 enzyme. By slightly amplifying the sol- Chemistry, University of Chicago, Chicago, IL, USA, Biochemistry and vation of polar protein sidechain groups with water, we found that the sampling Molecular Biology, University of Chicago, Chicago, IL, USA. of the energy landscape in SAM: SAM association is greatly enhanced without We have extended Upside, a near-atomic level model previously developed in applying any biasing energy potentials. We investigated the association process our group for fast folding of proteins in implicit solvent via Langevin dynamics, in detail by analyzing the encounter complex, the configurational transitions, the for predicting protein-protein interactions and binding affinities. Upside’s speed binding interface, as well as the variation in the estimated thermodynamic prop- arises from only explicitly accounting for the backbone N, Ca, and C atoms dur- erties. The study enhances our understanding of the binding process of SAM: ing the dynamics portion, while it infers the position of pendant groups represent- SAM protein and shed light on the protein association processes in general. ing side chains and propagates their forces onto the backbone. However, the reconstructed protein maintains detailed Rama maps, H-bonding, and side chain 1766-Pos Board B86 potentials, setting Upside apart from typical GG models. This provides a solid A Molecular Simulation Method to Predict the Self-Assembly of Helix foundation to study protein-protein interactions, and we have further improved Bundles in Biological Membranes Upside for this application with: 1) Monte Carlo jump moves to enhance diffu- Jingjing Huang1,2, Regis Pomes1,2. 1 2 sion of the individual protein chains, which benefits the Contrastive Divergence Biochemistry, University of Toronto, Toronto, ON, Canada, The Hospital parameter training methodology that we employ whereby decoys are generated for Sick Children, Toronto, ON, Canada. via simulation on the fly. 2) Retraining with a subset of protein-protein com- The association of helical trans-membrane (TM) segments defines the fold of plexes from a recent and popular benchmark set. We also discuss combining many integral membrane proteins. In addition, Membrane protein assembly Upside with all-atom molecular dynamics for further accuracy. via TM helix association is a key step in many cellular events. Although the structure of TM segments can be accurately predicted from the amino acid 1764-Pos Board B84 sequence using bioinformatic methods, the prediction of TM protein assembly A Structural View of the Dissociation of Escherichia Coli Tryptophanase and oligomerization state remains challenging. Here, we present a general, 1,2 1 1 1 Abraham H. Parola , Keren Green , Nasrin Qasem , Garik Gdalevsky , computationally-efficient simulation method for de novo prediction of the 1 3 4 4 Anna Kogan , Yehuda Goldgur , Ofra Lotan , Orna Almog . native oligomerization state and self-assembly of TM helix bundles. The 1Chemistry, Ben Gurion University of the Negev, Beer Sheva, Israel, 2 method is based on massively-repeated, all-atom molecular dynamics simula- Faculty of Arts and Sciences, New York Shanghai University, Shanghai, tions. To avoid sampling bottlenecks due to the high viscosity of lipid bilayers, China, 3Structural Biology Program, Memorial Sloan Kettering Cancer 4 we use a membrane mimetic consisting of a biphasic octane-water solvent. The Center, New York, NY, USA, Clinical Biochemistry and Pharmacology, approach is validated using a well-characterized tetrameric helix bundle, the Ben Gurion University of the Negev, Beer Sheva, Israel. 0 M2 proton channel from the influenza virus, as a model system. Unconstrained Tryptophanase (Trpase) is a pyridoxal 5 -phosphate (PLP)-dependent homote- simulations of multiple M2 helix monomers starting from arbitrary separations trameric enzyme which catalyzes the degradation of l-tryptophan. Trpase is and relative orientations lead to their spontaneous self-assembly into trimeric, known for its cold lability, i.e., a reversible loss of activity at low temperature tetrameric, or pentameric bundles. A systematic analysis of the structure and (2 C), associated with tetramer dissociation. Escherichia coli Trpase dissociates fluctuations of these helix bundles successfully identifies the correct oligomeric into dimers, while Proteus vulgaris Trpase dissociates into monomers. Accord- state (the tetramer) and the correct fold of the M2 channel. The average RMSD ingly, Trpase is an appropriate model to study the quaternary structure of pro- between this predicted fold and the NMR structure is 1.6 A˚ , underlining the ac- teins. We aimed at understanding the differences in the mode of dissociation curacy of the method. Furthermore, Competitive self-assembly simulations of between the E. coli and P. vulgaris Trpases. In particular, the effect of mutations mixed M2 and glycophorin A (GpA) helices yield the spontaneous formation of along the molecular axes of homotetrameric Trpase on its dissociation was stud- both M2 tetramer and GpA dimer, capturing the specificity of the self-assembly ied. Two groups of mutants of the E. coli enzyme were created to resemble the process. Calculations of the relative free energy of different oligomerzation amino-acid sequence of P. vulgaris Trpase. In one, residues 15 and 59 that are states and efforts to predict the open state of the M2 channel will also be pre- located along the molecular axis R (termed the noncatalytic axis) were mutated. sented. Taken together, these results show significant promise for the accurate The second group included a mutation at position 298, located along the molec- prediction of the structure of helical membrane proteins as well as and for eluci- ular axis Q (termed the catalytic axis). Replacing amino-acid residues along the dating the physico-chemical basis of TM helix assembly. R axis resulted in dissociation of the tetramers into monomers, similar to the P. vulgaris Trpase, while replacing amino-acid residues along the Q axis resulted 1767-Pos Board B87 in dissociation into dimers only. The crystal structure of the V59M mutant of pH Responsive Peptide Self-Assemblies: a Mechanism as old as Viruses E. coli Trpase was also determined in its apo form and was found to be similar Maite´ Paternostre1, Franck Artzner2. to that of the wild type. This study suggests that in E. coli Trpase hydrophobic 1Biophysics, CNRS, Gif-sur-Yvette, France, 2Institut of Physics of Rennes, interactions along the R axis hold the two monomers together more strongly, pre- CNRS, Rennes, France. venting the dissociation of the dimers into monomers. Mutation of position 298 External stimuli are powerful tools that naturally control protein assemblies and along the Q axis to a charged residue resulted in tetramers that are less suscep- functions. For example, during viral entry and exit changes in pH are known to tible to dissociation. The results indicate that dissociation of E. coli Trpase trigger large protein conformational changes. However, the molecular features into dimers occurs along the molecular Q axis. stabilizing the higher pH structures remain unclear. Here we elucidate the

BPJ 7843_7846 360a Tuesday, February 14, 2017 conformational change of a self-assembling peptide that forms either small or 1770-Pos Board B90 large nanotubes dependent on the pH. The sub-angstrom high-pH peptide struc- Structure Function Studies of a Novel Human TSH Beta Variant ture reveals a globular conformation stabilized through a strong histidine-serine Mihaly Mezei1, Ramkumarie Baliram2, Rejwan M. Ali3, Terry F. Davies4, H-bond and a tight histidine-aromatic packing. Lowering the pH induces histi- Rauf Latif4. dine protonation, disrupts these interactions and triggers a large change to an 1Pharmaceutical Sciences, Icahn School of Medicine at Mount Sinai, New extended b-sheet-based conformation. Re-visiting available structures of pro- York, NY, USA, 2Thyroid Research Unit and, IcahnSchool of Medicine at teins with pH-dependent conformations reveals both histidine-containing aro- Mount Sinai and the James J. Peters Veterans Affairs Medical Center, New matic pockets and histidine-serine proximity as key motifs in higher pH York, NY, USA, 3Pharmaceutical Sciences, St. Johns University, New York, structures. The mechanism discovered in this study may thus be generally NY, USA, 4Thyroid Research Unit, Icahn School of Medicine at Mount Sinai used by pH-dependent proteins and opens new prospects in the field of and the James J. Peters Veterans Affairs Medical Center, New York, nanomaterials. NY, USA. Thyroid stimulating hormone (TSH), a heteromeric (a/b) glycoprotein hor- 1768-Pos Board B88 mone binds to its receptor (TSHR) on the surface of thyrocytes and regulates Characterization of CHMP Polymers at the Mesoscale the production and secretion of thyroid hormones. Studies from our laboratory Maryam Alqabandi1, Nicola De Franceschi1, Nolwenn Miguet2, have shown that in addition to the heteromeric WT hormone there exists a Christophe Caillat2, Stephanie Mangenot1, Winfried Weissenhorn2, splice variant of the b subunit in mouse and human macrophages which is Patricia Bassereau1. 1 2 capable of binding to the TSH receptor and setting-off the receptor to produce UMR 168 Physico Chimie Curie, Curie Institute, Paris, France, Institut de G protein mediated signaling (cAMP). Molecular dynamics calculations were Biologie Structurale, Grenoble, France. performed on the complex of the TSHR ectodomain with the WT and splice- The ESCRT-III is an evolutionary conserved protein complex that mediates variant hormones. The analysis of the simulation highlights the binding modes membrane remodeling and scission in a variety of cellular contexts. The of the two hormone variants and the conformation preferences of the hormone ESCRT pathway has been extensively studied in vivo and reconstituted in vitro in the bound complexes. An additional simulation modeled the complex of the using yeast proteins. The consensus model emerging from these studies is that a follicle stimulating hormone receptor (FSHR) with the splice variant hormone þ sequential recruitment of two subcomplexes, formed by Vps20 Snf7 and to study the degree of selectivity against FSHR. Some of the analyses introduce þ Vps2 Vps24, results in membrane constriction and eventually scission. In novel ways of looking at simulation trajectories. Homo Sapiens, however, up to 17 ESCRT-III proteins exist, called Charged Multivesicular Body Protein (CHMP 1-7). Vps20 and Vps24 homologous are 1771-Pos Board B91 CHMP6 and CHMP3, respectively. Snf7 is present in three isoforms, namely Hijacking His-Tags to Make Functional Multi-Protein Complexes CHMP4A, B and C. There are two subunits sharing a relative high sequence Elizabeth R. Haglin1, Ariane Briegel2, Lynmarie K. Thompson1. homology with Vps2, called CHMP2A and CHMP2B. The increased number 1Department of Chemistry, University of Massachusetts Amherst, Amherst, of ESCRT-III subunits is paralleled by the functional diversification of the MA, USA, 2Institute of Biology Leiden, Leiden University, Leiden, complex. In Homo Sapiens it plays multiple roles in topologically equivalent Netherlands. membrane scission events: in nuclear envelope repair, plasma membrane Polyhistidine-tags have had a tremendous impact on biochemistry research by repair, nuclear envelope sealing after mitosis, cytokinesis, MVB formation enabling simple and robust protein purification. This technology uses the high and virus release. Several models have been proposed to explain how CHMPs affinity of the histidine imidazole for divalent metals such as Ni(II) or Zn(II) are spatially arranged in order to accomplish scission. On the contrary, the me- chelated to beads to separate the His-tagged protein from other impurities. chanical properties of ESCRT-III polymers have never been investigated so far. We have discovered that divalent metal binding to a His-tagged protein can We have characterized for the first time the mechanical properties of CHMP drive assembly of a functional multi-protein complex. Native-like complexes polymers at the mesoscale, providing strong experimental evidence indicating of the His-tagged cytoplasmic fragment of the aspartate chemoreceptor that modulation of membrane rigidity is an important function of CHMP poly- (CF4Q) with its cognate kinase CheA and adapter protein CheW can be assem- mers, and that this property plays an important role in the context of membrane bled in vitro, either by anchoring the chemoreceptor to Ni-NTA-functionalized scission. Moreover, we highlight the biological relevance of this property, pro- vesicles or by including a molecular crowding agent, PEG. These complexes posing a novel biological function for CHMP2. form extended hexagonal array structures, similar to the in vivo architecture. We have recently discovered that Ni(II) or Zn(II) can mediate similar array for- 1769-Pos Board B89 mation in the absence of vesicles or molecular crowding agents. Moreover, All Tubulins are Not the Same: Reversible Dissociation of AB-Tubulin deletion of the His tag prevents metal-mediated assembly: neither sedimenta- Dimers Differ Depending on the Source of Tubulin tion nor kinase activity are observed. We hypothesize the metals bind to the Felipe Montecinos-Franjola1, Sumit Chatuverdi2, Peter Schuck2, His-tag, leading to receptor dimer formation and ultimately array assembly. Dan L. Sackett1. 1 2 Metal-induced His-tag dimerization may prove a useful assembly method for NICHD-NIH, Bethesda, MD, USA, NIBIB-NIH, Bethesda, MD, USA. other multi-protein complexes. Microtubules are assembled from evolutionarily conserved a/b-tubulin dimers. Regulation of tubulin function involves expression of different isotypes of a- 1772-Pos Board B92 and b-tubulin and by posttranslational modifications (PTM) of the tubulin The Two Faces of Peptide Self-Assembly dimer. These two mechanisms are thought to modulate the heterogeneity of MinJun Lee, Young-Beom Jo, Jeseong Yoon, Seokmin Shin. tubulin dimers in the cytoplasm. We previously demonstrated the reversible Chemistry, Seoul National University, Seoul, Korea, Republic of. dissociation of mammalian brain tubulin dimers, by using state-of-the-art Self-assembly of peptide-based building blocks has been shown to be useful analytical ultracentrifugation and fluorescence spectroscopy methods, and in constructing artificial bionanostructures. Recently, a rational approach to measured the dissociation constant with unprecedented accuracy. However, construct b-barrel protein mimics from the self-assembly of peptide-based most studies of purified tubulin (including ours) used tubulin extracted from building blocks has been demonstrated. We performed MD simulations on brain tissue which is known to contain multiple isotypes of a- and b-tubulin the proposed nanoring structures formed by the self-assembly of b-sheet and also contain abundant PTM. Since we are able to measure tubulin dimer forming peptides. The stability of the nanoring structure with respect to dissociation with high accuracy we extended our studies by comparing the the size is investigated and several factors contributing to the stability are reversible dissociation of other tubulins extracted from brain and non-brain examined. We have also demonstrated that the sizes of nanorings can be sources. We obtained tubulin dimers from brains of non-mammalian animals controlled by introducing selective mutations. Formations of other toroidal containing multiple isotypes and high level of PTM, and also extracted tubulin nanostructures are also investigated by systematic simulations. Understanding dimers from other non-neural sources such as red blood cells, epithelial human strategies for rational designs of such structures will be discussed. Amyloid cell cultures and protozoan parasite cell cultures which are mostly single iso- formation, the formation of amyloid fibrils and prefibrilar aggregates of type with high or low levels of PTMs. We found that the reversible dissociation misfolded proteins and peptides, is very important to elucidate possible causes of tubulin dimers from these various sources are different from each other. for various neurodegenerative disorders. Computational studies can provide While tubulin dimers extracted from brain tissue readily dissociate in our ex- basic understanding concerning the detailed mechanism of self-assembly of periments, tubulin dimers obtained from some non-neural sources showed biomolecules. Systematic replica exchange molecular dynamics simulations tighter association without evidence of dissociation. Notably, human epithelial have been performed on the formation of the oligomers and protofibrils of cell tubulin dissociated as much as brain tubulin. These findings are novel since Ab peptide and peptides from a-synuclein. The mechanism of aggregation the tubulin dimer dissociation has never been determined before for such a va- and growth of these peptides is examined from detailed analysis of such riety of neural and non-neural tubulins. simulations. It was shown that the steric zipper contacts provided favorable

BPJ 7843_7846 Tuesday, February 14, 2017 361a interactions in the aggregation process and the structural flexibility might Intrinsically Disordered Proteins (IDP) and play important role for enhancing aggregation. Our results also suggested that amyloid formation is basically a hierarchical process and different Aggregates II topologies are favored in the different stages of aggregation. We proposed a new replica exchange scheme, Tq-REM, in which high temperature replicas 1775-Pos Board B95 in the conventional T-REM are reconstructed by q-replicas using effective Effect of Peptide Conjugates of Carboxylic Acids to the Aggregation of potentials with reduced barriers. It is expected that Tq-REM can provide use- Amylin ful tools to investigate systems where metastable states play very important Jayson Vedad1, Adam A. Profit2, Ruel Z.B. Desamero2. roles. 1Chemistry, Graduate Center, CUNY, New York, NY, USA, 2Chemistry, York College, CUNY, Jamaica, NY, USA. 1773-Pos Board B93 Human islet amyloid polypeptide (amylin or hIAPP) is the major protein Molecular Dynamics Studies of Non-Enveloped Virus Cell Entry component of amyloid fibrils in type 2 diabetes. In these insoluble fibers, the Mechanisms polypeptide exists in a structure known as the cross b motif in which extended Shivangi Nangia, Allyn R. Brice, Eric R. May. polypeptide chains run perpendicular to the long axis of the fibril in a parallel Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA. b-sheet conformation. Several small aromatic molecules and polyphenols are Cellular membranes are physical barriers that a virus must bypass, and the abil- known to serve as amyloid inhibitors and it has been postulated that many of ity of a virus to transport across these membranes is a critical stage of viral these compounds interact with their target polypeptide through p-stacking infection. Depending on the route of entry, a virus may face a barrier not interaction. Small peptide fragments derived from amylin also have been only at the exterior plasma membrane, but also inside the cell at the endosomal shown to serve as inhibitors of amyloid formation. The self-recognition ability membrane. There is a lack of understanding of the mechanisms employed by of these peptides directs them to corresponding regions of the target protein non-enveloped (membrane lacking) viruses to cross biological membranes. where they can exert their inhibitory effects. We prepared peptide conjugates Through molecular modeling methods we have investigated the model system with self-recognition elements designed to prevent self-assembly through a Flock House virus (FHV) by computing pathways for externalization of the charge repulsion mechanism by introducing benzene carboxylic acid groups membrane lytic gamma-peptides. We have found that changing protonation to hIAPP22-29 and found interesting trends with conjugates either serving as states of only a few residues in the FHV capsid leads to a disruption of the agonists or antagonist to amylin aggregation which can potentially pave new capsid collective dynamics, which in turn makes externalization of the peptides insights on to the development of newer aggregation inhibitors. a thermodynamically more favorable process. We go on to examine the inter- 1776-Pos Board B96 action of the gamma-peptides with lipid bilayers. These simulation studies are Aggregation Induced Conformation Changes Determine Amylin Mem- aimed at characterizing the membrane binding, folding and insertion properties brane Affinity of these peptides. We further examine the influence of lipid composition on the Barun K. Maity, Anoop Rawat, Bappaditya Chandra, Anirban Das, structural and energetic properties of these peptides and compare to previous Sudipta Maiti. experimental studies. The methods employed in this work involve a multi- Department of Chemical Science, Tata Institute of Fundamental Research, scale approach utilizing all-atom and coarse-grained models as well as Mumbai, India. enhanced sampling replica exchange methods. Amylin is a 37 residue intrinsically disordered protein whose aggregation is associated with Type II diabetes. For most amyloid aggregates, the intermedi- 1774-Pos Board B94 ate aggregations states (‘‘oligomers’’) are supposed to be more toxic than the Artificial Peptide-Based Membranes and their Interaction with Lipid mature fibrillar state, and interaction with the cell membrane is an important Systems component of the toxicity. It is likely that the origin of amylin toxicity is linked 1 1 2 3 Karin Kornmueller , Bernhard Lehofer , Gerd Leitinger , Heinz Amenitsch , to the conformational changes which occur as the peptide undergoes the aggre- Ruth Prassl1. 1 2 gation process. Here we probe the membrane affinity and the conformation of Institute of Biophysics, Medical University of Graz, Graz, Austria, Institute the peptide as a function of its aggregation state. We find that the affinity of the of Cell Biology, Histology and Embryology, Research Unit Electron peptide artificial lipid bilayer membranes is x times higher in the small oligo- Microscopic Techniques, Medical University of Graz, Graz, Austria, 3 meric state compared to the monomeric state. FRET studies show that the Institute of Inorganic Chemistry, Graz University of Technology, Graz, monomeric state is mostly disordered, while the oligomeric state has a smaller Austria. end to end distance, indicating folding of the protein. Raman and Infrared spec- Lamellar lipid structures are the fundamental structural motifs of biological troscopy of the oligomer shows a considerable presence of alpha helical confor- membranes. To mimic this feature we have engineered short amphiphilic mation, together with some beta sheet structure. The oligomers further develop designer peptides with the aim to create peptide-based membrane analogs. into larger aggregates, with mostly beta sheet character. We infer that the alpha As peptide scaffolds we have chosen six derivatives of V4WD2 - a peptide helical intermediate is primarily responsible for membrane binding. This is in composed of four hydrophobic valine residues, followed by tryptophan and contrast to amyloid beta, a similar sized peptide whose binding to the cell mem- two negatively charged hydrophilic aspartic acid residues. The hydrophile- brane is reported to be mediated by small oligomers containing an antiparallel lipophile ratio, tryptophan content, and level of hydrophobicity have been sys- beta-sheet conformation. Thus different disease causing protein oligomers tematically varied. Four peptide candidates readily self-assembled into interact with lipid membranes through different routes. membrane-like structures in aqueous solution: small angle X-ray scattering (SAXS) patterns revealed quasi Bragg peaks, which indicate a long range 1777-Pos Board B97 order with a lamellar repeat distance of ~4-5 nm. Transmission electron mi- Roles of Site-Specific Deamidation in Islet Amyloid Polypeptide Self- croscopy (TEM) images confirmed the presence of stacked sheets. ATR- Assembly and Toxicity Site-Specific Deamidation in Islet Amyloid Poly- FTIR spectroscopy suggested a parallel internal arrangement of the peptide peptide Self-Assembly and Cytotoxicity monomers. Two peptide derivatives (V3D and V4D2) remained monomeric Phuong Trang Nguyen, Ximena Zottig, Steve Bourgault. or as loose aggregates dispersed in solution; one peptide formed cylindrical Universite´ du Que´bec a` Montreal, Montreal, QC, Canada. structures. Asparagine deamidation is a spontaneous non-enzymatic post-translational To explore how these peptide systems respond when mixed with lipids, they modification resulting in the conversion of a asparagine to a mixture of aspartic were interfaced with dipalmitoyl phosphatidylcholine. At low concentrations acid (Asp) and iso-aspartic acid (iso-Asp). In alkaline condition, Asp/isoAsp is all peptides induced the formation of a heterogeneous mixture of vesicles: large deprotonated and could alter to the protein net charge. Otherwise, this chemical multilamellar vesicles (d-spacing ~6.3 nm) coexisted with oligo- or unilamellar conversion is known to influence the structure and the stability of protein and can vesicles (~50 nm in diameter) and a considerable fraction of bicelle-like struc- ultimately lead to protein misfolding and aggregation, two hallmark events in tures (~45 nm in length, ~18 nm in width). At high peptide-to-lipid molar ratios diverse amyloid related diseases including Alzheimer’s disease and diabetes (1/5) supramolecular peptide structures coexisted with highly uniform lipid- mellitus type 2 (DM-2). The aim of this study was to examine the effects of peptide mixed unilamellar vesicles (diameter ~260-280 nm). By SAXS a site-specific asparagine deamidation on the amyloidogenecity and the cytotox- headgroup-to-headgroup distance of dHH~3.6 nm was found at T=323K, while icity of the peptide Islet Amyloid Polypeptide (IAPP). IAPP is a natively disor- an interdigitated phase (dHH~3.0 nm) was induced at T=298K. dered 37-residue peptidic hormone that is co-expressed and co-secreted with Our results highlight the versatility of self-assembled peptide nanostructures insulin by pancreatic islet beta-cells. Its deposition as insoluble amyloid aggre- and indicate that subtle changes in the amino acid composition are key design gates correlates proportionally to the progression of DM-2. The kinetics of IAPP elements for both, the formation of artificial lamellar peptide membranes and amyloid formation and its post-translationally deamidated analogs at specific mixed lipid-peptide membrane systems. asparagine position were evaluated by a combination of Thioflavin T (ThT)

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fluorescence assay and transmission electron miscroscopy (TEM). We observed 1780-Pos Board B100 that site-specific asparagine deamidation to aspartic acid accelerates amyloid Comparison of Stabilities of Fibrils of the Amyloid-Beta Peptides formation, whereas asparagine deamidation to isoaspatic acid perseveres aggre- Timir Sil, Kanchan Garai. gation. The process of fibrillogenesis in absence or in presence of deamidation TIFR Centre for Interdisciplinary Sciences, Hyderabad, India. modifications was also monitored by circular dichroism (CD) spectroscopy Amyloid fibrils are known to be highly stable with respect to heat and chemical and we observed that the conformational transition disordered-to-beta-sheet denaturation. However the origin of the stability of amyloid fibrils are poorly associated with IAPP/IAPPs deamidated assembly was modulated. Using rat understood. We have investigated the stability of the amyloid fibrils of tetrame- pancreatic beta-cells, we observed that deamidation reaction does not prevent thylrhodamine labelled amyloid beta (TMR-abeta) fibrils which are fluores- IAPP-induced toxicity. These results show that site-specific asparagine deami- cently dark due to quenching of TMR fluorescence in self-assembled forms dation could potentially play a key role in the etiology of DM-2 by promoting (Garai and Frieden, PNAS, 2013). Hence we have used TMR fluorescence to IAPP amyloidogenic cascade and/or by stabilizing the resulting amyloid fibrils. measure the monomer-fibril equilibrium of TMR-abeta at various concentra- tions of urea and GdnCl. Our results indicate both kinetics and thermodynamics 1778-Pos Board B98 of dissociation of TMR-abeta fibrils is dependent on the concentration of the Strain-Specific Propagation by an Amyloid-Beta Dodecamer denaturants. The denaturation curves are independent of abeta concentrations 1 2 3 1 Dexter N. Dean , Pradipta K. Das , Pratip Rana , Ryan P. Campbell , indicating that folding-unfolding of the monomer being determining event of 3 2 1 Preetam Ghosh , Sarah E. Morgan , Vijay Rangachari . the dissociation of the fibrils. The data can be fit using a two-state model. 1Department of Chemistry and Biochemistry, University of Southern 2 The deltaG and the ‘‘m’’ values of unfolding for both abeta40 and abeta42 Mississippi, Hattiesburg, MS, USA, School of Polymers and High are measured to be about 4 kT and 1.4 kT respectively. In conclusion, we Performance Materials, University of Southern Mississippi, Hattiesburg, MS, 3 have established a protocol for measurement of stability of the amyloid fibrils, USA, Department of Computer Science, Virginia Commonwealth however, stability of the amyloid fibrils may not be correlated with the aggre- University, Richmond, VA, USA. gation propensity of amyloid peptides. Soluble oligomers of the amyloid-b (Ab) peptide have emerged as the primary neurotoxic agents in Alzheimer disease (AD). Increasing evidence implicates 1781-Pos Board B101 aggregates of Ab to propagate towards fibrils in a strain-specific manner. Morpho- Regulation of Bace1 Mrna Expression in Alzheimer’S Disease by Green logical differences observed within fibrils are known to arise due to micro- Tea Catechins and Black Tea Theaflavins structural differences among the smaller aggregates that form the fibril building Ryan J. Geiser, Shelby E. Chastain, Melissa A. Moss. blocks. Furthermore, polymorphic fibrils correlate with the clinically observed University of South Carolina, Columbia, SC, USA. phenotypes in AD pathology, cementing the idea that conformational strains of Every 67 seconds, someone in America develops Alzheimer’s disease (AD). oligomers may have significance in phenotypic outcomes. Given the existing Among the top 10 causes of death, AD is the only disease that cannot be cured, diversity among Ab oligomers, it becomes imperative to determine the ability treated, or prevented. The amyloid cascade hypothesis states that amyloid-b of various oligomeric strains to faithfully propagate their structure. However, (Ab) protein follows a nucleation-dependent pathway to form aggregates that dearth in a molecular understanding of Ab oligomers has confounded the insights induce nerve damage and deterioration. Ab monomers originate from the pro- into such a mechanism. We have previously established that a distinct dodecamer teolytic cleavage of the amyloid precursor protein (APP) via sequential b-sec- of Ab42, called large fatty acid-derived oligomers (LFAOs), are formed as an retase-1 (BACE1) and g-secretase cleavage. In vitro models show that the off-pathway species under interfacial conditions. Here we demonstrate that at presence of Ab leads to an upregulation of BACE1 mRNA expression, result- high concentrations, LFAOs seed Ab towards fibrils containing distinct morpho- ing in a feed-forward mechanism for the amyloidogenic cleavage of APP. logical features different from those generated in the absence of the seed. More Epidemiological studies have correlated diets high in green tea catechins and importantly, the fibril structure seems to contain units of parent LFAO seeds, black tea theaflavins with a reduced incidence of AD. indicating a biophysically faithful propagation of the seed. The observations Previous studies in our lab demonstrated the varying inhibitory capabilities of reported here may have profound significance in deciphering the emerging catechins (epicatechin, epigallocatechin, epigallocatechin gallate) and theafla- roles of Ab oligomer phenotypes in prion-like propagation and dissemination vins (theaflavin, theaflavin monogallate) towards Ab oligomerization, the most of toxicity in AD. toxic Ab aggregate. This study further characterized these changes in oligomer 1779-Pos Board B99 formation as well as the antioxidant capabilities of these compounds and sub- Building Bigger: Modeling Large-Scale Protein Interactions in Amyloidosis sequently utilized SH-SY5Y neuroblastoma cells to determine whether cate- Michael Bergman. chins and theaflavins could reduce Ab-induced upregulation of BACE1 Biochemistry & Molecular Biology, Mayo Clinic Rochester, Lake Geneva, mRNA expression. While catechins had little effect on oligomerization, thea- WI, USA. flavins altered both the size distribution and conformation of oligomers. Aggregation of mutant immunoglobulin light chains leads to a disease called When SH-SY5Y cells were treated with oligomers made in the presence of light chain (AL) amyloidosis. Studies have been done to characterize the bio- compounds, epicatechin and epigallocatechin reduced BACE1 expression rela- physical properties of these amyloid proteins; AL-09 is one of the most char- tive to treatment with native Ab oligomers. When SH-SY5Y cells were treated acterized. When compared to its non-mutated counterpart, I O18/O8, AL-09 instead with native oligomers and antioxidant-capable concentrations of com- fibrils present different levels of clustering (using EM imaging). Structural in- pounds, both epicatechin and theaflavin reduced Ab-induced BACE1 expres- formation about the oligomeric and pre-fibrillary species could hold insight into sion. These results indicate that the antioxidant characteristic of catechins the aggregation mechanisms. Differences could be resultant of the different and theaflavins may be more essential in down regulating BACE1 mRNA dimeric conformations. Previous experiments have analyzed the contribution expression than their capability to inhibit Ab oligomerization. of mutations in AL-09. Y87H was identified as having the most amyloidogenic effect, followed by N34I. Through computational methods, we investigated the 1782-Pos Board B102 structural possibilities behind the changes in dimeric interface and the mecha- Secondary Structure Flipping Connected to Salt-Bridge Formation nism for fibril formation. Converts Toxic Amyloid-b40 Oligomers to Fibrils UCSF Chimera was used for most of the molecular modeling and structural Bappaditya Chandra1, Debanjan Bhowmik1, Barun Kumar Maity1, analysis. This includes monomers, dimers, tetramers, and hexamers of both I Kaustubh R. Mote2, Debabrata Dhara2, Kallol Bera1, Anirban Das1, O18/O8 VL and AL09 VL. The ZDOCK server was used to predict interactions Ravindra Venkatramani1, Sudipta Maiti1, Perunthiruthy K. Madhu2. between dimeric units. Molecular Flipbook was used to model possible aggre- 1Chemical Science, Tata Institute of Fundamental Research, Mumbai, India, gation pathways, using the predictions from ZDOCK, and visually explore how 2Chemical Science, TIFR Centre for Interdisciplinary Sciences, Hyderabad, secondary nucleation may be contributing to fibril formation. India. Y87 is normally buried in the I O18/O8 VL dimer interface, but the mutated res- Amyloid beta (Ab) aggregation is the likely initiator of Alzheimer’s disease idue, H87, interferes and may cause the altered dimeric interface. This confor- (AD), but a molecular level understanding of this process is still lacking. A mational change creates a groove on the AL09 VL dimer, which ZDOCK complicating factor is that aggregation intermediates may be more neurotoxic predicts is the most likely site for protein-protein interaction. Several exposed than the aggregates, but they are difficult to characterize due to their transient tyrosine residues and the mutant I34 lie along this groove, likely contributing to nature. Major structural features (dominated by b-sheets) appear to remain aggregation. Other differences include: residue interactions, biophysical prop- conserved from small oligomers to fibrils, providing no specific answers to erties, and fibril formation mechanisms. Modeled decamers of both AL09 VL the puzzle. Single mutations in Ab linked to early onset AD can potentially pro- and I O18/O8 VL reveal structural patterns that can be compared to lab- vide some clues, but are yet to do so. Here, using optical and NMR techniques, formed fibrils. In larger-scale models (60-mers or bigger), these structures and flash-freezing the specimen at different stages of aggregation, we find that can even be compared to EM images. the apparently conserved b-sheet character1 hides a major secondary structure

BPJ 7843_7846 Tuesday, February 14, 2017 363a transition. The small oligomers have an intramolecular anti-parallel b-sheet 1785-Pos Board B105 structure containing a beta turn (consistent with a porin like architecture). Alzheimer’s Protective Cross-Interaction Between Wild-Type and A2T We have confirmed the presence of intramolecular hydrogen bonding in small Variants Changes Ab42 Dimer Structure oligomers by measuring the backbone distance between two arms of the peptide Payel Das1, Anita Chacko1, Georges Belfort2. with REDOR experiments. This hydrogen-bonding network flips to give rise to 1IBM T J Watson Research Center, Yorktown Heights, NY, USA, 2RPI, the well-known intermolecular in-register parallel beta sheet structure in the Troy, NY, USA. mature fibrils. This transition is connected to, and possibly driven by the forma- Whole genome sequencing has recently revealed the protective effect of a sin- tion of a salt bridge between residues D23 and K28. Molecular dynamics sim- gle A2T mutation in heterozygous carriers against Alzheimer’s disease (AD) ulations also show a strong correlation between changes in the D23-K28 and age-related cognitive decline. The impact of the protective cross- distance and the evolution of the secondary structure. This is significant in interaction between the wild-type (WT) and A2T variants on the dimer struc- the context of a clear linkage between early onset AD and mutations in the ture is therefore of high interest, as the Ab dimers are smallest known salt bridge region. We have further investigate the Ab conformation in mem- neurotoxic species. Toward this goal, extensive atomistic replica exchange brane bound form with the help of SERS and ssNMR2. Overall, our studies un- molecular dynamics simulations of the solvated WT homo- and A2T hetero- cover a comprehensive set of structural changes with signatures spanning from Ab1-42 dimers have been performed, resulting into a total of 51 ms of sampling the atomic to the mesoscopic scales, and suggest mechanisms which likely for each system. Simulated heterodimer reveals a striking lowering of transient determine the putative toxic to non-toxic transition in Ab. interactions formed between central and C-terminal hydrophobic regions, when 1. Sarkar et al. Angew. Chem. Int. Ed. 2014, 53, 6888–6892. compared to the WT homodimer. Interestingly, the A2T N-terminus forms 2. Bhowmik et al. ACS Nano, 2015, 9, 9070. intra- and inter-peptide contacts with the central and C-terminal residues. This atypical involvement of the N-terminus within A2T heterodimer revealed 1783-Pos Board B103 in our simulations implies possible interference on Ab42 aggregation and toxic Effect of a Paramagnetic Spin Label on Amyloid-Beta Structural oligomer formation, which is consistent with experiments. In conclusion, the Ensemble present study provides detailed structural insights onto the AD protective effect 1 1 2 Sukanya Sasmal , James Lincoff , Teresa Head-Gordon . of the A2T mutation in the heterozygous state. 1Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA, 2Department of Chemistry, 1786-Pos Board B106 Bioengineering, Chemical and Biomolecular Engineering, University of Sequence Specific Quantitative Hydroxyl Radical Footprinting Reveals California, Berkeley, Berkeley, CA, USA. Structural Details of Amyloid-ß (1-42) Peptide Oligomerization Paramagnetic relaxation enhancement (PRE) experiments are a widely used Janna Kiselar1, Andrew Nix2, Anant Paravastu3, Rosenberry L. Terrone2, NMR technique, probing into the long-range dynamics of proteins. In this Alexandra L. Klinger4. study, we examine the usefulness of such experiments for characterizing the 1Case Western Reserve University, Cleveland, OH, USA, 2Mayo Clinic, structural ensembles of intrinsically disordered proteins (IDPs). We compare Jacksonville, FL, USA, 3Georgia Institute of Technology, Atlanta, GA, USA, different ensemble-averaged structural properties of the computationally gener- 4DecipherBio, Philadelphia, PA, USA. ated structural ensembles of the IDP amyloid-b42 (Ab42) to the tagged Ab42 Synchrotron based hydroxyl radical footprinting (HRF) was employed to study peptide in which a nitroxide spin label has been attached to a cysteine intro- the formation of Ab oligomers, believed to be the neurotoxic species in Alz- duced at its N-terminus. We find that the tagged peptide induces a strong pop- 42 heimer’s disease. Rates of oxidative modification for seventeen individual res- ulation shift in a subset of the original Ab42 structural sub-populations. idues (F4, Y10, H13, H14, L17, F19, F20, V24, K28, I31, I32, M35, V36, V39, Through back-calculation of NMR observables from the two structural ensem- V40, I41) were determined for monomer, 2-4mer, and oligomer Ab relative bles, differences would be evident in local residual dipolar couplings, and 42 1 15 to intrinsic rates of modification determined for the same residues in fragment possibly differences in heteronuclear H N NOEs if the paramagnetic tag peptide. The ratio of measured and intrinsic rates reveals protection from hy- is quenched in the real experiments. We conclude that PRE experiments can droxyl radical attack due to reduced side chain solvent accessibility for struc- be used to study untagged IDP ensembles only when aided by additional exper- tured Ab . Protection in the intrinsically disordered monomer peptide is iments and molecular simulations during the ensemble characterization. 42 consistent with transient C-terminal ß-strand formation and partial folding in 1784-Pos Board B104 the hydrophobic core region. Further, HRF data are also consistent with pub- Disordered Amyloid-BETA Protein Probed by Single-Molecule Fret lished solid state NMR work showing C-terminal antiparallel ß-sheet formation Fanjie Meng, Jae-Yeol Kim, Hoi Sung Chung. in the Aß42 oligomer. Implications with respect to recent atomic-resolution Laboratory of Chemical Physics, National Institute of Diabetes and Digestive structures of Ab42 fibrils will also be discussed. and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA. Alzheimer’s disease is a neurological disorder characterized by extracellular ag- 1787-Pos Board B107 gregation of amyloid-b protein (Ab) 40/42 and tau. Solid-state NMR studies have Membrane Bilayers Help to Stabilize and are Affected by Ab-Fibrils on shown that the fibril structures of Ab40 and Ab42 are different and they exhibit the Surface: A Molecular Dynamics Study polymorphism depending on the aggregation seed. Given difficulties arising from Sachin R. Natesh1, Karl F. Freed2, Esmael J. Haddadian1. the intrinsic heterogeneity and limitation of workable concentrations, however, 1Biological Sciences Collegiate Division, University of Chicago, Chicago, experimental characterizations of monomer and small soluble oligomers have IL, USA, 2Department of Chemistry and James Frank Institute, The been difficult. In this study we exploited the power of single-molecule FRET University of Chicago, Chicago, IL, USA. to characterize the monomer conformations of Ab40 and Ab42 at room temper- Alzheimer’s Disease (AD) is an important and increasingly prevalent neurode- ature. Purified proteins from bacteria contain biotin for immobilization and generative disease. Aggregation of Ab-peptides is important in etiology of AD unnatural amino acid (4-acytelphenylalanine) and cysteine at the N- and and the interaction of these peptides with cell membranes is probably one of the C-termini for site-specific labeling of Alex488 and Alexa647. In the urea dena- key pathological events responsible for neuron cell death in AD. Using the su- turation experiment, only a single FRET peak was observed in all denaturant con- percomputer Anton, we modeled the interaction of unstructured Ab-monomers centrations and the mean FRET efficiency gradually decreased with increasing and Ab-peptides in the fibril structure (residues 1-40, PDB ID 2M4J, based on denaturant concentration,suggesting proteins are unfolded even at the native con- an AD brain-seeded fibril structure) with different lipid bilayers (POPC/Choles- dition. The donor-acceptor cross-correlation analysis of the single-molecule tra- terol, ratio 4:1; POPC/POPG/Cholesterol, 1:1:1; POPC/POPE/POPG 1:1:1). jectories obtained from immobilized molecules and the maximum likelihood Our 20 m-second long simulations starting with unstructured Ab-monomers analysis of photon trajectories showed no evidence of significant dynamics other close to the lipid surface showed that the bilayer greatly accelerated the rate than acceptor blinking on the time scale between microsecond to millisecond. of structural conversion from a random coil to a b-structure as well as the ag- However, the two-dimensional (2D) FRET efficiency-donor lifetime analysis gregation of Ab from monomers to interacting oligomers in comparison to our (Chung, Louis, Gopich. JPhysChemB, 2016) showed that the 2D distributions bulk simulations. This was the case regardless of the lipid composition. The are shifted upward from the diagonal, which results from large distance fluctua- six-layer brain-seeded fibril on the lipid surface partially unraveled, mainly, tions between the donor and acceptor dyes. These results strongly suggest that at its end facing away from the lipid; however, it maintained its overall shape both Ab40 and Ab42 are intrinsically disordered with the conformational fluctu- and matched the structural stability of an infinitely long fibril model that we ations on the nanosecond time-scale, which have also been observed for a number previously simulated. The brain-seeded fibril structure interacted strongly of intrinsically-disordered proteins by Schuler and coworkers. The similar mean with lipid head-group atoms and constrained the motion of the lipid molecules FRET efficiencies of Ab40 and Ab42 also suggest the similar dimension of both under the structure; the diffusion of these molecules was reduced by more than unfolded proteins, consistent with the recent solution NMR study by Bax and 40%. Our simulations suggest that Ab fibrils change the fluidity of the lipid coworkers (Roche el al. Biochemistry, 2016). bilayers and therefore may affect cellular function.

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1788-Pos Board B108 of Pennsylvania, Philadelphia, PA, USA, 4Department of Physics, Drexel Probing the Mechanism of Glycosaminoglycan-Mediated Amyloid Assem- University, Philadelphia, PA, USA. bly with a Non-Amyloidogenic Peptide Model Formation of oligomers of b-amyloid (Ab) peptides is a pivotal initial event Mathew Sebastiao1, Isabelle Marcotte1, Steve Bourgault1,2. associated with the pathogenicity of Alzheimer’s disease (AD). Using the 1Chemistry, Universite´ du Que´bec a` Montre´al, Montreal, QC, Canada, single-molecule fluorescence resonance energy transfer (FRET) microscopy 2Quebec Network for Research on Protein Function, Structure, and technique, we have detected the very beginning of the formation of Ab peptide Engineering (PROTEO), Quebec, QC, Canada. oligomers in physiological solutions. The existence of oligomers is identified Over the last two decades, numerous studies have reported that glycosamino- from the FRET between dyes individually bonded to the N-terminus of the glycans (GAGs) accelerate amyloid assembly of peptides and proteins whose 40-unit peptides. Several dimers and oligomers identified by the varying aggregation is associated with amyloid-related diseases. GAGs are long, linear, FRET efficiency have been detected. The dimer with the highest stability is unbranched polysaccharides that are abundant at the cell surface and in the characterized to have a distance of 43 A˚ between its two N-termini, in an extracellular matrix. These sulfated polysaccharides have been associated anti-parallel structure that is similar to that of the dimer unit (anti-parallel with virtually all amyloid extracts analyzed from patients afflicted with protein hairpin) that has been previously identified in the fibrils. misfolding diseases. Strikingly, GAGs not only mediate amyloid assembly of aggregation-prone polypeptides but also the fibrilisation of numerous non- 1791-Pos Board B111 amyloidogenic protein. The exact mechanism by which GAGs favor peptide Statistical Thermodynamic Modeling of Early Ab Oligomer Formation self-assembly, however, is still a matter of active debate. In this context, we Nicholas P. van der Munnik1, Tao Wei2, Melissa A. Moss1, Mark J. Uline1. 1Chemical Engineering, University of South Carolina, Columbia, SC, USA, used a non-amyloidogenic and highly soluble peptide, PACAP27, as a model 2 to investigate this mechanism. PACAP27 is a 27-residue neurohormone that Chemical Engineering, Lamar University, Beaumont, TX, USA. is non-pathogenic, stable in solution, and does not aggregate. In presence of Alzheimer’s disease (AD) is the most common neurodegenerative disease. A low molecular weight heparin (LMWH) however, it readily forms amyloid- mounting body of research implicates amyloid-b (Ab) oligomers as an etiolog- like fibrils. Interestingly, PACAP27 rapidly adopts an a-helical structure ical factor in AD. However, the exact structure and stability of these species re- upon binding to sulfated GAGs and this conformation can be observed during mains unclear. We have implemented an approach that uses Ab conformations the lag phase of the amyloid reaction. After a prolonged incubation, secondary- obtained from replica exchange molecular dynamics (REMD) simulations in structural transitions into a b-sheet-rich conformation are observed. The amy- conjunction with a statistical thermodynamic model to derive thermodynamic loid nature of these assemblies were confirmed by atomic force microscopy, information on Ab oligomer formation. Fully atomistic REMD simulations of transmission electron microscopy, circular dichroism spectroscopy and Ab were performed using Gromacs, the Charmm 36 force field and the TIP3P thioflavin-T fluorescence. In order to study whether a-helical structures are water model. An Ab monomer was simulated using REMD at two different ionic essential to GAG-mediated amyloidogenic pathways, two synthetic variants strengths. Additionally, an Ab dimer was simulated at room temperature and with reduced helical folding propensity were designed. Conformationally physiological ionic strength. These simulations were used to derive an ensemble restricted peptides were able to form amyloid fibrils despite increased resis- of conformations for both monomers in isolation and during the binding process. tance to a-helix formation. Lag phases from aggregation kinetics were observ- A self-consistent field theory has been developed to model the thermodynamics ably reduced in both variants, suggesting that the formation of helical structures of interacting Ab molecules. The theory consists of formulating the appropriate in the presence of GAGs is not an obligatory step in the mechanism of amyloid thermodynamic potential for Ab molecules in a bath of solvent and ionic species fibril formation. in terms of the energy and entropy associated with all species. In this study, the centers of mass of three Ab monomers were assigned to positions in space, and 1789-Pos Board B109 the model was used to calculate the properties of equilibrium under those con- Probing the Binding of Ab Peptides to Lipid Bilayers straints. This procedure was repeated over a range of relative positions to eluci- Christopher Lockhart, Dmitri K. Klimov. date the free energy landscape of the trimer formation process. This theory School of Systems Biology, George Mason University, Manassas, VA, USA. provides a rich description of the molecular organization and physics that guide Alzheimer’s disease pathogenesis has been related to the accumulation of Ab Ab interactions that cannot be determined experimentally. The unique flexibility peptides on cellular membranes. In this work, we investigate the interactions of this theoretical framework to treat solution conditions and systems of arbitrary of Ab peptides with zwitterionic DMPC bilayers in the absence or presence geometries makes this a promising method to both further fundamental under- of calcium salt and with an anionic DMPS bilayer using isobaric-isothermal standing of Ab oligomerization and serve as an inhibitor design tool. all-atom explicit-solvent replica-exchange molecular dynamics simulations. Importantly, these simulation systems probe the thermodynamic binding of 1792-Pos Board B112 Ab peptides to lipid bilayers and provide a detailed characterization of Ab Strain Specific Propagation of an Amyloid Beta Oligomer in Alzheimer conformational ensembles. We have found that both zwitterionic and anionic Disease bilayers lead to a promotion of Ab helical conformations, although the anionic Vijay Rangachari. DMPS bilayer produces less helix than zwitterionic bilayers. When Ab binds to University of Southern Mississippi, Hattiesburg, MS, USA. the anionic bilayer, it forms fewer intrapeptide contacts and is subsequently Low-molecular weight oligomers of amyloid-b (Ab) have now been under- more expanded than when bound to zwitterionic DMPC bilayers. Ab penetrates stood as the primary neurotoxic agents responsible for synaptic dysfunction into the bilayer hydrophobic core of zwitterionic bilayers in the absence or and neuronal abnormalities in Alzheimer disease (AD) patients. Alongside presence of calcium salt; however, in the latter system, binding is enhanced cellular toxicity, aggregates of Ab are also involved in proliferation and due in part to electrostatic cross-bridging of anionic amino acids, calcium spreading of toxicity, the mechanism of which remains unclear, especially ions, and negatively-charged lipid phosphate groups. Insertion of Ab into zwit- the role of oligomers in such a process. Emerging pathological evidence indi- terionic DMPC bilayers indents the bilayer structure, resulting in bilayer thin- cate prion-like propagation among Ab aggregates, which may govern their abil- ning and a lipid density depression. For comparison, the anionic DMPS bilayer ity to proliferate. Furthermore, polymorphism observed within the aggregation prevents deep Ab insertion localizing amino acids to the bilayer surface via end products of fibrils are known to arise due to microstructural differences strong electrostatic interactions between charged amino acids and polar lipid among the oligomers. Diversity in aggregate morphology correlates with the headgroups. Not surprisingly, Ab binding to the DMPS bilayer results in less observed phenotypes in AD, cementing the idea that conformational strains bilayer thinning than binding to DMPC bilayers. In all systems, the effect of of oligomers could be significant in phenotypic outcomes. Therefore, it is Ab on the bilayer structure is limited to its immediate binding footprint. Ab imperative to determine the ability of oligomeric strains to faithfully propagate peptides also fail to enhance the permeation of water through the bilayer. their structure. We have identified a 12-mer of Ab42 called, large fatty acid- Collectively, these results provide a mechanistic explanation for the status of derived oligomer (LFAOs), which replicate upon interacting with monomers monomeric Ab peptides as an inert species. to generate quantitative amount of LFAOs. Replication is efficient at low con- centrations (< 10 mM), where LFAOs is predominantly a 12mer. At higher con- 1790-Pos Board B110 centrations, LFAOs are predominantly 12-24mers that are less efficient for Beta-Amyloid Oligomer Formation in Physiological Solutions - a Study by replication. However, 12-24mers undergo propagation towards morphologi- Single-Molecule Fluorescence Resonance Energy Transfer cally distinct fibrils, one that is made of discrete LFAO building blocks. These Jun Han1, Erwen Mei2, Mei-Ping Kung3, Hank F. Kung3, Jian-Min Yuan4, results establish that strain-specific propagation of oligomeric Ab takes places Hai-Lung Dai1. in two distinct steps of amplification and propagation in low and high concen- 1Department of Chemistry, Temple University, Philadelphia, PA, USA, trations of the seed, respectively. These data have opened doors to understand- 2Regional Laser and Biotechnology Laboratories, University of ing underlying mechanisms of proliferation as well as strain-specific Pennsylvania, Philadelphia, PA, USA, 3Department of Radiology, University phenotypic outcomes in AD.

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1793-Pos Board B113 to be effective at preventing the formation of Ab fibrils. In this work we use Aggregation of Amyloid Proteins at the Surface-Liquid Interface discontinuous molecular dynamics (DMD) simulations to learn how four Siddhartha Banerjee1, Mohtadin Hashemi1, Zhengjian Lv1, naturally-occurring phenolic compounds, resveratrol, vanillin, curcumin, and Sibaprasad Maity1, Jean-Christophe Rochet2, Yuri Lyubchenko1. epigallocatechin-3-gallate (EGCG), bind to Ab(17-36) monomers and affect 1Department of Pharmaceutical Sciences, University of Nebraska Medical oligomerization and fibrillation. We use a coarse-grained model/force field Center, Omaha, NE, USA, 2Medicinal Chemistry and Molecular for inhibitors which has geometric and energetic parameters that are compatible Pharmacology, Purdue University, West Lafayette, IN, USA. with the coarse-grained protein model PRIME20. Preliminary results show that Aggregation of amyloid proteins leads to the neurodegenerative diseases like the U-shaped protofibril structure formed by Ab(17-36) is similar to the corre- Alzheimer’s and Parkinson’s disease. Though there are structural similarities sponding part of the Ab(1-42) fibril model, based on solid state NMR data. The of the amyloid-b protein (Ab) aggregates found in amyloid plaque with those order of peak heights in the peptide-inhibitor radial distribution function shows assembled in vitro but there is a serious complication in translating the that the strength of the inhibitor binding affinity is resveratrol > curcumin and knowledge of in vitro studies to the in vivo one due to dramatic difference EGCG > vanillin. Simulations of 8 Ab(17-36) peptides aggregating in the pres- in concentration of Ab in two environments. For in vitro, the concentration ence of 30 inhibitors show that EGCG, resveratrol and curcumin inhibit Ab(17- required for aggregation is in micromolar range, whereas the physiological 36) fibril formation while vanillin only retards the lag phase. Instead of forming concentration of Ab is in low nanomolar range. This raises the question of an ordered b sheet structure (as occurs in the absence of inhibitors), the peptides which factors trigger in vivo Ab aggregation in such a low concentration. remain random coils and form complexes with the inhibitors, binding mainly to We hypothesize that the interaction of Ab with surfaces allows dramatic in- the hydrophobic residues near the peptide termini. The relative ability to inhibit crease in aggregation within in vivo range. To test this, we have investigated Ab(17-36) fibrillation is EGCG > resveratrol > curcumin > vanillin, consis- the on-surface aggregation of full-length Ab-42, a peptide Ab(14-23) and tent with experimental findings on full length Ab(1-42) aggregation. Our sim- a-synuclein at nanomolar concentration by atomic force microscopy ulations provide molecule-level insights into the mechanisms by which small approach. 1-(3-aminopropyl) silatrane functionalized mica surface has been molecules inhibit Ab aggregation. employed. Significant aggregation of all these proteins has been observed on surface, although essentially no aggregation has been found in solution 1796-Pos Board B116 over the same time scale. Importantly, the on-surface aggregation has been Cross-Reactivity of Alpha-Synuclein with Other Cellular Components Can a dynamic process where the aggregates formed on the surface can dissociate Dramatically Modulate Amyloid Formation from the surface and come to the bulk. Molecular dynamics simulation re- Pernilla Wittung-Stafshede. sults show that the interaction of the Ab(14-23) monomer with surface leads Biology and Biological Engineering Department, Chalmers University of to the conformational change facilitating the formation of the dimer on the Technology, Gothenburg, Sweden. surface. Overall, the discovery on acceleration of the amyloid aggregations The aggregation process, going from monomers to amyloid fibers, of the pro- by surfaces suggests that interaction with cell surface plays a significant tein a-synuclein (aS) somehow causes degeneration of dopaminergic neurons role in initiating the aggregation process. in Parkinson’s disease patients. The exact activities of aS are not yet identified, The work supported by NIH grants R01 GM096039 and R01GM118006 but synapse vesicle formation and transport have been proposed. aS binds to YLL. strongly to vesicles made of negatively charged lipids in vitro and such inter- actions may thus play a role in vivo. Probably because of variations in exper- 1794-Pos Board B114 imental conditions, vesicle binding has been reported to both trigger and block Biophysical Insight into the Anti Amyloidogenic Behavior of Cysteine aS amyloid formation in vitro. As the cell environment is highly crowded with Masihuz Zaman, Rizwan Hasan Khan. biomolecules, in addition to membranes, interactions with other aggregation- Biotechnology, Aligarh Muslim University, Aligarh, India. prone proteins may modulate aS amyloid formation in vivo. To better under- Protein misfolding and aggregation is associated with various human disorders stand how interactions with other biomolecules, that may take place in vivo, and thus inhibition of such occurrence has been center for intense research ef- may modulate aS amyloid formation, we have employed an array of biophys- forts against such neurological disorders. We investigated the thermal aggrega- ical tools (circular and linear dichroism, fluorescence, light scattering, and im- tion/amyloid formation of stem bromelain at molten globule state as well as aging methods) to probe aS amyloid formation as a function of lipid vesicles anti-fibrillation activity of cysteine and its effect on amyloid fibril formation (of different composition and lipid-to-protein ratios) and the amyloid- kinetics against stem bromelain. Thermal treatment of stem bromelain at pH forming peptide in type-2 diabetes, IAPP (in pro-peptide and processed forms; 10.0 (where it encountered in gut epithelium during adsorption) favors the ag- as monomer or amyloid seed). Our results underscore the importance of consid- gregation process. The extent of aggregation increases with increase in protein ering cross-reactivity effects in Parkinson’s disease. For example, we revealed concentration as confirmed by increased turbidity and Rayleigh light scattering that at lipid-to-protein ratios where only a fraction of aS is bound, vesicles can measurements. Thioflavin T binding as well as microscopic techniques completely remove the lag time of aS amyloid formation and the resulting am- confirmed that aggregates were fibrillar in nature. In addition, structural yloid fibres have unique properties (thin and fragile). Moreover, we discovered changes were monitored by circular dichroism (CD). Further, we investigated that IAPP speed up aS amyloid formation and, at some conditions, the two pro- anti-fibrillation activities of cysteine against stem bromelain by using similar teins make mixed amyloids with distinctive features. The latter result offers a approaches. Decreased turbidimetric and light scattering values as well as possible explanation why type-2 diabetes patients are pre-disposed for Parkin- dye binding (ThT and ANS) in presence of cysteine indicate that it has anti- son’s disease. fibrillar property. Cysteine restores native like structures of stem bromelain as confirmed by dynamic light scattering studies. Hydrogen bonding between 1797-Pos Board B117 cysteine and stem bromelain plays a momentous role through inhibition of Using the SCN Vibrational Probe Group to Determine the Membrane- stem bromelain aggregation as established by isothermal titration calorimetric Bound Structural Distribution of N-Terminally Acetylated Alpha- measurements. Additionally, a specific non- covalent interaction (thiophilic) Synuclein between aromatic amino acid residues of stem bromelain and thiol group of Kavita D. Shroff, Kristen E. Fiore, Casey H. Londergan. cysteine may also have notable role in inhibition of amyloid formation. The Chemistry, Haverford College, Haverford, PA, USA. outcome of the current study may provide a new guide into inhibition of protein The intrinsically disordered neural protein a-synuclein (aS) is a major compo- fibrillation (that are responsible for various neurodegenerative diseases), pave nent of the Lewy bodies associated with Parkinson’s Disease (PD) and other the way for breakthrough of novel anti-amyloidogenic agents and other small neurological diseases. While disordered in solution, once in contact with a lipid molecules that may exert similar effect against amyloidogenesis and its related membrane the N-terminus forms an amphipathic helical structure along the neurodegenerative diseases. membrane interface while the C-terminus remains unstructured. Site specific vibrational spectroscopy using a thiocyanate probe group attached to a number 1795-Pos Board B115 of single-cysteine variants of aS was performed to elucidate the site-specific Amyloid Beta Aggregation in the Presence of Naturally-Derived Inhibitors local dynamics of aS in contact with different lipid systems. The binding dy- Carol K. Hall, Yiming Wang, David C. Latshaw II. namics of non-N-acetylated aS were characterized in POPC/POPA vesicles Chemical and Biomolecular Engineering, North Carolina State University, and SDS micelles. However, in vivo aS is primarily N-terminally acetylated, Raleigh, NC, USA. which encourages a-helix formation of the N-terminal region and influences Alzheimer’s Disease (AD) is a neurodegenerative disease that causes dementia, the conformation of aS when in contact with a lipid system. To document nervous system degradation, and death. Currently there are no therapeutic the effects of both N-terminal acetylation and vesicle curvature on the binding agents available for the treatment of AD despite great efforts by the research dynamics of aS, IR results from N-terminally acetylated aS with single-site community. Many small phenolic compounds have been shown in experiment probe groups and varying lipids will be presented.

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1798-Pos Board B118 tion of CaN contributes to disorders such as Alzheimer’s disease, Down Determining the Role of N-Terminal Acetylation on a-Synuclein Function syndrome, autoimmune disorders and cardiac hypertrophy. We have previously Siobhan Toal1, Adam Trexler2, David DeWitt3, Mark Brown4, shown that the regulatory domain within CaN is intrinsically disordered. As Elizabeth Rhoades1. intracellular calcium levels rise, calcium-loaded calmodulin (CaM) binds to 1Chemistry, University of Pennsylvania, Philadelphia, PA, USA, 2National the CaN regulatory domain. This binding causes the regulatory domain to Heart, Lung, and Blood Institute, National Institute of Health, Bethesda, MD, fold around CaM, and in turn causes an autoinhibitory domain from CaN’s USA, 3Integrative Physiology and Neuroscience, Washington State active site. We are exploring how environmental factors such as crowding University, Pullman, WA, USA, 4Chemistry, Yale University, New Haven, and salt concentration modulates the conformational ensemble of the disor- CT, USA. dered regulatory domain, and how that in turn effects the association with CaM. a-Synuclein (aS) is an abundant neuronal protein implicated in Parkinson’s dis- ease. Acetylation of the N-terminal methionine of aS is a ubiquitous modifica- 1801-Pos Board B121 tion of aS derived from healthy brain tissue as well as from Parkinson’s Chemical Shift Assignment of the Regulatory Domain of Calcineurin Using disease patients. In vitro, it has a minor impact on both the solution structure NMR Spectroscopy and membrane-binding properties of aS and its role in aS function is unknown. John Hunt. In order to gain greater insight into the role N-terminal acetylation in aS func- Mississippi State University, Starkville, MS, USA. tion, we created an acetylation mimic, where the N-terminal methionine is re- Calcineurin (CaN) is a serine/threonine phosphatase that is universally ex- pressed in eukaryotic cells. In humans, CaN participates in cardiovascular and placed with asparagine (aSasn) and contrasted it with unmodified (aSunmod) central nervous system development, and is known for its role in T-cell activa- and N-terminally acetylated (aSacetyl ) aS. Chemical shift analysis suggests that both N-terminal modifications increase helicity of the first z10 residues tion. Because of its signaling activity, regulatory problems that occur with CaN have been associated with several human diseases, including Alzheimer’s of aS in solution, with aSasn showing intermediate helicity. Moreover, both disease and cardiac hypertrophy. CaN is activated by increased levels of intracel- aSacetyl and aSasn purified via the non-ionic detergent octyl-beta-glucoside 2þ (OG) are a mixture of monomeric and oligomeric species, with OG bound to lular calcium ions (Ca ). These ions bind to a regulator protein, Calmodulin the oligomers. We used smFRET to examine the structural changes that in mono- (CaM), which activates CaN by binding to CaN’s calmodulin binding region (CaMBR). The binding site is located in the regulatory domain (RD) of CaN. mer aS upon binding OG. We find that there are significant differences in the to- 2þ pology of aS bound to OG that propagate into the C-terminus and which are The RD is intrinsically disordered until Ca -bound CaN binds, whereupon dependent upon the specific N-terminal modification. Interestingly, we are the RD folds and releases the previously bound autoinhibitory domain (AID). able to discern three intermediate conformations of the N-terminus of aSasa Our research is centered on understanding the molecular interactions that facil- function of OG concentration. Taken together, our work suggests that N-terminal itate binding of CaN to the RD. To accomplish this, we used 2D and 3D NMR acetylation may play a role in aS recognizing relevant cellular binding partners. techniques to examine the structural determinants of binding and completed the protein’s backbone and sidechain assignments in the solution phase. Our pro- 1799-Pos Board B119 tein production methods included expressing protein from transformed E. coli Generating a Model for Calmodulin Induced Folding in the Disordered and purifying the cell lysate on a Ni-NTA column followed by a CaM- Regulatory Domain of Calcineurin Sepharose column. Purity was affirmed using SDS-PAGE and LC-MS. Dinesh K. Yadav. Chemistry, Mississippi State University, Mississippi State, MS, USA. RNA Structures and Dynamics Calcineurin (CaN) plays an important role in the T-cell activation, cardiac system development and nervous system function. Previous studies have suggested that 1802-Pos Board B122 the regulatory domain (RD) of CaN binds Calmodulin (CaM) towards the N-ter- Pressure Effects on Self-Assembly of GMP, Guanosine 50-Monophosphate minal end. Calcium-loaded CaM activates the serine/threonine phosphatase ac- Balasubramanian Harish1, Mimi Gao2, Roland Winter2, Catherine Royer1. tivity of CaN by binding to the regulatory domain, although the mechanistic 1Center for Biotechnology & Interdisciplinary Studies, Rensselaer details of this interaction remain unclear. It is thought that CaM binding at the Polytechnic Institute, Troy, NY, USA, 2Physical Chemistry l - Biophysical RD displaces the auto inhibitory domain (AID) from the active site of CaN, acti- Chemistry, Faculty of Chemistry and Chemical Biology, Technical vating phosphatase activity. In the absence of calcium-loaded CaM, the RD is at University Dortmund, Dortmund, Germany. least partially disordered, and binding of CaM induces folding in the RD. Previ- Nucleic acid self-assembly is fundamental to many biological functions and may ous studies have shown that an a-helical structure forms in the N-terminal half of have played a role in the origins of life on Earth. The self-assembly of guanosine the RD, but organization may occur in the C-terminal half as well. Here, we are 5’-monophosphate (GMP) can serve as a model system in understanding the self- interested in the structural transition of the full length RD as it binds to CaM. Us- assembly mechanisms of more complex systems. GMP is known to form ing nuclear magnetic resonance (NMR) spectroscopy, we have successfully as- different stacking superstructures, where the stacking units are monomers, di- signed >85% of the 15N, 13Ca, 13Cb and HN chemical shifts of the unbound, mers or tetramers. The size and relative population of these superstructures þ regulatory domain of CaN. While the protein is disordered, secondary chemical are affected by temperature and cations. In particular, K ions have been shown shifts indicate that some regions possess a-helical propensity, even in the un- to favour tetramer stacks (also known as G-quartets) by occupying the channel bound state. Our study of the CaM and CaN interaction supports the formation cavity between G-quartets in the stack, whereas other monovalent cations like þ þ of a distal helix in the region between the AID and calmodulin-binding region. Na and Rb ions preferentially bind to the surface in proximity to the phosphate Heat capacity changes upon binding predict that 54 residues fold when CaM groups. The effect of high pressure on these superstructures can be used to gain binds to CaN, consistent with the formation of this distal helix. Paramagnetic insights into the self-assembly mechanism. Moreover, life may have originated relaxation enhancement (PRE) studies of this interaction suggest a potential bind- in extreme environmental conditions similar to those encountered in deep sea ing mode where the distal helix binds to CaM near residues I9-A15. Mutagenesis thermal vents, such that understanding the effects of both temperature and pres- in the distal helix disrupts PREs, further supporting this hypothesis. Together, sure on these assemblies is important. High-pressure NMR, FTIR and SAXS ex- these data suggest that the interactions between CaM and the distal helix of periments reveal dissociation of tetrameric GMP stacks to monomer stacks at CaN can be important in regulation of phosphatase activity. higher pressures. The effect of pressure at different temperatures and in the pres- ence of different cations will be presented. To better understand the stacking 1800-Pos Board B120 mechanism, diffusion NMR under high pressure is used to estimate the effect Role of Disordered Protein Ensembles in Protein-Protein Interactions: of pressure on the size of tetrameric and monomeric stacks. Calcineurin as a Model System Trevor Creamer. 1803-Pos Board B123 Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Preferential Interactions of Charged and Neutral Cosolutes with a Model KY, USA. RNA Hairpin The relationship between the conformational ensembles adopted by intrinsi- Jacob C. Miner1,2, Angel E. Garcia2. cally disordered regions (IDRs) within proteins and their functions is of 1Theoretical Biology and Biophysics (T-6), Los Alamos National Laboratory, some importance. For example, the extent to which a protein binding site within Los Alamos, NM, USA, 2Center for Nonlinear Studies, Los Alamos National an IDR is accessible should play a role in the rate of association. Given that Laboratory, Los Alamos, NM, USA. many signaling proteins possess disordered regions that include binding sites, Cosolutes, including neutral salts and denaturants, can profoundly stabilize and that signaling processes often need to be initiated rapidly, this hypothesis different configurations of RNA or induce denaturation. Salient preferential in- is worth exploring. Calcineurin (CaN) is a highly-conserved, heterodimeric teractions between RNA and solution species can be assessed through all-atom Ser/Thr phosphatase that plays vital roles in memory development and reten- molecular dynamics (MD) simulations. At equilibrium, these simulations allow tion, cardiac growth, and immune system activation. Alterations in the regula- preferential interactions to be linked to experimental observations including

BPJ 7843_7846 Tuesday, February 14, 2017 367a cosolute distributions in vapor pressure osmometry and Donnan equilibrium all 252 different RNA 3WJs labeled with Alexa488 and Cy5 fluorescent dyes. measurements. The analysis toolkit included probability distribution analysis (PDA) for In this work we describe preferential interactions between charged and neutral FRET distance determination and FRET position and screening (FPS) toolkit cosolute species (Kþ/Cl- and urea) and their interactions with an RNA for structural model generation. We detected only one predominant conformer sequence that forms a hyperstable GCAA tetraloop using isothermal (NVT) for RNAs 3WJ. Furthermore we report that bulges in the junction determine MD simulations. The RNA-KCl preferential interactions (Donnan coefficients) orientation and rotation of helices, inducing coaxial stacking. We provide a are determined at cellular (~100 mM) and high (1-3 M) salt concentrations, and geometrical description of the junction geometry and our results show that small show an unexpected linear concentration dependence. At 3 M KCl, the Donnan changes in the sequence make dramatic changes in RNA 3WJ tertiary structures coefficient is zero. In high (2-6 M) concentrations of urea, KCl preferentially which are expected to have significant impact on the functionality. As the accu- excludes urea, while RNA preferentially attracts urea. Interestingly, despite racy of FRET restrained modeling depends on the correct description of the dye the depletion of urea around KCl, the RNA-KCl preferential interaction in- behavior, we characterized the position dependent fluorescent properties of the creases. These results offer new insight into the interactions between RNA coupled dyes and statistically analyzed the systematic deviations of the calcu- and many of the cosolute species that affect RNA folding in vivo. lated FRET distances from the model generated distances. This allowed us to identify the problematic labeling positions. Thus we could improve the accuracy 1804-Pos Board B124 of the FRET method by careful selection of labeling sites. Rna Compaction in the Presence of Polyvalent Cations Liya F. Oster1, Richard Sportsman1, Christian Beren1, Hongcheng Yao2, 1807-Pos Board B127 Jae-Ho Shin3, Charles Knobler1, William Gelbart1. FRET, SAXS and Molecular Simulations Resolve the Solution Structures 1University of California, Los Angeles, Los Angeles, CA, USA, 2The of Three Coexisting Conformers of Flexible RNA Four-Way Junction University of Hong Kong, Hong Kong, China, 3Chalmers University of Hayk Vardanyan1, Simon Sindbert1, Stanislav Kalinin1, Technology, Gothenburg, Sweden. Christian A. Hanke1, Tomasz Soltynski2, Grzegorz Lach2, The effects of polyvalent cations on the effective size and charge of double- Danilo Springstubbe3, Bettina Apel3, Edward Snell4, Thomas D. Grant5, stranded DNA (dsDNA) have been well studied. In the presence of polyvalent Jan Lipfert6, Sabine Muller€ 3, Janusz M. Bujnicki2, Holger Gohlke7, cations, dsDNA in dilute solution undergoes a single-molecule, first-order phase Claus A.M. Seidel1. transition, otherwise called condensation: more explicitly, upon onset of 90% 1Department of Molecular Physical Chemistry, Heinrich-Heine-Universit€at, neutralization of the phosphate backbone, the DNA undergoes discontinuous Dusseldorf,€ Germany, 2International Institute of Molecular and Cell Biology, compaction into tightly wound toroids. However, the effects of these cations Warsaw, Poland, 3Department of Biochemistry, Ernst-Moritz-Arndt- on long single-stranded RNAs (ssRNA) have not been well characterized. In Universit€at, Greifswald, Germany, 4Hauptman-Woodward Medical Research this study we use centrifugation methods to examine the effective size of long Institute, Buffalo, NY, USA, 5Department of Structural Biology, University ssRNAs in solutions of increasing concentration of the tetravalent cation sper- at Buffalo, Buffalo, NY, USA, 6Department of Physics, Ludwig-Maximilian- mine. In contrast to the case of dsDNA, we find only a continuous decrease in University, Munich, Germany, 7Department of Pharmaceutical and the size of ssRNA upon increase in spermine concentration. However, the Medicinal Chemistry, Heinrich-Heine-Universit€at, Dusseldorf,€ Germany. decrease is significant enough to suggest that RNA molecules longer than viral RNAs play multiple roles in the storage, transmission, and regulation of genetic genomes can be packaged in vitro into virus-like vectors for gene delivery. information. They commonly exhibit helical junctions as main architectural building blocks of RNA tertiary arrangements. However, the knowledge about 1805-Pos Board B125 equilibrium structures and tertiary conformational changes of large RNAs, not How Counterion Identity Impacts the Pathway of RNA Tertiary Folding restrained by external or tertiary interactions, is rather limited. A typical Robb Welty, Kathleen B. Hall. example is the hairpin ribozyme where helical four-way junction (RNA4WJ) Biochem & Mol. Biophys, Washington University, St. Louis, MO, USA. is an essential structural motif. The number of known RNAs that influence biological systems is quickly In order to rationalize the fundamentals of ribozyme architecture, we performed growing. This influence is exerted via binding interactions, which are exqui- high-precision Fo¨rster resonance energy transfer (hpFRET) measurements at sitely tuned by conformational state (or equilibrium of states) of RNA tertiary the single-molecule level and small angle X-ray scattering (SAXS) in connec- structure. The conformational equilibria of RNA, as well as the tertiary folding tion with all-atom and coarse-grained molecular simulations to map the confor- process, are modulated differently by different cations. To identify changes in mational space and determine structural models of a large RNA4WJ (z 45 kD) the RNA tertiary folding process we investigated the cation initiated folding of as a prototypic system. the prokaryotic rRNA GTPase center (GAC), a well characterized 60mer RNA, We resolved the structures of three coexisting conformers of a fully Watson-Crick in the presence of different cations. Robust kinetic and equilibrium measure- base paired RNA4WJ. 51 donor-acceptor-pairs were measured using single- ments were made by taking advantage of a site specific fluorescence labeling molecule multi-parameter fluorescence detection (smMFD). For each dataset, scheme, and multiple spectroscopic techniques. These data were used to refine the single-molecule approach allowed for the simultaneous extraction of three dis- a theoretical model of the folding pathway of this RNA. Our model consists of tances (and their corresponding errors) belonging to one major FRET state and five temporally resolved kinetic steps whose rates are tuned uniquely by two minor states. Distinct Mg2þ-affinities of the two minor states were used for different cations. This work was funded by the NIH R01-GM098102 to the FRET peak assignment. Rigid body models for the major and both minor con- KBH. Labeled RNA molecules were contributed by Agilent. formers were obtained by docking rigid ds A-RNA helices explicitly taking into account dye position distributions. The three rigid body models were refined by 1806-Pos Board B126 all-atom MD simulations and coarse-grained RNA folding using FRET restraints. Accurate Determination of the RNA Three-Way Junctions Via Single- Our results demonstrated that FRET-restrained molecular modeling can distin- Molecule High-Precision Fret Measurements guish different conformational states of a complex RNA junction and deter- Olga Doroshenko1, Hayk Vardanyan1, Aiswaria Prakash1, mine their respective structures with a resolution of a few A˚ ngstro¨m. Our Stanislav Kalinin1, Simon Sindbert1, Oleg Opanasyuk1, Christian Hanke1, broadly applicable method provides a powerful tool to probe the conforma- Sabine Muller€ 2, Holger Gohlke3, Claus A.M. Seidel1. tional ensembles of functional RNAs. 1Molecular Physical Chemistry, Heinrich Heine University, Dusseldorf, Germany, 2Ernst-Moritz-Arndt-University Greifswald, Greifswald, 1808-Pos Board B128 Germany, 3Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Single-Molecule Electric Snapshots of RNA Tertiary Pseudoknot Folding Heine University, Dusseldorf, Germany. Pathway In nature RNA helices are often linked via various junctions and bulges that Xinyue Zhang, Ruicheng Shi, Andrew J. Burcke, Li-Qun Gu. determine overall 3D structure which are used as building blocks and functional Bioengineering, University of Missouri, Columbia, MO, USA. components in nanotechnology applications. Hence visualization of RNA con- RNAs can fold into various conformations in their biological mechanisms. formations is crucial for understanding their biological functions. Fo¨rster-Reso- Elucidation of RNA folding processes is important to the understanding and nance-Energy-Transfer (FRET) restrained high-precision structural modeling regulation of RNA biological functions. Here we developed a nanopore electric can be used to determine the structure of these junctions. Multi-parameter fluo- snapshot technology for probing the folding pathway of RNA tertiary structures rescence detection (MFD) of single molecules and ensemble Time-Correlated at room temperature. Our target is the gene 32 mRNA pseudoknot of bacterio- Single Photon Counting (eTCSPC) measurements were applied to perform phage T2. Driven by a transmembrane voltage applied, the target RNA can be FRET study on systematic series of 6 RNA three- way-junctions (3WJs) derived captured by the nanopore from one side, and immobilized in the pore via the from the hairpin ribozyme. Bulge and sequence variations were considered as attached streptavidin. Upon capture, the precisely designed spacer between dominant factors influencing junction, two of which have bulge (two and five un- streptavidin and RNA allows RNA to be released as an unfolded free chain paired nucleotides) in the junction region. We have generated a database of over- to the other side the pore and re-fold. When a reverse potential is applied to

BPJ 7843_7846 368a Tuesday, February 14, 2017 draw the T2 RNA back, the re-folded RNA must be unfolded prior to returning. troscopic study of the conformational dynamics of this paradigmatic riboswitch Importantly, the detected unfolding time (stability marker) can clearly report with three stable macrostates. We demonstrate that NMR and smFRET revealed the identity of each folding state, from the free chain to the hairpin, and to complementing folding transitions of the add Asw on the second to minute time- the final pseudoknot. The time-dependent occurrence probabilities of all the scale for which we could map individual ligand-dependencies. Our results suggest folding states help us to map the entire stepwise folding pathway in the time that the adenine-sensing riboswitch thermostat operates via a ligand-induced equi- scale from 1 second to several minutes. This RNA single-molecule folding librium shift in persistent multi-state conformational dynamics. approach would lead to broad applications in detecting RNA structure- determined biological functionalities and their regulation by ligands. 1812-Pos Board B132 Dynamic Equilibrium of the TPP Riboswitch as Observed by MFD Fret 1809-Pos Board B129 Junyan Ma1, Soheila Rezaei2, Feng Ding2, Hugo Sanabria2. Folding Heterogeneity in HIV-1 Frameshifting Hairpin 1Chemistry, Clemson University, Clemson, SC, USA, 2Physics and Dustin B. Ritchie1, Collin Tittle1, Negar Rezajooei1, Logan Rouleau1, Astronomy, Clemson University, Clemson, SC, USA. Tonia R. Cappellano1, William Sikkema1, Michael T. Woodside1,2. Antibiotic-resistant infections are one of the widely known health problems that 1Physics, University of Alberta, Edmonton, AB, Canada, 2National Institute threaten over 2 million people each year in United State, and over 23,000 people for Nanotechnology, National Research Council, Edmonton, AB, Canada. died due to antibiotic resistant bacteria. Recent studies have proposed an innova- Ribosomes translate mRNA in 3-nucleotide steps, maintaining an open reading tive approach to fight antibiotic resistant bacteria using mRNA as targets for new frame until a stop codon is reached. Programmed 1 ribosomal frameshifting potential drugs. Riboswitches are one of the most studied messenger RNAs that (PRF), whereby the ribosome is forced backward by 1 nt to shift the reading control gene function and respond to second messengers such as small molecules frame, is essential for propagation of many viruses. Frameshifting depends on and proteins and could be used as targets. However, riboswitches are highly dy- two mRNA structures: a slippery sequence and a downstream stimulatory struc- namic structures and evade most common methods of characterization. Using a ture. PRF in HIV-1 is thought to be stimulated by a hairpin, although controversy structure-guided drug design rationale, our first goal is to determine the around the role of a potential pseudoknot exists. The conformational dynamics of structure-function relationship of riboswitches upon binding of effector mole- the stimulatory structure under tension applied by the ribosomal helicase during cules. We utilize Fo¨rster Resonance Energy Transfer (FRET) at a single mole- translation may play an important role in PRF, therefore we used optical twee- cule level in Multiparameter Fluorescence Detection mode to understand the zers to apply tension to the HIV-1 frameshift signal and monitor the folding relationship between structure and dynamic of the TPP Riboswitch. We compare and unfolding dynamics. The folding and unfolding kinetics and energy land- our results with Discrete Molecular Dynamic (DMD) simulations and find that scape of the hairpin were measured by ramping the force on the hairpin up the TPP-riboswitch is in equilibrium between two conformational states, which and down, providing a detailed biophysical characterisation of the hairpin. are potential targets to bind new small molecules. We alter the dynamic equilib- Whereas the unfolding reflected the simple two-state behavior typical of many rium by the presence TPP, MgCl2 and TPPþMgCl2. hairpins, unexpectedly, refolding was not just a reversal of the two-state unfold- ing, but instead displayed more heterogeneous kinetics. Evidence was found for 1813-Pos Board B133 Glms multiple refolding pathways as well as previously unsuspected, partially folded Folding and Catalysis of the Ribozyme Riboswitch Studied at the intermediates. A longer HIV-1 frameshifting-signal construct with the potential Single-Molecule Level 1 2 to form the suspected pseudoknot was also measured. This construct typically Andrew Savinov , Steven M. Block . 1Biophysics Program, Stanford University, Stanford, CA, USA, displayed very similar mechanical properties to the hairpin alone, perhaps 2 with more heterogeneous unfolding. Our observations of the HIV-1 frameshift- Departments of Applied Physics and Biology, Stanford University, Stanford, ing hairpin under tension suggest a possible functional role in PRF similar to the CA, USA. dynamics observed for frameshifting pseudoknots. We present findings from optical trapping experiments performed on the glmS ribozyme riboswitch. Found in many Gram-positive bacteria, the glmS ribos- 1810-Pos Board B130 witch down-regulates GlmS expression in the presence of the cell wall precur- Structural Insights to the 30 UTR of Gait Elements sor—and enzymatic product of GlmS—glucosamine-6-phosphate (GlcN6P). In Nancy Wells1, Blanton S. Tolbert2. response to GlcN6P, the riboswitch site-specifically cleaves itself near its 50 1Case Western Reserve, Cleveland Hts, OH, USA, 2Case Western Reserve, end, which targets the glmS mRNA for subsequent degradation1. We performed Cleveland, OH, USA. self-cleavage assays on optically trapped ribozyme molecules in the presence In the Interferon-Gamma-Activated Inhibitor of Translation (GAIT) system, a sin- of either GlcN6P or its catalytically inactive analog, Glc6P (glucose 6-phos- gle inflammatory signal Interferon g (IFN-g) activates both transcriptional on and phate). The assays demonstrate that our experimental construct, consisting of off-switches in human myeloid cells through the use of structured mRNAs. There aminimal ribozyme sequence, is enzymatically active and strictly dependent is current data suggesting that these pro-inflammatory mRNAs may regulate the upon GlcN6P. We also performed single-molecule force spectroscopy on the GAIT complex via structures in their 3’ UTRs. Several studies have demonstrated ribozyme catalytic core, measuring its unfolding/folding dynamics under that these structured RNAs are capable of recognition and regulation of the GAIT external loads. The data reveal a series of intermediate folding states occupied system for multiple pro-inflammatory mRNAs and thus forming a post transcrip- during mechanically-induced denaturation and renaturation. Analysis of the tional regulon. At present there is no structural information on these GAIT ele- data leads to a model for the folding pathway, involving sequential passage ments. To understand the role that GAIT structure plays in regulating through a series of discrete substructures. We have measured kinetic and ther- inflammation preliminary NMR experiments have been performed. The following modynamic parameters for substructure formation, and are using these values four GAIT elements have been examined: Cp, CCL22, CCR4, and CXCL13. All to reconstruct a folding-energy landscape for the ribozyme. We are also deter- four fold into thermodynamically stable stem loops despite sequence variation. mining the effect of cofactor binding on the folding-energy landscape, using the Each RNA folds into stable stem loop structures containing an internal loop, apical inactive analog Glc6P. Single-molecule self-cleavage activity measurements hairpin loop, and stably defined upper and lower stem. Various temperature under a range of controlled loads allow us to determine the profile of catalytic studies have revealed that the GAIT element for a stable helical region. activity in response to destabilizing forces. By combining results from mea- surements of folding and catalytic activity, we gain further insights into how 1811-Pos Board B131 specific structural features of this ribozyme relate to its catalytic function. Ligand-Directed Conformational Dynamics of the Adenine-Sensing Ribos- Footnotes witch Thermostat 1 Collins, J.A., Irnov, I., Baker, S., and Winkler, W.C. (2007) Genes Dev € Sven Warhaut, Klara Rebecca Mertinkus, Philipp Ho¨llthaler, Boris Furtig, 21(24), 3356-68 Mike Heilemann, Martin Hengesbach, Harald Schwalbe. Goethe University Frankfurt, Frankfurt am Main, Germany. 1814-Pos Board B134 Bacterial gene expression can be regulated by translational operating ribos- Mechanistic Insights into Functional Protein-RNA Interactions Involved witches, cis-acting mRNA elements that exhibit ligand-dependent conformational in Viral Replication switching between gene-OFF and gene-ON states with trapped or exposed ribo- Blanton S. Tolbert. some binding site, respectively. The adenine-sensing riboswitch (Asw) located Case Western Reserve University, Cleveland, OH, USA. inthe 5’ leader sequence of the add gene of the Gram-negative, human pathogenic, To replicate their genomes, viruses use conserved RNA structures that usurp host marine bacterium Vibrio vulnificus is the first discovered temperature- RNA binding proteins. The mechanisms by which viral RNA structure and compensated riboswitch, a riboswitch thermostat, that adjusts the relative popula- sequence contribute to functional host interactions are still poorly understood. tions of an adenine-binding competent and a binding incompetent apo conforma- Here, we used High Throughput Sequencing Equilibrium (HTS-EQ) binding ex- tion to compensate the inherent temperature-dependence of ligand binding. We periments combined with independent biophysical measurements to describe the have performed an integrated NMR and single-molecule FRET (smFRET) spec- complete affinity distribution of human hnRNP A1 bound to the HIV ESS3 stem

BPJ 7843_7846 Tuesday, February 14, 2017 369a loop. HTS-EQ involves competitive binding of hnRNP A1 to a completely ran- 3D structure prediction at present. Previously, we’ve developed a method domized pool of ESS3 apical loop variants (n=16,384). Quantitative analysis of 3dRNA for RNA 3D structure prediction, and it gave a performance of mean the resulting affinity distribution reveals RNA sequence; motif copy number; motif RMSD 3.95 A˚ and INF 0.88 for a test set of 32 RNAs with lengths from 12 spacing; and secondary structure determine specificity by modulating rates of pro- to 101 nt. Recently, we developed a new version of 3dRNA, 3dRNA v2.0, ductive hnRNP A1-RNA encounters. To investigate if these rules can be extended which improved the tree representation of the 2D structures, extended 3D tem- to other viral systems, we determined a hybrid SAXS/NMR structure of hnRNP A1 plates library, included a bias sampling method, incorporated newly developed in complex with an IRES element from Enterovirus 71. Indeed, the structure and scoring function, and most importantly, added the feature of predicting the 3D biophysical studies of the complex reveal a common set of mechanistic principles structure of ssDNA. 3dRNA v2.0 gives a mean prediction accuracy of RMSD determine functional hnRNP A1-RNA interactions. Thus, our work provides sig- 3.24 A˚ and INF 0.91 for ncRNA if 10000 candidates are sampled. The website nificant insights into the combinatorial factors that determine how viral RNA ele- address is: http://biophy.hust.edu.cn/3dRNA. ments recruit host RNA binding proteins to control gene expression. 1818-Pos Board B138 1815-Pos Board B135 Homology Modeling of RNA Using Hierarchical Natural Moves Redox Editing of RNA: A Novel Endogenous Modification Type with Tran- Xiong An Lee, Adelene Y.L Sim. sition Metal Cofactor (COPPER) Biomolecular Modeling and Design Division, Bioinformatics Institute, Josef H. Wissler. A*star, Singapore, Singapore. ARCONS Institute for Applied Research & Didactics, D-61231 Bad Like proteins, RNAs adopt diverse and intricate three-dimensional structures in Nauheim, [email protected], Germany. order to function. However, high-resolution experimental RNA structures are Objective: Beside splicing in transcriptional processes for removal or recombi- difficult to obtain due the intrinsic flexibility of RNA molecules. While ab nation of different parts of RNA [introns and exons], point or serial modifications initio RNA structure prediction is computationally expensive, homology of individual RNA nucleotides are known by substitution editing and inser- modeling can be adopted in cases where there is substantial sequence similarity tion or deletion editing. Entitled redox editing was investigated in the main to an existing RNA structure. Nonetheless, the significant drawback is that cur- RNA transcript fraction [noncoding ncRNA] of activated cells which escaped rent RNA homology modeling approaches are either not general enough1-3 or consideration as potential targets of therapy, so far. Methods and further links are not designed to handle large insertions and deletions 4. To circumvent are in Ann.N.Y.Acad.Sci.1137:316-342,2008. Results: Several spliced non- this, we present an alternative homology modelling approach that leverages Mendelian ncRNA [<200n] with bioactivity in epigenetic vascular phenotype on the hierarchial folding of RNA. Namely, we build on our previously devel- biology [vasculo-, angio-, lymphangio-, arteriogenesis] were found exuded oped Hierarchical Natural Move Monte Carlo5 protocol, and pull (through har- by reticuloendothelial cells when activated by intrinsic and extrinsic envi- monic springs) the target sequence to the template structure by controlling the ronmental chemical [metabolic, hypoxic] and physical [mechanosensitive, hierarchical degrees of freedom of the RNA molecule. In this way, we are able exercise, hemodynamic, shear stress] chance factors. In focus is a RNP [angio- to make large conformational changes while preserving the physical geometry tropin] which was sequenced after isolation from extracellular fluids where of any insertions and deletions, and retaining atomistic representation of all it is active. It is built up of angiotropin-related RNA [ARNA, 75n] as non- atoms. Additionally, because we control the molecular degrees of freedom Mendelian episcription product which by metal ions [Cu,Ca,Na,K] folds with based on RNA secondary structure, our protocol is general and easily scalable a Mendelian-coded angiotropin-related protein [ARP, S100-A12]. A metal to larger RNA systems. We benchmark the performance of our approach to 5’ 3’ ion-structured CUG -hairpin loop of ARNA fits to folded ARP protein other existing techniques and also illustrate how we are able to successfully 5’ groove. ARNA is a functional, end-phosphorylated modified, edited, redox- handle large insertions using SAM riboswitch as an example. By preserving and metalloregulated small hairpin non-coding RNA with ~80% base homology atomistic representation of the RNA, our homology models can be directly 5’ at its end of 75n to microRNA-126-5p [miR 126-5p, 21n] and other portions of used in RNA-based computational small-molecule drug design. its Mendelian 85bp MIR126 gene. Biosynthetically, its adenosine-N1-oxide/ isoguanosine-crotonoside constituents of 151Da base family is formed from 1819-Pos Board B139 adenosine by Fenton-type redox-[OH*-]radical RNA chance modification in Coarse-Grained Modeling of RNA for Biology and Nanotechnology tautomeric/mesomeric complex with copper-[þ,þþ] ions by environmental Petr Sulc1, Flavio Romano2, Thomas Ouldridge3, Jonathan Doye4, factors [hypoxia, metal ions, reductones/vitamins, shear stress]. Ard Louis5. Conclusions: The results suggest novel mechanisms and therapeutic targets in 1Center for Studies in Physics and Biology, The Rockefeller University, New epigenetic vascular phenotype biology. Metabolically and shear stress- York, NY, USA, 2Universita’ Ca’ Foscari, Venice, Italy, 3Imperial College, switchedvascular adaptation [angiogenesis/arteriogenesis], like collateral safe- London, United Kingdom, 4Department of Chemistry, Oxford University, guard circulation, is shown up subject to redox editing of ncRNA with copper Oxford, United Kingdom, 5Department of Physics, Oxford University, ions by modification of some adenosines to isoguanosine-crotonoside via Oxford, United Kingdom. disproportionation of adenosine-N1-oxide. We present a recently developed nucleotide-level model for RNA, oxRNA. The 1816-Pos Board B136 model is designed to reproduce structural, mechanical and thermodynamic prop- Distinct Chain Organizing Principles of Proteins and RNA erties of RNA, and the coarse-graining level aims to retain the relevant physics Lei Liu, Changbong Hyeon. for RNA hybridization and the structure of single- and double-stranded RNA. Korea Institute for Advanced Study, Seoul, Korea, Republic of. We test the model in a range of nanotechnological and biological settings. Ap- plications explored include the folding thermodynamics of a pseudoknot, the Chain organizations of RNA and proteins differ even though both biopolymers formation of a kissing loop complex, the structure of a hexagonal RNA nanor- are densely packed in their native states. Motivated by the recent discoveries that interphase chromosomes have territorial organization related to metasta- ing, the unzipping of a hairpin motif, RNA overstretching, and RNA plecto- bility, we analyzed biomolecular structures and showed that the intrachain con- neme formation. Where experimental data are available, we obtain tact probabilities, P(s) as a function of the arc length s, decay in power-law ~ s-Y quantitative or seminquantitative agreement with our model. over the intermediate range of s,10% s % 110. The contact probability scaling We argue that the model can be used for efficient simulations of the structure of systems with thousands of base pairs, and for the assembly of systems of up to exponent Y z 1.11 for large RNA (N > 110), Y z 1.41 for small sized RNA hundreds of base pairs. (N < 110), and Y z 1.65 for proteins. It is visually clear that folded structures of large sized RNA (N R 110) adopt crumpled structures, partitioned into We further use the model to study the thermodynamics and kinetics of an RNA modular multi-domains assembled by proximal sequences along the chain, toehold-mediated strand displacement reaction, during which an invading sin- whereas the polypeptide chain of folded proteins intermingles with the rest gle RNA strand displaces incumbent RNA strand that has been previously of the structure. We explained Y z 1 for large RNA as a result of the hierar- bound in a duplex with a substrate strand. This reaction is an essential mecha- chical ordering of the secondary to tertiary elements in the folding process. nism in active nucleic acid nanotechnology and has also been hypothesized to occur in vivo, for instance during spliceosome assembly. Recent experiments 1817-Pos Board B137 have also demonstrated promising application of RNA strand displacement sys- 3D Structure Prediction of Noncoding RNA and Single Stranded DNA tems to synthetic biology. We use oxRNA to obtain the rate of the displacement Jian Wang, Yi Xiao. reactions as a function of the length of the binding region of the invading strand Huazhong University of Science and Technology, Wuhan, China. and temperature. These results can be used as bounds for estimation of Nonconding RNA (ncRNA) and single stranded DNA (ssDNA) are important displacement reaction rates in vivo and for design of RNA displacement cas- biological nucleic acid molecules but the number of experimentally solved 3D cades for bionanotechnological applications. structure is very limited at present. Some computational methods have been The source code implementing our model on CPU and GPU is released for pub- proposed for RNA 3D structure prediction but there are no methods for ssDNA lic use at dna.physics.ox.ac.uk.

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1820-Pos Board B140 several recent RNA force-fields including Amber-99, Chen-Garcia 2013, and Coarse-Grained Model of RNA Captures Excess Ion Atmosphere Between our newest RNA force-field. We find that our newest base-pairing calibrated Conformational Basins model achieves the best agreement with experiment, and that the choice of wa- Udayan Mohanty. ter model significantly affects the resulting base-pairing stabilities. Chemistry, Boston College, Chestnut Hill, MA, USA. 1823-Pos Board B143 We have developed a coarse-grained structure based model to investigate the Spatial Modeling of Cytosolic Diffusion and Degradation of Influenza a excess ion atmosphere around an irregular RNA structure in aqueous solution Virus Explains Measured Nuclear Entry Efficiency containing MgCl2 and KCl under physiological ionic strength. The structure þ Andreas Herrmann, Edda Klipp. based model captures the dynamics of outer sphere Mg2 population. Conden- þ þ Institute of Biology, Humboldt-University Berlin, Berlin, Germany. sation of K and Cl- is described implicitly, while Mg2 ions are treated explic- Influenza A virus carries its segmented genome inside a lipid envelope. Since itly. The implicit model takes into account electrostatic heterogeneity of genome replication occurs inside the nucleus, the main goal of virus infection is phosphates, electrostatic and mixing free energy of the screening ions, and en- to deliver all genome segments through the cytoplasm into the nucleus. After forces ion accessibility near the RNA. We couple the electrostatics to the struc- endocytic uptake, influenza viruses transit early endosomal compartments ture based coarse-grained model and probe fluctuations by molecular dynamics and eventually reach late endosomes. There, the viral glycoprotein hemagglu- simulations. KCl condensation dynamically responds to conformational fluctu- tinin (HA) triggers fusion between endosomal and viral membrane, a critical ations of the RNA. The RNAs investigated are 58-mer from the large subunit ri- step that leads to release of the viral segmented genome destined to reach the bosomal RNA and a 26 nucleotide pseudoknot from Beet Western Yellow Virus cell’s nucleus. Endosomal maturation is a complex process involving acidifica- RNA. We predict the excess ion atmosphere of the native and mimics of inter- tion of the endosomal lumen as well as endosome motility along microtubules. mediate state conformations of the RNAs. We determine the effects of chelated þ þ þ While the pH drop is clearly critical for the conformational change and mem- ions (Mg2 and K ) on the excess ion atmosphere and the Mg2 -RNA interac- brane fusion activity of HA, the effect of intracellular transport dynamics on the tion free energy. We show the that excess ion atmosphere is sensitive to small progress of infection remains largely unclear. In this study, we developed a native basin fluctuations, i.e., RNA dynamics must be included for a quantitative comprehensive mathematical model accounting for the first steps of influenza description of the full conformational ensemble. For both RNAs, we compare virus infection. We calibrated our model with experimental data and challenged our predictions against fluorescence titration data of Draper and coworkers. its predictions using recombinant viruses with altered pH sensitivity of HA. We Work done in collaboration with A. Wang, S. Roy, R. Hayes, K. Streu, and J. N. identified the time point of virus-endosome fusion and thereby the diffusion Onuchic. Work supported by John Simon Guggenheim Memorial Foundation distance of the released viral genome to the nucleus as a critical bottleneck Fellowship (UM). for efficient virus infection. Further, we concluded and supported experimen- 1821-Pos Board B141 tally that the viral RNA is subjected to cytosolic degradation strongly limiting Multistep Folding Kinetics of Group I Intron RNA Studied by Mg2D-Con- the probability of a successful genome import into the nucleus. centration Jump Simulations 1824-Pos Board B144 Naoto Hori1, Natalia A. Denesyuk2, D Thirumalai1. 1 2 Nucleic Acids on Graphene Nanoparticle Surfaces Department of Chemistry, University of Texas, Austin, TX, USA, Institute Srivathsan V. Ranganathan. for Physical Science and Technology, University of Maryland, College Park, The RNA Institute, University at Albany, ALBANY, NY, USA. MD, USA. Graphene oxide-nucleic acid nanoassemblies hold promise for development of RNA molecules can function as enzymes forming specific tertiary structures. biosensors. Graphene oxide (GO) quenches fluorescence of fluorescently tagged Since they are highly charged molecules, mono- and divalent cations are essen- 2þ single stranded RNA or DNA upon binding, which can be recovered by desorp- tial for the folding reaction. Mg ions are also often important at specific po- tion of the strand by its complementary strand or DNA-binding proteins in sitions for their catalytic activities. Here, we study multistage folding kinetics 2þ solution. To design nucleic acid sequences for targeting biomolecules with of Azoarcus group I intron, which is mediated by Mg cations, by Brownian high-specificity, it is important to get a fundamental understanding of the inter- dynamics simulations using a coarse-grained RNA model with explicit ions [1]. action of GO with individual nucleobases (A, T, G & C) as well as small oligo- From unfolded structures prepared in the absence of divalent ions, 300 indepen- 2þ nucleotides. A quantitative understanding of graphene-nucleic acid interaction is dent folding reactions were triggered by adding 5 mM Mg , and tracked until needed to build a platform towards designing graphene based biosensors. To this they reach the native state. The folding reaction takes place through, at least, three end, we performed molecular dynamics simulations, guided by dispersion- phases: (i) Rapid collapse from the unfolded state to an intermediate state in which corrected Density Functional Theory (DFT) and Isothermal Titration Calorim- the RNA forms relatively small conformations. Formations of most secondary etry (ITC) experiments, to accurately quantify and understand the molecular structures are completed in this phase. (ii) In the second stage transition to an in- mechanism of nucleobases and nucleosides binding to graphene in water. We termediate state that is nearly compact as the native structure occrs, but certain have employed an thermodynamically calibrated force-field to characterize catalytically important tertiary interactions are not formed. (iii) In the last stage structure and interaction of nucleic acids at graphene surfaces. From single nu- folding to the native structure takes place. Formations of some tertiary interac- cleosides to oligos, we show characteristic differences in binding modes, loca- tions are rate-limiting from the second to third step. The changes in the radius tion and free-energies between DNA, RNA and other modified forms, such as of gyration are in good agreement with previous SAXS experiments in which unlocked nucleic acids. Our results provide a quantitative understanding of also three-steps folding has been observed [2]. We discuss kinetics of key tertiary nucleobase-graphene interactions, and also build a platform to understand single interactions, by comparing with previous equilibrium study [1]. For instance, the stranded nucleic acid binding to graphene surfaces, duplex formation at the triple helix (TH) is folded distinctively in the initial phase of the second stage, graphene-water interface and their subsequent release to the bulk. which is consistent with a similar finding in the equilibrium study [1] demon- strating that TH scavenges for [Mg2þ] even at low ion concentration. Protein: DNA Interactions: Chromatin and [1] Denesyuk, N.A. and Thirumalai, D., Nature Chem. 7, 793 (2015) [2] Roh, J.H. et al., J. Am. Chem. Soc. 132, 10148 (2010) Other Structures 1822-Pos Board B142 1825-Pos Board B145 Simulating the Melting of the Salmonella 4-U RNA Thermometer Using Asymmetric DNA Unwrapping Drives Sequential Dimer Release in Nucle- Replica Exchange Molecular Dynamics to Assess Force-Field Accuracy osomes Parisa Ebrahimi, Alan Chen. Yujie Chen1, Joshua Tokuda1, Traci Topping2, Steve Meisburger1, Chemistry, SUNY Albany, Albany, NY, USA. Suzette Pabit1, Lisa Gloss2, Lois Pollack1. The Salmonella 4U RNA thermometer is a non-coding regulatory RNA capable 1School of Applied and Engineering Physics, Cornell University, Ithaca, NY, of regulating gene expression as a function of temperature. At low temperature, USA, 2School of Biomolecular Sciences, Washington State University, four uridines are base paired with the SD sequence and inactivate the transla- Pullman, WA, USA. tion of mRNA. Temperature increase makes this RNA duplex unfold, exposing The fundamental structures of genome packaging in the nucleus are nucleo- the SD sequence for translation. We simulated the melting of the Salmonella somes, whose stability and dynamics provide key mechanisms for the epigenetic 4U thermometer over a broad temperature range using Replica Exchange Mo- control of genes. The canonical nucleosome consists of 147 basepairs of DNA lecular Dynamics (REMD) to study the opening rate of base pairs and deter- tightly wrapped around an octamer of histone proteins. In vitro and in vivo mine the free energy of base pairing as a function of temperature. As these studies reveal a growing diversity of nucleosomal structures with varying site-specific opening rates have recently been measured by NMR, this system DNA conformations and histone compositions that facilitate epigenetic activity. is ideal for comparing the fidelity of different RNA force-fields for describing Since characterization of the kinetic pathways between different biologically base-pairing stability. We use this model system to compare the accuracy of relevant nucleosomes species remain elusive, we developed a novel biophysical

BPJ 7843_7846 Tuesday, February 14, 2017 371a method to probe the coordination between changes in DNA conformation and acids of the histone proteins and the DNA minor groove. At an elevated salt reconfiguration of the histone core. We used time-resolved small angle x-ray condition, however, the ends of the nucleosomal DNA were observed to scattering (TR-SAXS) to visualize DNA unwrapping and time-resolved Fo¨rster spontaneously detach from and reattach to the histone core in discrete 10.5 resonance energy transfer (TR-FRET) to monitor dimer release during the salt- basepair steps. We found the breakage of histone-DNA contacts to occur induced disassembly of nucleosomes. Applying this approach to study the for- in several steps: a positive histone side chain first abruptly exits the minor mation of the hexasome and tetrasome (missing one or two H2A-H2B dimers, groove, forms a salt bridge with a DNA backbone phosphate for an extended respectively), we found that the asymmetric unwrapping of the DNA guides period of time, and then the contact breaks completely. Reattachment of the the sequential release of proteins. This link between DNA conformation and his- DNA proceeds through the same steps in the reverse order. Finally, we find tone composition may provide new mechanistic insight into the activity of chro- the sequence of the DNA to influence the likelihood of spontaneous disasso- matin remodelers and histones chaperones. ciations, suggesting that certain segments of DNA could be more likely to initiate transcription and more accessible to chromatin binding factors. Our 1826-Pos Board B146 studies demonstrate the utility of large, microsecond timescale atomistic sim- Sequence Dependence in Salt Based Nucleosome Unwrapping Using Saxs ulations for characterizing spontaneous processes that are beyond the resolu- 1 1 1 2 Alexander Mauney , Joshua Tokuda , Yujie Chen , Lisa Gloss , tion of single-molecule experimental methods. Traci Topping2, Oscar Gonzalez3, Lois Pollack1. 1Cornell University, Ithaca, NY, USA, 2Washington State University, 1829-Pos Board B149 Pullman, WA, USA, 3University of Texas at Austin, Ithaca, NY, USA. DNA in Tight Spaces: Linking Structure, Stability and Protection in Prot- Wrapping and unwrapping of DNA from nucleosome core particles is a major amine Packaged DNA part of DNA dynamics in vivo, though there is much about it that is still unknown. Jason E. DeRouchey. As DNA availability is a key component of transcription regulation, understand- Chemistry, University of Kentucky, Lexington, KY, USA. ing how variations in DNA sequence and histone structure or composition Packaged DNA is ubiquitous in nature and the laboratory with examples impact their binding is crucial for understanding the overall transcription pro- ranging from chromatin, viruses, sperm cells, bacterial nucleoids, artificial vi- cess, and how DNA is organized into chromatin. In this work, small angle ruses and gene therapy constructs. Sperm nuclei are one of the best examples of x-ray scattering (SAXS) was used to examine multiple combinations of DNA in vivo maximum DNA compaction and therefore an ideal model system to sequence and histone variants at different salt concentrations, and in solution study biophysically. Despite intense research, the physical mechanisms under- where there are no geometric constraints. By using salt to destabilize the lying tight packaging of DNA remain poorly understood especially at the mo- nucleosome-DNA complex multiple unwrapped states are accessible, allowing lecular level. Spermiogenesis is a unique multi-step process resulting for many possible pathways to be explored by the complexes. We use novel ultimately in the replacement of histones by protamines in sperm nuclei to a Monte-Carlo methods to generate realistic sequence-dependent unwrapped final volume roughly 1/20th that of a somatic nucleus. The near crystalline or- structures for the nucleosomal DNA. These structures are used in ensemble opti- ganization of DNA in mature sperm is thought crucial for both DNA delivery mization methods to determine which ones are represented in the SAXS data1. and the protection of genetic information due to the absence of DNA repair. In We determine differences in both the unwrapping paths and equilibrium con- this talk, I will first discuss our past studies on understanding how cations stants characteristic of the different histone/DNA combinations. The changes modulate DNA-DNA forces in the condensed phase and the interrelationships in the pathway are related to structural differences between the DNA sequences. between cation chemistry, packaging densities and compaction. The last half of 1. Chen Y., Tokuda J.M., Topping T., Sutton J.L., Meisburger S.P., Pabit S.A., my talk will discuss recent experiments aimed at understanding the various bio- Gloss L.M., Pollack L. Revealing transient structures of nucleosomes as DNA logical implications for both protamine-DNA packaging and correlations to unwinds. Nucleic Acids Res. 2014;42:8767-8776. infertility and oxidative stress in sperm chromatin. 1827-Pos Board B147 1830-Pos Board B150 Investigating the Histone Replacement Pathway in Sperm Using TPM Investigating the Mechanics of Protamine-Induced DNA Condensation in Elizabeth D. White, Adam Smith, Obinna Ukogu, Hilary Bediako, Sperm Moumita Dasgupta, Ashley Carter. Hilary Bediako, Adam D. Smith, Obinna Ukogu, Moumita Dasgupta, Physics, Amherst College, Amherst, MA, USA. Elizabeth White, Ashley R. Carter. DNA is folded in drastically different ways in somatic cells and sperm cells. In Physics & Astronomy, Amherst College, Amherst, MA, USA. somatic cells, DNA is wrapped around histone proteins and remains accessible We examine how protamine, a positively-charged nuclear protein, causes DNA for transcription and replication. However, in sperm cells, DNA is dramatically compaction within the nucleus of a sperm cell. Beyond the basic science of un- condensed into a toroid structure when the histones are replaced by protamine derstanding the mechanics behind DNA condensation, DNA folding in sperm proteins. Here, we investigate the process of histone replacement by protamine has implications in epigenetics, fertility, and material science. In order to study using tethered particle motion (TPM). TPM measurements are ideal for looking how DNA folds in sperm cells, we perform an in vitro Tethered Particle Motion at DNA folding since measurements are taken at zero force. In TPM, one end of (TPM) assay to look at the folding of individual DNA molecules. In TPM, a the DNA is attached to a glass coverslip and the other to a 1-micron-diameter polystyrene bead is attached to a DNA molecule, which is tethered to a cover- bead to form a tether. We record the motion of the bead using video microscopy slip. By observing the position of the bead over time with video microscopy, we and relate this motion to the length of the DNA with about 10 nm precision. The can measure the length of the DNA to a precision of about 10 nm. When prot- length of the DNA changes as histones are unwrapped from the DNA and re- amine binds to the DNA, it forms approximately 60-nm-diameter loops, which placed by protamine, allowing us to reconstruct the folding pathway for histone then condense into toroids. Here, we see individual looping events that indicate replacement, as well as the energy landscape. This pathway is important in that protamine-induced toroid formation is a stepwise mechanism. research on fertility, epigenetics, and biomaterials research. 1831-Pos Board B151 1828-Pos Board B148 Holliday Junction Structure Development for Single-Molecule Visualization The Molecular Mechanism of Nucleosome Breathing Mate Gyimesi, Zoltan Kovacs, Mihaly Kovacs. David Winogradoff, Aleksei Aksimentiev. Biochemistry, Eotvos University, Budapest, Hungary. Center for the Physics of Living Cells, Department of Physics, University of A double Holliday junction (DHJ) DNA structure is formed as a key intermediate Illinois at Urbana-Champaign, Urbana, IL, USA. during homologous recombination (HR), supporting error-free somatic DNA Meters of DNA wrap around histone proteins to form nucleosomes and fit in- repair of double-stranded DNA breaks and the meiotic formation of gametes. side the micron-diameter nucleus. In order for DNA to become available for In HR, the broken DNA ends are processed and homologous DNA serves as tem- template-directed processes—such as transcription, replication, and repair— plate. DHJ structure is formed when both processed ends are engaged. This struc- nucleosomes must unwrap and disassemble. Single-molecule studies indicate ture can either be resolved by nucleolytic cleavage (prominent during meiosis) or that the outer stretches of nucleosomal DNA spontaneously unwrap and re- dissolved by the concerted action of RecQ-family helicases (e.g. RecQ, Sgs1, wrap from the histone core, a process known as ‘‘DNA breathing.’’ Using BLM), type I topoisomerases (Top3a, TOP3A) and eukaryotic regulatory pro- the explicit solvent molecular dynamics method, we observed, for the first teins (RMI1 and RMI2), called the BTR complex in humans. The BTR complex time, spontaneous unwrapping of DNA at ultimate, atomic-scale resolution. migrates the HJ branches and decatenates the final structure.This process is neces- Starting from a crystal structure of a nucleosome particle, we built several sary to avoid chromosomal rearrangements in somatic cells as it solely results in variants of solvated nucleosome systems that differed from one another non-crossover products. Although the protein components necessary for DHJ only by the nucleotide sequence of the DNA and/or the ionic conditions. dissolution have been described, little is known about the molecular/biophysical At physiological salt conditions, the DNA was observed to maintain its high- mechanism of HJ migration. Investigation of these mechanisms requires single- ly bent conformation, stabilized by the contacts between the charged amino molecule techniques to reveal otherwise hidden properties (e.g. processivity,

BPJ 7843_7846 372a Tuesday, February 14, 2017 directionality, pausing, subunit composition specificity). Therefore we prepared RPA is capable of disrupting DNA secondary structure up to ~7 base pairs (bp). and fluorescently labeled all BTR complex components and developed a novel We have previously shown using a novel single molecule total internal reflection l-DNA based HJ structure, which contains a 2x10-kb plasmid doublet ligated fluorescence FRET assay that ssDNA translocases can push an E. coli SSB in a head-to-head orientation and inserted into the middle region of lgt11. This tetramer along ssDNA (Sokoloski et.al, PNAS. 2016). We used this approach to structure enables us to follow HJ migration in a microfluidic flow-cell coupled to- test the hypothesis that RPA can also be pushed by an ssDNA translocase. S. cer- tal internal reflection fluorescence (TIRF) microscope. Simultaneous detection of evisiae Pif1pushes humanCy5-RPAalong (dT)60 towards a 3’-ssDNA end labeled the DNA structure and differently labeled BTR complex components reveals the with Cy3 donor and ultimately displaces the RPA from the ssDNA in an ATP mechanism of HJ migration. The developed structure is also suitable for investi- dependent manner. This result indicates translocase pushing is a general feature gating the alternative processes of dissolution and resolution that are prominent in of SSB proteins. We also assessed the possibility that monomeric Pif1 can push somatic DNA repair and meiosis, respectively. RPA into a duplex causing unwinding in a state where Pif1 alone is not an active helicase. A 18 bp DNA hairpin with an internal Cy3 label 9 bp inand a 5’ (dT)45 tail 1832-Pos Board B152 shows only low FRET efficiency in the presence of sub-nanomolar concentrations Mitochondrial Protein ABF2P Intercalates, Bends, Loops, and Compacts of Cy5-hRPA indicating that the RPA cannot melt sufficient dsDNA to reach the DNA internalCy3. Similarly,whenPif1is addedtothis DNAunderconditions wherethe Divakaran Murugesapillai1, Arka Chakraborty2,Se´bastien Lyonnais2, bound population is entirely monomeric (<1 nM), there’s no sign of duplex un- Maria Sola`2, Mark C. Williams1. winding in the presence of ATP. However, when Pif1 and ATP are added to this 1 Department of Physics, Northeastern University, Boston, MA, USA, Cy3-DNA/Cy5-hRPA complex, high FRET signals are observed, indicating inva- 2 Department of Structural Biology, Molecular Biology Institute of Barcelona sion of the Cy5-hRPA into the duplex. These results suggest a new mechanism for (CSIC), Barcelona, Spain. duplex unwinding where an ssDNA translocase can push RPA into dsDNA Abf2p is an abundant yeast mitochondrial high-mobility group box (HMGB) pro- causing base pair melting and strand separation. (TML: NIH GM030498/ tein, containing two HMGB domains, or boxes, facilitating binding and bending of GM045948, RG: NIH GM098509, JES: ACS PF-15-040-01-DMC). DNA. Like most HMGB proteins, Abf2p is an architectural DNA binding protein, and it also plays an essential role in maintaining the mitochondrial genomic DNA. 1835-Pos Board B155 To understand how Abf2p proteins interact with DNA, we use optical tweezers and How Protein Deformation Affects DNA Loop Topology and Geometry atomic force microscopy in a liquid environment to probe these interactions. With Pamela J. Perez, Wilma K. Olson. optical tweezers, we characterize the binding of these proteins to DNA. We observe Center for Quantitative Biology, Rutgers University, Piscataway, NJ, USA. that the effective contour length of the DNA has increased, likely due to DNA inter- Genomes are much more complex than a linear sequence of four different nucle- calation by one or more of its abundant aromatic residues. The force-extension otides. DNA organization in 3-D space is fundamental to its function. Partitioning curves resemble those from DNA-intercalator complexes. Furthermore, by quanti- long segments of DNA into topological domains by protein-mediated loops is fying the force-dependent binding we determine an equilibrium dissociation con- often utilized to control its spatial arrangement. A simple experimental model of stant of 150 nM. The strong intercalation and bending of DNA leads to a highly this is the formation of a nucleoprotein complex resulting from the binding of flexible DNA-Abf2p complex with a persistence length of 2 nm at saturating con- the Lac repressor protein to two distant operator sites on a short closed circular centration. Also, Abf2p induces DNA loop formation when DNA is held at low DNA molecule. The resulting mini-circle is separated into two topologically inde- forces. When pulled quickly, we are able to break these loops. Pulling rate depen- pendent DNA loops such that the level of supercoiling in one domain is indepen- dence measurements reveals a low loop dissociation timescale of ~ 20 s. Moreover, dent of the other. We have developed a computational method to predict a AFM experiments show dynamic formation of loops on the surface in real time. The preferred spatial arrangement for each isolated domain. The model system is con- timescale for loop formation is on the order of minutes. To dissect the mechanism structed by attaching two DNA loops to opposing ends of each of two operators by which the protein performs its function, we perform experiments with Abf2p bound to the Lac repressor protein. The model is further modified to investigate mutants. We observe that the properties of Box1 with N-terminal domain mutant the role of protein deformation on the constrained circular DNA. An abundance is similar to wild-type Abf2p. However, in the presence Box 2 mutant, we don’t of studies of the Lac repressor suggest that it can undergo wide fluctuations, ex- observe intercalation but a decrease in persistence length. AFM images confirm tending angle between the two dimeric arms of the V-shaped protein to as much looping for Box1 with N-terminal domain, and we show that Box2 alone does as 180 deg. in the absence of bound DNA. Simulating the distortion of the protein not mediate looping. Taken together, our results suggest a unique mechanism to providesnew spatial constraints on eachunbound DNA segmentofthe mini-circle. compact the mitochondrial genomic DNA through DNA intercalation and looping. Analysis of the predicted looping architecture provides insight into understanding the sensitivity of loop topology and geometry to changes in protein structure. 1833-Pos Board B153 Towards Rolling-Circle Replication at the Single-Molecule Level 1836-Pos Board B156 Cesar L. Pastrana1, Carolina Carrasco1, Parvez Akthar2, Sanford H. Leuba2, Topoisomerase Poison Disturbs Relaxation of Mechanical Stresses During Saleem A. Khan2, Fernando Moreno-Herrero1. Gating Motion of DNA-Binding Core 1,2 1 1CNB-CSIC, Madrid, Spain, 2University of Pittsburgh, Pittsburgh, PA, USA. Nan-Lan Huang , Michael K. Gilson . 1Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Many bacterial plasmids replicate by an asymmetric rolling-circle mechanism that 2 requires sequence-specific recognition for initiation, nicking of one of the template California, San Diego, La Jolla, CA, USA, Academia Sinica, Taipei City, DNA strands and unwinding of the duplex prior to subsequent leading strand DNA Taiwan. synthesis. Nicking is performed by a replication-initiation protein (Rep), which Topoisomerases II (Top2) are enzymes that relieve the excessive supercoiling also recruits PcrA helicase to the plasmid origin to initiate the unwinding. Here, that accompanies DNA replication and gene transcription. They do this by we have used magnetic tweezers to directly measure DNA nicking-religation ac- generating a transient double-strand break, passing through another DNA tivity of RepC and the unwinding activity of PcrA, the replication initiator protein segment, and resealing the cleavage afterwards. Anticancer agents such as eto- and the helicase involved on the replication of plasmid pT181, respectively. We poside work by blocking the resealing step, leading to chromosome breakage found that nicking by RepC occurred only in negatively supercoiled DNA and and cell death. These drugs, known as topoisomerase poisons, generate serious this activity was force- and twist-dependent. This suggests that the supercoiling side-effects due to their non-selective action against different top2 isoforms. state of the DNA is a switch for RepC nicking activity and thus for PcrA loading In order to elucidate the mechanism and selectivity of these drugs, we previ- and unwinding. We measured PcrA translocation rates in the presence of single- ously carried out long time-scale molecular dynamics (MD) simulations of stranded DNA binding proteins (SSB), finding that PcrA is a slow and processive drug-entrapped and drug-free complexes of Top2 bound to a cleaved DNA helicase in the presence of RepC capable to unwind plasmid-sized substrates. segment. We observed ‘‘frozen’’ behaviours of the Top2-DNA complex in These results constitute a promising starting point to fully recapitulate the the presence of drugs, in contrast with a closing motion in the drug-free com- rolling-circle replication mechanism at the single molecule level. plex. In order to delineate how Top2 relaxes stresses generated during DNA transformation in resealing, and how drug binding impacts on this function, 1834-Pos Board B154 we have used the CAMS package to compute mechanical stresses at the atom- Superfamily 1 Helicase PIF1 Can Push Replication Protein a Along Single istic level for the drug-bound and drug-free complexes. We inspect the occur- Stranded DNA and into Duplex DNA rence of compressive and tensile stresses over time, and decompose their Joshua E. Sokoloski, Roberto Galletto, Timothy M. Lohman. difference into contributions from potential terms in an additive force field Biochemistry and Molecular Biophysics, Washington University in St. Louis, including bond, angle, dihedral, van der Waals, and electrostatic potentials. St. Louis, MO, USA. By studying stress distributions at the sub-molecular level, we anticipate a Superfamily 1 helicase monomers are single stranded (ss) DNA translocases, but deeper understanding of the mechanism of DNA resealing by Top2, as well poor helicases. Replication protein A (RPA) is an ssDNA binding protein (SSB) as insights that to guide experimental design and the development of new com- that binds ssDNA with high affinity and can diffuse along ssDNA. On its own, pounds for isoform-selective inhibition of these enzymes.

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Chromatin and the Nucleoid II tail truncated structures exhibited that existing of either tails can stabilize nucleosome in closed conformation, but the truncation of both tails disrupts 1837-Pos Board B157 both DNA-tail-DNA bridges and induces the transition from a closed to an Compaction and Segregation of Sister Chromatids by Loop-Extruding open conformation. Enzymes In addition, we studied the impact of acetylation on H3 tail using an enhanced Anton Goloborodko1, Maxim Imakaev1, John F. Marko2, Leonid Mirny3. sampling method. The results show that irrespective of the presence or absence 1MIT, Cambridge, MA, USA, 2Department of Physics and Astronomy, of the acetylation, the H3 tail remains in contact with the DNA. Acetylation Northwestern University, Evanston, IL, USA, 3Institute for Medical slightly weakened the interaction between the tail and DNA, and enhanced Engineering and Science, MIT, Cambridge, MA, USA. a-helix formation, resulting in a more compact tail conformation. This trend During cell division, two copies of each chromosome are segregated from each became stronger with increasing number of acetylation within the H3 tail. other and compacted more than hundred-fold into the canonical X-shaped We inferred that this compaction induces unwrapping and exposure of the structures. According to earlier microscopic observations and the recent Hi-C linker DNA, enabling DNA-binding proteins (e.g., transcription factors) to study, mitotic chromosomes are compacted into arrays of consecutive loops bind to their target sequences and/or changing chromatin dynamics and of ~100 kb. Mechanisms leading to formation of such loop arrays are largely conformation. unknown. It has been proposed that SMC complexes involved in chromosome 1840-Pos Board B160 condensation can extrude chromatin loops, leading to formation of arrays of Extent of Nucleosome Destabilization Governs Yeast HMGB Cellular consecutive loops. Here, we use computer simulations to test whether the pro- Function posed loop-extruding activity of SMCs could compact and segregate sister Micah J. McCauley1, Ran Huo1, Nicole Becker2, Molly Nelson Holt2, chromatids. First, we model the system of loop-extruding SMCs on a chromatin Uma Muthurajan3, Ioulia Rouzina4, Karolin Luger3, L. James Maher2, fiber and show that it self-organizes into an array of consecutive dynamic loops. Nathan Israeloff1, Mark Williams1. The structure of this array depends on microscopic properties of loop-extruding 1Physics, Northeastern University, Boston, MA, USA, 2Biochemistry and SMCs and can be predicted with a simple analytical model. Second, we model Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA, the process of loop extrusion in 3D and show that, coupled with the topo II 3Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA, strand-passing activity, it leads to robust compaction and segregation of sister 4Chemistry and Biochemistry, The Ohio State University, Columbus, chromatids. We show that loop-extruding proteins transform individual chro- OH, USA. matids into bottle-brush structures consistent with the microscopic observations As essential constituents of chromatin, nucleosome core particles package eu- and Hi-C-based models. These bottle-brush chromatids repel each other allow- karyotic genomic DNA. Nucleosome disruption plays a key role in many nu- ing topo II to disentangle them. This mechanism of chromosomal condensation clear processes including transcription, DNA repair and recombination. and segregation does not require additional proteins or specific DNA markup Although high mobility group (HMGB) proteins are known to facilitate these and is robust against variations in SMC number and properties. processes, the specific interactions between nucleosomes and HMGB proteins, 1838-Pos Board B158 and how these interactions determine protein function in the cell, are not well Polymer Models Integrate Inverted Nuclear Geometry with Conventional understood. This work combines atomic force microscopy (AFM) and optical HI-C Compartmentalization tweezers (OT) experiments to determine the extent to which HMGB proteins Martin J. Falk1, Natalia M. Naumova2, Geoffrey Fudenberg1, disrupt a chain of 12 core particles separated by 60 base pair segments of linker Yana N. Feodorova3, Job Dekker2, Leonid A. Mirny1, Irina Solovei4. DNA. We find that both the double box yeast Hmo1 and the single box yeast 1Physics, Massachusetts Institute of Technology, Cambridge, MA, USA, Nhp6A show a strong binding preference for nucleosomes and that both 2UMass Medical School, Worcester, MA, USA, 3Medical University of HMGB proteins destabilize nucleosomes. Furthermore, core particle dispersion Plovdiv, Plovdiv, Bulgaria, 4LMU Munich, Munich, Germany. is driven by disruption of the outer turn as DNA is unwound from the core par- The organization of interphase nuclei differs dramatically across cell types in a ticle up to strong binding regions that bookend the two halves of the inner turn. functionally-relevant fashion. A striking example is found in the rod photore- However, unlike Nhp6A, Hmo1 completely breaks one of these regions and ceptors of nocturnal mammals, where the conventional nuclear organization also releases half of the inner turn. The significantly different extent of nucle- is inverted. In particular, in murine rods, constitutive heterochromatin is osome destabilization for these two yeast HMGB proteins explains why they packed into a single chromocenter in the nuclear center, which is encircled are localized to different types of genomic sites. Thus, HMO1 proteins are by a shell of facultative heterochromatin and then by an outermost shell of found primarily in highly transcribed genomic regions that code for ribosomal euchromatin. This arrangement converts rod nuclei into microlenses, reducing proteins, which likely require nucleosomes to be actively destabilized by light scattering and thereby facilitating nocturnal vision. Surprisingly, Hi-C HMO1, as observed here. In contrast, Nhp6A is not localized to such regions, maps of conventional and inverted nuclei display remarkably similar compart- pointing to a significant difference in cellular function that correlates with the mentalization. Here, we simulate a de novo polymer model that is capable of protein’s measured nucleosome destabilization capability. replicating both conventional and inverted geometries while preserving the pat- terns of compartmentalization as observed by Hi-C. In this model, chromatin is 1841-Pos Board B161 a polymer composed of three classes of monomers arranged in blocks repre- As a Nucleus Enters a Small Pore, Chromatin Stretches and Maintains senting constitutive heterochromatin, facultative heterochromatin, and euchro- Integrity Even with DNA Breaks 1 1 1 2 matin. Different classes of monomers have different levels of attraction to each Jerome Irianto , Yuntao Xia , Charlotte R. Pfeifer , Roger A. Greenberg , Dennis E. Discher1. other and to the nuclear lamina. Our results indicate that preferential interac- 1 2 tions between facultative heterochromatin and constitutive heterochromatin SEAS, University of Pennsylvania, Philadelphia, PA, USA, Cancer provide a possible mechanism to explain nuclear inversion when association Biology, Abramson Family Cancer Research Institute, Perelman School of with the lamina is lost. Medicine, University of Pennsylvania, Philadelphia, PA, USA. As a cell enters a small constriction and pulls its nucleus through, the chromatin 1839-Pos Board B159 must distort and somehow maintain genomic stability despite ever-present dou- Distinct Roles of H3 and H2A Tails in Linker DNA Dynamics ble strand breaks in the DNA. Here we visualize within a living cell the pore- Zhenhai Li1, Jinzen Ikebe1, Shun Sakuraba2, Hidetoshi Kono1. size dependent deformation of a specific locus engineered into chromosome-1 1National Institutes for Quantum and Radiological Science and Technology, and cleaved. An mCherry-tagged nuclease targets the sub-micron locus, Kizugawa, Japan, 2The University of Tokyo, Kashiwashi, Japan. causing DNA cleavage and recruiting repair factors such as GFP-53BP1 to a Nucleosome breathing potentially increases the DNA exposure, which in turn large region around the locus. Aspiration of a cell and its nucleus into a micro- recruits DNA-binding protein and regulates gene function. Numerous studies pipette shows chromatin aligns and stretches parallel to the pore. Extension is have shown the critical roles of N-terminal tails of histones H3 and H4 in largest in small pores, increasing more than 10-fold but remaining 30-fold gene expression; however, few studies have focused on the H2A C-terminal shorter than the DNA contour length in the locus. Brochard and de Gennes’ tail. Here we present thorough computational studies on a single nucleosome blob model for tube geometry fits the data, with a simple modification for particle showing the linker DNA closing and opening, which is thought to be chromatin crowding. Continuity of the highly extended, cleaved chromatin is nucleosome breathing. With our simulation, the H2A C-terminal and H3 N-ter- also maintained, consistent with folding and cross-bridging of the DNA. minal tails were found to modulate the nucleosome conformation differently. Surprisingly, extensional integrity is unaffected by an inhibitor of the DNA The H2A C-terminal tail regulates nucleosome conformation by binding to repair scaffold. Nevertheless, large extensions in small pores were not observed linker DNA at different locations, whereas the H3 N-terminal tail regulates with ‘‘dead’’ nuclease, suggestive of a compromised or modified structure in linker DNA by binding to it in different patterns. Further MD simulation on damaged DNA.

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1842-Pos Board B162 the effect of the binding and reaction is included through the bead shape, which Interphase Chromatin Dynamics in Response to Local DNA Damage depends on the phase. Processive reactions e.g. by RNA polymerases along the Jonah Eaton, Alexandra Zidovska. DNA can be also modeled in this scheme. We observed both positive- and Department of Physics, New York University, Center for Soft Matter negative-feedback effects of the reaction-dependent conformational changes Research, New York, NY, USA. to the reaction rate, depending on the conditions. In this presentation, we partic- The dynamic organization of chromatin inside the eukaryotic cell nucleus is ularly focus on the cases where the reactivity of the chain is not uniform. Rele- essential to the regulation of the genome such as maintaining its integrity, vance to such heterogeneity in actual chromatin structures, such as DNA gene expression and replication. However, the physical picture of chromatin sequences and histone modifications, will be also discussed. structure and dynamics remains unclear and requires identifying the dynamical and structural signatures of specific biological processes. In this work, we study 1845-Pos Board B165 the chromatin dynamics in interphase, time between two cell divisions, in Investigating Physical Interactions of the Liquid-Like Nucleolus with response to DNA damage. As shown previously, chromatin dynamics across Interphase Chromatin the whole nucleus change dramatically after DNA damage; chromatin’s Christina Caragine, Shannon Haley, Alexandra Zidovska. coherent motion is eliminated, while its local mobility increases and its Center for Soft Matter Research, Department of Physics, New York compaction decreases [1]. Here, we explore the local and global chromatin dy- University, New York, NY, USA. namics in response to local DNA damage in human cells. To do so, we induce Recently, it has been reported that nucleoli and other subnuclear bodies such as double stranded DNA breaks (DSBs), which we visualize using 53BP1- p-granules in the cell nucleus form by liquid-liquid phase separation in frog oo- mCherry, and observe the spatiotemporal evolution of their dynamics using sin- cytes and C. elegans embryos (1,2). Such studies in mammalian cells are lack- gle particle tracking. Simultaneously, we use histone H2B-GFP as a reporter on ing. While the fusion of nucleoli in human cells has been observed before, its chromatin position to monitor the global chromatin dynamics using displace- origin remains unclear (3,4). Moreover, the role of the chromatin polymer in the ment correlation spectroscopy (DCS, [1]). To elucidate the dynamic signature facilitation of nucleolar fusion is completely unknown. In this work, we studied of the local and global DNA damage response, we vary the number of DSBs as nucleoli and their physical interaction with chromatin in human cells. First, we well as explore the ATP-dependence of these processes. Our preliminary re- investigated the aggregation process dictating the assembly of nucleoli and sults show that coherent motion of chromatin is lost with an increasing DSB found it to be consistent with fusion of liquid droplets found previously in other count, while the local subdiffusive dynamics of the DSBs remains unchanged. cells. Further, we analyzed nucleolar fusion events and found that after fusion, [1] Zidovska, Weitz, Mitchison (2013). PNAS, 110( 39), 15555-60. the eccentricity of the new nucleolus decreases, indicating the nucleolus un- dergoes a structural change and behaves as a fluid-like structure. Historically, 1843-Pos Board B163 the nucleolar assembly in mammalian cells was believed to complete within Analyzing DNA Double-Strand Break Repair Processes with High- two hours after mitosis, when the final number of nucleoli forms via de novo Resolution Localization Microscopy and Persistent Topology assembly as well as fusion events (3). However, we found that fusion events Andreas Hofmann1, Matthias Krufczik2, Michael Hausmann2, can also occur later in the cell cycle. To explore the interaction of nucleoli Dieter W. Heermann1. with chromatin, we altered chromatin state using biochemical perturbations. 1Institute for Theoretical Physics, Heidelberg University, Heidelberg, We found that chromatin is indeed involved in the positioning, shape and align- Germany, 2Kirchhoff Institute for Physics, Heidelberg University, ment of nucleoli inside the cell nucleus. Heidelberg, Germany. 1. Brangwynne, CP, et al. Science 2009, 1729 In the past years, research in the field of genome dynamics has revealed that the 2. Brangwynne, CP, et al. Proc. Natl. Acad. Sci. 2009, 4334 genome and its underlying constituents are not randomly structured. Novel 3. Savino, TM, et al. J. Cell Biol. 2001, 1097 fluorescence light microscopic techniques enable optical resolutions down to 4. Farley, KI, et al. Chromosoma 2015, 323 the order of 10nm and open new insights into genome structure and organiza- 1846-Pos Board B166 tion. We irradiated cells of breast cancer cell lines with different doses. Specific Influence of Local DNA and Protein Features on Large-Scale Properties of fluorescently labeled antibodies were used to target histone modifications Chromatin which are indicators for heterochromatin (H3K9me3). In addition g-H2AX Stefjord Todolli, Nicolas Clauvelin, Wilma K. Olson. was tagged by antibodies. Localization microscopy uses two different spectral Center for Quantitative Biology, Rutgers University, Piscataway, NJ, USA. states of dye molecules to achieve optical separation and thus spatial isolation DNA in eukaryotic cells is packaged into a hierarchical chromatin structure, of single molecule positions. Spectral Position Determination Microscopy with assistance from proteins, in order to fit inside the nucleus. While highly (SPDM) applied here works with conventional fluorophores in 3D conserved compacted, this chromatin structure is quite dynamic and local variations at cell nuclei and does not require any additional staining or cumbersome sample the smallest scale have a compounding effect on higher-order structures. Nucle- preparation. With this super-resolution microscopy, we are able to determine osome spacing, the presence of nucleosome-free DNA regions and linker his- the positions of specifically labeled molecules and to calculate density and dis- tones contribute to the structure and behavior of chromatin, and consequently tance distributions thereof. This data allows to quantify the complex changes of have an impact on biological processes such as gene expression. We have the chromatin which we find after irradiation caused by the damage and the used a meso-scale representation of chromatin in Monte Carlo simulations of arising repair mechanism. Our aim is to firstly identify regions of active repair precisely positioned nucleosomal arrays to understand the effect of nucleosome processes and subsequently, to analyze both the heterochromatin density and its spacing and the presence of nucleosome-free gaps on large-scale properties of morphology around these clusters in order to gain new insights on the interplay chromatin. Our simulations show a highly diverse chromatin fiber, beyond the of the repair mechanism and heterochromatin. To this end, we developed a new widely referenced solenoid and zigzag models. The presence of nucleosome- analytical methodology to also characterize the morphology of heterochromat- free regions and small fluctuations in the positioning of single nucleosomes in by means of persistence topology. The advantage of looking at topological in an otherwise regular structure have dramatic effects on these fibers. Under- properties of the heterochromatin structure is the intrinsic scale invariance standing these effects can have significant biological implications, in that chro- that allows to compare structures without being restricted to a certain length matin fibers in vivo do not have a regular structure. Furthermore, our scale. simulations offer a glimpse into how the presence and positioning of linker his- tones may affect chromatin structure. 1844-Pos Board B164 An Active Chain Model to Consider the Crosstalk between the Structure 1847-Pos Board B167 and Function of Chromatin: Effects of Heterogeneity Linking the Active Undulations of Nuclear Envelope with Surface Fluctu- Yuichi Togashi. ations of the Chromatin Globule Research Center for the Mathematics on Chromatin Live Dynamics Fang-Yi Chu, Shannon Haley, Alexandra Zidovska. (RcMcD), Hiroshima University, Higashi-Hiroshima, Japan. Center for Soft Matter Research, Department of Physics, New York There exist a wide variety of macromolecules binding to DNA, many of which University, New York, NY, USA. work as molecular machines, e.g. RNA polymerases in transcription. The bind- The nuclear envelope (NE) presents a physical boundary between the cyto- ing and operation of these macromolecules may affect the structural organiza- plasm and the nucleoplasm. NE is comprised of two lipid bilayers supported tion of chromatin fibers. In turn, the local structure of chromatin may alter its from inside by nuclear lamina, a network formed by intermediate filaments accessibility and affinity for macromolecules. Thus the crosstalk between the called lamins. The shape and size of the cell nucleus change during the cell cy- structure and function (reaction) of chromatin may arise. To study such cross- cle. It has been shown previously that both lack of lamins as well as inhibition talk, we modeled the complex of DNA and other macromolecules working on it of cytoskeletal filaments activity lead to changes in nuclear shape and stiffness as a bead-polymer chain. Each bead has an internal state variable (phase), and (1,2). In this work, we study nuclear shape fluctuations at short time scales. We

BPJ 7847_7850 Tuesday, February 14, 2017 375a used spinning disk confocal microscopy and observed the fluctuations of the 1850-Pos Board B170 chromatin globule surface (CGS) underneath the NE, visualized by H2B- Mixing of Oxidized and Bilayer Phospholipids GFP, and the NE itself, visualized by LMNA-GFP in human cells. We moni- Radha Ranganathan, Jasmeet Singh, Melissa Ghafarian. tored the shape fluctuations of the cell nucleus and characterized both CGS Physics, California State University Northridge, Northridge, CA, USA. and NE fluctuations during the cell cycle. Remarkably, we found that while Aggregation states of mixtures of the oxidized phospholipid (OxPL), 1-palmi- CGS and NE typically fluctuate in phase, they do exhibit sites, where they toyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) and 1,2-Dipalmitoyl-sn- move out of phase, thus effectively locally separate. To explore the mechanism glycero-3-phosphocholine (DPPC), and 1,2-Dioleoyl-sn-glycero-3-phospho- behind these shape fluctuations, we employed biochemical perturbations. We choline (DOPC), were characterized using a fluorescence quenching assay, found the shape fluctuations of CGS and NE to be ATP-dependent and that dynamic light scattering, time-resolved fluorescence quenching, and electron both the cytoskeletal filaments, specifically actin and microtubules, as well spin resonance in the temperature range 5-60 C. PGPC when present alone as chromatin, are involved in the dynamics of the shape fluctuations of the forms 3.5 nm radii micelles of aggregation number 33. When incorporated cell nucleus. Based on our observations we have developed a model describing into the bilayer forming lipid vesicles, PGPC forms mixed aggregates only their dynamic contributions. in the gel phase and only within a limited composition. In the liquid phase 1. Dahl, KN et al (2008) Circ Res. 102(11): 1307-18. and in the gel phase at higher PGPC mole fractions, complete separation 2. Makhija, E et al (2015) Proc Natl Acad Sci U S A. 13(1): E32-40. into PGPC micelles and lipid vesicles is observed. Results indicate four types of aggregation states depending on temperature and composition. In the 1848-Pos Board B168 gel phase, DPPC and PGPC form mixed vesicles for PGPC molar fraction, How to Open a Nucleosome XPGPC % 0.3 and coexisting vesicles and micelles at higher XPGPC. Data sug- Katalin Toth1, Yoriko Lill2, Kathrin Lehmann1, Alexander Gansen1, gest that liquid phase DPPC at 50 C forms mixed vesicles with segregated or Jo¨rg Langowski1. hemi fused DPPC and PGPC for XPGPC % 0.3. At 60 C, DPPC and PGPC do 1Biophysics of Macromolecules (B040), German Cancer Research Center, 2 not mix, but form coexisting vesicles and micelles. DOPC and PGPC do not Heidelberg, Germany, Single-Molecule Biophysics Group, Purdue mix in any proportion in the liquid phase. Two dissimilar aggregates of University, West Lafayette, IN, USA. the sizes of vesicles and PGPC micelles were observed for all XPGPC for DNA accessibility in the cell nucleus depends on its packaging. The stability of T R 22 C. DOPC-PGPC and DPPC-PGPC mixing is non-ideal for the basic compaction unit, the nucleosome, is regulated through the interactions XPGPC > 0.3 in both gel and fluid phases resulting in complete exclusion of between DNA and histones. Natural sequence variation along the DNA PGPC from the bilayer. Formation of mixed vesicles is favored in the gel strongly modulates nucleosome stability. Histone variations, mutations and phase but not in the liquid phase for XPGPC % 0.3. For XPGPC % 0.3, aggre- posttranslational modifications alter intra- and internucleosomal interactions. gation states change progressively from mixed vesicles in the gel phase to We are investigating interactions which hold nucleosomes together by component segregated mixed vesicles in the liquid phase close to the chain measuring distances in reconstituted samples using single molecule Fo¨rster en- melting transition temperature to completely separated coexisting vesicles ergy transfer. By increasing the salt concentration, thus reducing intramolecular and micelles at higher temperatures. electrostatic interactions, we could study the effect of DNA sequence variation and histone acetylation on nucleosome stability (1,2). Here we present investi- 1851-Pos Board B171 gations on the non-electrostatic interactions influenced by the type of anion. We Direct Imaging of Liquid Domains by Cryotem in Submicron Vesicles found that the stabilizing effect of anions corresponds to the Hofmeister series: Caitlin E. Cornell1, Long Gui2, Kelly K. Lee2, Sarah L. Keller1. kosmotropic ions stabilize strongly, while chaotropic ones destabilize. Using 1Chemistry, University of Washington, Seattle, WA, USA, 2Medicinal different labeling sites, we had previously identified intermediate states upon Chemistry, University of Washington, Seattle, WA, USA. nucleosome opening (3). The chaotropic anion perchlorate increases the pro- The distribution of lipids and proteins within the plasma membranes of living pensity of such an intermediate opening state, probably due to a disruption cells appears to be heterogeneous on short time and length scales. In model of histone-histone interactions. The same intermediate state was promoted by membranes containing ternary or quaternary mixtures of lipids, the presence a histone mutation at the interface between two histones. These results help of submicron domains has been inferred by indirect methods such as FRET, us understanding the dynamics of DNA packaging in nucleosomes. neutron diffraction, and NMR. In cases when the indirect methods rely on References: model-dependent assumptions, it is ideal to verify the results through direct 1) Toth et al 2013 CytometryA 83: 839-846 microscopy. However, direct imaging of submicron membrane domains pre- 2) Gansen et al 2015 NAR 43(3): 1433-43. sents significant experimental challenges. Here we present results from exper- 3) Bo¨hm et al 2011 NAR 38(8): 3093-3102. iments in which we preferentially label lipid domains with electron-dense probes, and we image the domains in submicron vesicles by cryo electron Membrane Physical Chemistry I microscopy.

1849-Pos Board B169 1852-Pos Board B172 Oxidation of Polyunsaturated Phospholipid Decreases the Cholesterol Probe Partition between Liquid-Disordered (LD) and Liquid-Ordered Content at which Cholesterol Bilayer Domains Start to form in Phospho- (Lo) Phases and Investigation of Nanodomain Sizes lipid-Cholesterol Membranes Thais A. Enoki1, Frederick A. Heberle2, Gerald W. Feigenson1. Laxman Mainali1, Mariusz Zareba2, Witold K. Subczynski1. 1Department of Molecular Biology & Genetics, Cornell University, Ithaca, 1Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA, NY, USA, 2Oak Ridge National Laboratory, Oak Ridge, TN, USA. 2Ophthalmology, Medical College of Wisconsin, Milwaukee, WI, USA. If the plasma membrane has coexistence of liquid-disordered (Ld) and liquid- Saturation-recovery electron paramagnetic resonance and differential scanning ordered (Lo) phases, then proteins can be sorted between these phases. This calorimetry were used to determine the cholesterol content at which pure partition of molecules in Ld and Lo phases can be examined with fluorescent cholesterol bilayer domains (CBDs) and cholesterol crystals begin to form in probes in model lipid mixtures that have coexisting domains. Here, we 1-palmitoyl-2-arachidonoylphosphochatidyline (PAPC) membranes. To pre- measured the partition coefficient, Kp, of Bodipy-PC and tryptophan octadecyl serve compositional homogeneity of the membrane suspension at a high choles- ester (TOE) between Ld and Lo phases for three-component lipid mixtures that terol content, multilamellar liposome suspensions were prepared using the exhibits macroscopic phase separation, 1,2-distearoyl-sn-glycero-3-phospho- rapid solvent exchange method. The cholesterol content in membranes was choline (DSPC)/ 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/ Choles- changed from 0 mol% to 66 mol%. With spin-labeled cholesterol analogs, it terol (Chol) and brain sphingomyelin (bSM) /DOPC/Chol, and for mixtures was shown that in these membranes the CBDs begin to form at ~33 mol% that exhibit nanoscopic phase separation, DSPC/ 1-palmitoyl-2-oleoyl-sn-glyc- cholesterol, preceding formation of cholesterol crystals (formed at 50 mol% ero-3-phosphocholine (POPC)/Chol and bSM/POPC/Chol. We observed that cholesterol). Membranes were subjected to oxidative modification through the Kp of Bodipy-PC smoothly changes for the four component system, the iron-induced lipid peroxidation, and the extent of oxidative modification DSPC/DOPC/POPC/Chol, as DOPC is replaced by POPC. In contrast, along (formation of phospholipid hydroperoxides) was monitored using an iodomet- this trajectory we observed an abrupt transition in the Ld and Lo domain sizes. ric assay. It was shown that oxidative modification of polyunsaturated PAPC Here we discuss three different methods to measure the partition coefficient: molecules significantly decreases the cholesterol content at which CBDs and (i) Single-dye fluorescence; (ii) FRET experiments along a thermodynamic tie- cholesterol crystals start to form. Biological and biomedical relevancies of line; and (iii) dye partitioning on GUVs. FRET experiments are a powerful the obtained results are discussed. method to detect the coexistence of phase Ld and Lo, even with domain sizes Acknowledgments: This work was supported by grants EY015526, EB001980, below optical resolution. We modeled the experimental FRET data with Monte and EY001931 from the National Institutes of Health. Carlo simulations, assuming monodisperse sizes of domains, and known Kp

BPJ 7847_7850 376a Tuesday, February 14, 2017 values of donor and acceptor probes. Since FRET depends on the distance be- 1856-Pos Board B176 tween donors and acceptors, and this distance depends upon the domain size, Physical Chemical Properties of Silver Nanoparticles Stabilized with Poly- this study makes possible the measurement of nanodomain size. We found ether-Block-Amide Interacting with Cellular Membrane Models at the domain sizes of 10.0 5 2.5 nm of radius for nanodomains in DSPC/POPC/ Air-Water Interface Chol and bSM/POPC/Chol. Luciano Caseli, Gustavo B. Soriano, Roselaine S. Oliveira, Fernanda F. Camilo. 1853-Pos Board B173 Ciencias Exatas e da Terra, Universidade Federal de Sao Paulo, Diadema-SP, Chemical Gradients Trigger and Guide Movement of Giant Lipid Vesicles Brazil. Baharan Ali Doosti, Tatsiana Lobovkina. It is known that silver nanoparticles can be applied as antimicrobial agent, used Chalmers, Gothenburg, Sweden. in hygiene and surgical products. However, the molecular mechanism by which Directed cell movement is one of the central biological processes, and is they act in the plasma membrane and in the cell wall is not completely under- vital for early embryonic development, wound healing, and spreading stood in detail. For this reason, it is relevant to find models by which studying of cancer. Migration of cells is often directed by chemical signals and such interactions is possible. One of these models is the formation of lipid involves various cell components. The cytoskeleton and proteins in the mem- Langmuir monolayers at the air-water interface, in which half a membrane brane are featured as the key players. However, little is known about the can be mimicked with good control of the chemical composition, surface den- response of lipids to chemical cues. To address this question, we used giant sity and viscoelasticity parameters. In this present work, we investigated the lipid vesicles as the cell membrane model system, that were deposited on a interaction between silver nanoparticles stabilized with polyether-block-amide glass substrate. We show that chemical gradients trigger and guide the move- (PEBA) dispersed in 1-butyl alcohol with Langmuir monolayers of dipalmitoyl ment of entire lipid vesicles, mimicking the cell body translocation. Our results phosphatidyl choline (DPPC). The interaction was investigated with surface are important for understanding the membrane response during migratory cell pressure-area isotherms, polarization modulation infrared reflection-absorption behavior. spectroscopy (PM-IRRAS), and Brewster angle microscopy (BAM). The nano- particles expanded the DPPC monolayers, and changed the hydrophobic bands 1854-Pos Board B174 of the lipid observed in the vibrational spectra. Data of BAM show the forma- Modeling Tissue Specific Plasma Membranes in Silico tion of aggregates due to insertion of the nanoparticles. These behaviors found Helgi I. Ingolfsson, Timothy S. Carpenter, Felice C. Lightstone. for the mixed monolayer are probably attributed to the high surface activity of Lawrence Livermore National Laboratory, Livermore, CA, USA. the nanoparticles. However, in the absence of the lipid, there is no formation of The detailed lipid organization of cellular membranes remains rather elusive. real Langmuir film of the nanoparticles, being its surface activity governed by a A typical plasma membrane contains hundreds of different lipid species that dynamic process of adsorption and desorption during the compression and the are actively regulated by the cell. This is far greater diversity than is needed rearrangement of the molecular system of the metallic nanoparticle and the to maintain bilayer barrier properties and to solvate membrane proteins. stabilizing agent (PEBA), leading the formation of aggregates. We believe Marked differences are found in the lipids composition of different cells and these results can provide an interesting clue to understand the role of the nano- tissue types. How do these tissue specific differences affect the overall lipid particle and stabilizing agent on its possible action against microbial cellular organization or the average bilayer properties? membranes. We start to address these questions by modeling biologically realistic plasma membranes (PM) from different tissues using coarse-grained Martini molecular 1857-Pos Board B177 dynamics simulations. Lipidomics literature for both whole cell and PM frac- Membrane Permeation of Gasotransmitters tions was explored and characteristic differences between different tissues Christopher N. Rowley. identified. Biologically realistic idealized PMs for a few tissue types were con- Department of Chemistry, Memorial University of Newfoundland, St. John’s, structed based on their identified differences and previous simulation work on NL, Canada. an idealized mammalian membrane (Ingo´lfsson, et al. Lipid organization of the NO, CO, and H2S are endogenous gasotransmitters. Using the solubility-diffu- plasma membrane. JACS, 136:14554-14559, 2014). Multiple large-scale simu- sion model [1], we calculated the membrane permeability of these molecules lations (multi microsecond long and ~0.1 square micron large) were preformed; across model bilayers using molecular dynamics simulations. Replica ex- providing a high-resolution view of the lipid organization of these different change MD [2] and Generalized Langevin methods [3] were used to calculate bilayers. We analyzed a variety of membrane physicochemical properties, the potential of mean force and diffusion coefficient profile of permeation, including: lipid-lipid interactions, bilayer bulk material properties, domain for- respectively. The membrane permeability of these compounds were all pre- mation and coupling between the bilayer leaflets, and evaluate the main differ- dicted to be very high (> 1 cm/s) [4], indicating these molecules can cross ences between the different tissue types. membranes freely. Hydrophobicity and high diffusivity are consistent features This work performed under the auspices of the U.S. Department of Energy of the gasotransmitters, which allows them to reach their intracellular targets by Lawrence Livermore National Laboratory under Contract DE-AC52- quickly. 07NA27344. Release number: LLNL-ABS-703500. [1] Awoonor-Williams, Rowley BBA - Biomembranes, 2016, doi: 10.1016/ j.bbamem.2015.12.014 1855-Pos Board B175 [2] Lee, C., et al. J. Chem. Inf. Model. 2016 doi: 10.1021/acs.jcim.6b00022 Phospholipid Head Groups Provide Differentially Stable Membrane Envi- [3] Gaalswyk, K., Awoonor-Williams, E., Rowley, C. N., J. Chem. Theory ronments for Vitamin E Comput. 2016, doi: 10.1021/acs.jctc.6b00747 Andres T. Cavazos, Michaela E. Bell, Zachary L. Leach, Jacob J. Kinnun, [4] Riahi, S., Rowley C.N. J. Am. Chem. Soc. 2014, doi: 10.1021/ja508063s Stephen R. Wassall. Department of Physics, IUPUI, Indianapolis, IN, USA. 1858-Pos Board B178 Vitamin E (a-tocopherol) is a lipid-soluble antioxidant that has a primary role Effects of Ester and Ether Linkage in Phospholipids on Ld D Lo Domain of protecting phospholipids from oxidation in membranes. Whether there is Size Transition for a Four-Component Lipid Bilayer Mixture preferential interaction between a-tocopherol and specific phospholipids to Wen-Chyan Tsai, Gerald W. Feigenson. optimize this function has been a longstanding question. Here we compare Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA. the effect of a-tocopherol on the molecular organization of two oleic A coexisting liquid disordered (Ld) and liquid ordered (Lo) region in the four- acid-containing phospholipids, 1-palmitoyl-2-oleoyl-sn-glycerophosphatidyle- component bilayer mixture, distearoyl-phosphatidylcholine/dioleoyl-phospha- thanolamine (16:0-18:1PE, POPE) and 1-palmitoyl-2-oleoyl-sn-glycerophos- tidylcholine/stearoyl-oleoyl-phosphatidylcholine/cholesterol (DSPC/DOPC/ phatidylcholine (16:0-18:1PC, POPC) in mixtures with 5, 10 and 20 mol % SOPC/CHOL), displays a transition from nanoscopic to macroscopic lipid a-tocopherol. By solid-state 2H NMR spectroscopy, we directly observed domains. Giant unilamellar vesicles (GUVs) were generated using the electro- POPC-d31 and POPE-d31 (analogs of POPC and POPE with a perdeuterated formation technique and visualized with an inverted fluorescent microscope. sn-1 chain) in the mixed membranes. The spectra observed with POPE-d31 C12:0-DiI (0.02 mol%), partitioning into the Ld phase, was used for visualiza- in the presence of a-tocopherol consist of a superposition of two spectral com- tion of lipid domain patterns. At mole fraction 0.45/0.31/0.24 = DSPC/ ponents that we ascribe to a-tocopherol promoting a transition from lamellar (DOPCþSOPC)/CHOL, a transition was observed by tuning the compositional (La) to inverted hexagonal (HII) phase. This transition is not observed in mix- ratio of DOPC to SOPC from 0.25 to 0.6. The influence of phospholipid ether tures of POPC-d31 with a-tocopherol where only a single spectral component linkages on the domain size transition is elucidated using ether-linked DSPC due to the La phase was recorded. The molecular origin of the differential in and DOPC in the four-lipid mixture. Our goal is to evaluate the influence bilayer stability between POPE and POPC will be discussed in terms of head due to varying dipole-dipole repulsion in the phospholipid membrane contain- group spacing and the ability to accommodate a-tocopherol. ing ester vs ether-linked phospholipids. The results presented in the study

BPJ 7847_7850 Tuesday, February 14, 2017 377a provide a more detailed mechanism of how a cell controls its raft -nonraft impact of asymmetry on the liquid-liquid demixing phase transition, on order, compartmentalization. and on clustering. Partitioning and clustering across from ordered and disor- dered phases are studied. The effect of varying cholesterol concentration is 1859-Pos Board B179 explored. Oxidation of Cholesterol Changes the Permeability of Lipid Membranes Waldemar T. Kulig1,2, Heikki Mikkolainen2, Agnieszka Olzynska3, 1862-Pos Board B182 Piotr Jurkiewicz3, Lukasz Cwiklik3,4, Tomasz Rog1,2, Martin Hof3, The Mechanism of Copper-Induced Peroxidation Ilpo Vattulainen1,2, Pavel Jungwirth2,4. Dov A. Lichtenberg, Ilya Pinchuk. 1Department of Physics, University of Helsinki, Helsinki, Finland, Tel Aviv University, Tel Aviv, Israel. 2Department of Physics, Tampere University of Technology, Tampere, In spite of the great interest and intensive research effort, the actual mechanism Finland, 3J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of copper-catalyzed peroxidation is debatable. It might have been initiated via of the Czech Republic, v. v. i., Prague, Czech Republic, 4Institute of Organic consecutive reduction of Copper(II) by lipid hydroperoxide (r.1) followed by a Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Fenton-like reaction of Copper(I) with hydroperoxide (r.2). Prague, Czech Republic. The sum of reactions r.1 and r.2 gives r.3 , , The function of living systems is inherently based on cells, which are the key 2LOOH / LO2 þ LO þ H2O building blocks of living organisms. Each cell in a human body is surrounded r.3 by a cell membrane, which essentially is a functional interface separating the However, reaction r.1 is ‘‘thermodynamically unfavorable’’ because the reduc- , cell from its surroundings. Biological membranes consist of lipid bilayers, in tion potential of LO2 (0.77V - 1.44V), is higher than the reduction potential of which a plethora of different proteins is embedded. Cholesterol molecules Cu2þ/Cu1þ (0.16V). The commonly accepted paradigm is therefore that the 2 commonly present in lipid bilayers are largely susceptible to oxidation. This steps mechanism (r.1; r.2} is inconsistent with the experimentally observed per- oxidation leads to oxysterols which play a crucial role in many regulatory pro- oxidation and that some additional agent(s) are involved in reducing Copper(II) cesses in a human body. They are produced naturally from cholesterol during to Copper(I). Nevertheless, this argument is not valid for systems far from equi- either enzymatic side chain hydroxylation (catalysed by cytochrome P450) or librium state. Furthermore, the overall dismutation process (r.3) is thermody- non-enzymatic oxidation (due to direct exposure of cholesterol to reactive ox- namically favorable, so that the reaction may occur either via the two steps ygen species). Oxysterols differ from cholesterol by the presence of additional mechanism or through an intermediate complex that undergoes intramolecular polar groups that are typically hydroxyl, keto, hydroperoxy, epoxy, or carboxyl rearrangement without formation of intermediate Copper(I). moieties. Like cholesterol, many oxysterols are hydrophobic and hence Notably, the two mechanisms of r.3 may be expected to result in different confined to cell membranes. However, small chemical differences between overall reaction orders. Initiation via consecutive reactions r.1 and r.2 is ex- the sterols can significantly affect how they interact with other membrane com- pected to result in a first order kinetics of initiation with respect to hydroper- ponents, and this in turn can have a substantial effect on membrane properties. oxide concentration, whereas formation of copper-hydroperoxide complex(es) Using several experimental techniques, including dynamic light scattering and may result in higher order initiation reactions. Experimentally, the depen- time-resolved fluorescence spectroscopy, together with atomistic molecular dy- dence of peroxidation on the concentration of hydroperoxides accords with namics simulations, we characterized the behaviour of oxysterols in phospho- second order kinetic law far better than with the first order. This is consistent lipid membranes and compared the resulting data to that of cholesterol. We with the observation that continuous peroxidation of lipids within the given found that the permeability of lipid bilayers changes drastically (as compared lipoprotein particle requires the presence of two hydroperoxide molecules to cholesterol) when tail-oxidized sterols are present, meanwhile this effect per lipoprotein. It is also consistent with the effects of deuteration of LDL was not observed in systems containing ring-oxidized sterols. Here, we ratio- on its peroxidation. nalize the different behaviour of various oxysterol classes based on both exper- imental data and molecular dynamics simulations. 1863-Pos Board B183 F4,5GWALP Partitioning, Orientation, and Effect on Bending Moduli in 1860-Pos Board B180 Models of the Plasma Membrane Pre-Transition Effects Mediate Forces of Assembly between Transmem- Rebecca D. Usery1, Thais A. Enoki1, Vanessa P. Nguyen2, brane Proteins: Recent Results on the Orderphobic Effect Barrera N. Francisco2, Gerald W. Feigenson1. Shachi Katira, Kranthi K. Mandadapu, David Chandler. 1Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA, Chemistry, University of California, Berkeley, Berkeley, CA, USA. 2Biochemistry & Cellular and Molecular Biology, University of Tennessee, We have recently shown a mechanism for a generic and powerful force of as- Knoxville, TN, USA. sembly and mobility for transmembrane proteins in model lipid bilayers, Ternary mixtures of high-melting lipid, low-melting lipid, and cholesterol referred to as the orderphobic effect [1]. The force is a pre-transition effect exhibit a region of liquid-ordered (Lo) þ liquid-disordered (Ld) phase coexis- related to the first-order order–disorder transition in lipid bilayers and is tence analogous to raft þ non-raft behavior in cells. Curvature in vitro can modulated by proximity to the phase transition and structural features of trans- induce sorting of both protein and lipid components resulting in accommoda- membrane proteins such as hydrophobic mismatch. We present recent develop- tion by the less rigid phase. However a number of highly-curved membranes ments related to the orderphobic effect in the liquid-disordered phase. We also in vivo have more rigid, raft-like composition. We have measured membrane discuss the implications of this effect on the physical mechanism of formation rigidity by fluctuation analysis of giant unilamellar vesicles (GUVs) with phase of lipid rafts. contrast microscopy. We find that including transmembrane helical WALP [1] Katira, S., Mandadapu, K. K., Vaikuntanathan, S., Smit, B., & Chandler, D. peptides rigidifies the liquid-disordered phase. This rigidifying effect has (2016). Pre-transition effects mediate forces of assembly between transmem- been examined at various cholesterol fractions. A change in rigidity was not brane proteins. Elife, 5, e13150. observed for Lo phases. Electron paramagnetic resonance (EPR) and oriented circular dichroism (OCD) studies of the location and orientation of the peptide 1861-Pos Board B181 show that it is largely excluded from the Lo phase at high concentrations. Based Molecular Dynamics Simulations Reveal the Impact of Compositional on the partition coefficient of the peptide determined by FRET-based methods, Asymmetry in Lipid Membranes on Phase Behavior and Leaflet the effect of WALP on Lo rigidity has been examined for low but relevant frac- Interactions tions. Our findings indicate the disordered phase could be more rigid than the 1 2 Michael D. Weiner , Gerald W. Feigenson . raft phase when transmembrane proteins are present. 1Field of Physics, Cornell University, Ithaca, NY, USA, 2Field of Biophysics, Cornell University, Ithaca, NY, USA. 1864-Pos Board B184 The plasma membrane of eukaryotic cells is compositionally asymmetric, Non-Monotonic Effect of Solutes on Miscibility Transition Temperatures; meaning that different lipids are found in the cytosolic and exoplasmic leaf- Simple Explanation of Closed-Loop Phase Diagrams lets. Since compositionally asymmetric bilayers are difficult to form in vitro, Michael Schick1, David W. Allender2. studies in silico play an especially important role. Model membranes embody 1Physics, University of Washington, Seattle, WA, USA, 2Physics, Kent State the behavior of the exoplasmic leaflet with coexisting liquid-ordered and University, Kent, OH, USA. liquid-disordered phases. We use Molecular Dynamics simulations to study There has been renewed interest in the effect of adding a solute to a lipid the effects of compositional asymmetry and to examine the interactions be- mixture which can undergo a miscibility transition to coexisting liquid-ordered tween the two leaflets. Coarse-grained simulations are employed to produce (lo) and liquid-disordered (ld) phases. Most solutes in sufficient quantity phase separation. In the outer leaflet, a phase-separating mixture of phospha- decrease the transition temperature, Tc, simply by diluting the components tidylcholines and cholesterol is chosen, while the inner leaflet contains phos- that bring about the phase separation. Differences in solutes are manifest at phatidylethanolamines, phosphatidylserines, and cholesterol. We consider the low and intermediate concentrations. If the solute is an amphiphile that likes

BPJ 7847_7850 378a Tuesday, February 14, 2017 to sit at the interface of such phases then, as is well known, the amphiphile 1867-Pos Board B187 lowers the transition temperature at all concentrations. More interesting is Progress Towards a Membrane-Bound Structure of the ITK/NEF the solute which prefers one phase or the other. If it prefers the ld phase, which Complex has the larger entropy per particle of the two phases, the solute initially lowers Rebecca Eells1, Kindra Whitlatch2, Frank Heinrich1,3, Mathias Lo¨sche1,3, Tc. At intermediate concentrations, the solute can increase Tc. At large concen- Thomas E. Smithgall2. trations it must lower Tc by simple dilution of the system. On the other hand, if 1Department of Physics, Carnegie Mellon University, Pittsburgh, PA, USA, the solute prefers the lo phase, then its initial addition raises Tc. This continues 2Department of Microbiology and Molecular Genetics, University of at intermediate concentrations. At large concentrations it must, again, lower Tc. Pittsburgh School of Medicine, Pittsburgh, PA, USA, 3Center for Neutron This latter case provides a very simple explanation for the occurrence of closed Research, National Institute of Standards and Technology, Gaithersburg, loop ternary phase diagrams in systems in which the solute is cholesterol. The MD, USA. above behavior is illustrated in a simple model calculation. Human immunodeficiency virus-1 (HIV-1) Nef is a membrane-associated accessory protein essential for viral replication, immune evasion, and AIDs 1865-Pos Board B185 progression. Nef functions via interactions with numerous host cell proteins Physiochemical Characterization of Archaeal Exosomes involved in signal transduction and trafficking. Nef interaction with Src family Alexander P. Bonanno, Michelle Jiang, Noah Gilly, Parkson L.-G. Chong. kinases in HIV-1 target cells leads to constitutive kinase activity that enhances Temple University, Philadelphia, PA, USA. infectivity and viral replication while down-regulating immune receptors. Exosomes (microvesicles) secreted from thermoacidophilic archaea are Recently, Nef was also shown to interact selectively with Tec kinases in believed to play an important role in cell division and biofilm formation and HIV-1 target cells. In particular, interaction with Itk promotes infectivity and hold great promise for technological applications. However, there have been replication (Tarafdar et al, J. Biol. Chem. 2014). Engagement of the two pro- very few studies characterizing the exosomes from thermoacidophiles. In the teins at the inner plasma membrane is mediated by Itk’s SH3 domain, but present study, we have isolated exosomes (~130-170 nm in diameter) from the detailed mechanism and contribution of other domains to Nef engagement the thermoacidophilic archaeon Sulfolobus acidocaldarius (optimum growth: o remain unclear since most studies so far are solution-based thus do not account 78-80 C, pH 2-3) and used dynamic light scattering, fluorescence spectroscopy for the influence of the membrane on the interaction between the binding part- and conventional biochemical methods to characterize them. Proteomics and ners. In addition, detailed structures of the membrane-associated complexes lipidomics studies by Ellen et al. (2009 Extremophiles 13:67-79) showed have yet to be elucidated, and little is known about the structure of active > that exosomes from S. acidocaldarius were made of 29 proteins and tet- Itk. Sparsely-tethered bilayer lipid membranes (stBLMs) are resilient biomi- raether lipids. Using the steady-state polarization and the average lifetimes metic bilayers for the surface-sensitive characterization of protein binding to of intrinsic protein fluorescence (excited at 285 nm from an LED) obtained functionalized membranes and structural determination of the resulting pro- by phase-modulation fluorometry, we found that the rotational relaxation tein-membrane complexes. We use surface plasmon resonance and neutron time of proteins in S. acidocaldarius exosomes (buffer: 50 mM Tris containing reflection to characterize Itk/Nef interactions at the membrane interface, both 10 mM EDTA and 0.02% NaN3 at pH 7.2) underwent a steady decrease from o in terms of the conformation of the individual proteins on the bilayer and, 0.30 to 0.06 ns when the temperature increased from 20.0 to 50.4 C. There- eventually, as a complex. Here we will discuss the binding of Nef to anionic after, to our surprise, the trend was reversed and the rotational relaxation o membranes and the specific interaction of Itk with stBLMs that contain phos- time increased with increasing temperature, to 0.14 ns at 63 C. The biphasic phoinositolphosphates (PIPs). change in exosome protein fluorescence at 50.4oC also occurs in the cooling mode and could be attributed to temperature-induced protein conformational 1868-Pos Board B188 changes within the exosomes or a temperature-induced phase transition in exo- Viscoelastic Measurements of Hydroxycholesterol Phospholipid Mono- some tetraether lipid membranes. The possible mechanism underlying the layers decrease in rotational rate with increasing temperature above 50.4oC will be Nikki Hoang1, Lyle H. Nyberg2, Benjamin L. Stottrup1. discussed. (Support by NSF). 1Physics, Augsburg College, Minneapolis, MN, USA, 2Chemistry, Augsburg College, Minneapolis, MN, USA. 1866-Pos Board B186 The rheological characteristics of multi-component cholesterol/phospholipid Water Dynamics at the Bilayer Interface is Similar to that within the SecY monolayers have received less attention than measurements of phase behavior, Translocon domain morphology, and pressure-area isotherms. The liquid-liquid coexis- Venkatramanan Krishnamani1, Sara Capponi2, Stephen H. White3. 1 tence observed in these systems is a compelling model to investigate both bio- Department of Molecular Physiology and Biophysics, University of Iowa, logical membranes as well as for the physical chemistry of two dimensional Iowa City, IA, USA, 2Cardiovascular Research Institute, University of 3 phase separated systems in their own right. We use a pulsating drop module California in San Francisco, San Francisco, CA, USA, Department of for an optical tensiometer to measure the elastic and viscous moduli for lipid Physiology and Biophysics and Center for Biomembrane Systems, University monolayers exhibiting unique liquid-liquid phase behavior. Robustly, hydrox- of California, Irvine, Irvine, CA, USA. ycholesterol isomers induce a kink in pressure-area isotherms for monolayers The membrane bilayer interface is a chemically complex region composed of over a wide range of compositions and phospholipids. Likewise, monolayers water, lipid head groups, and acyl chain carbonyls. This interface facilitates of 27-hydroxycholesterol and 1,2-Dimysristoyl-sn-glycero-3-phosphocholine the formation of secondary structure, which is a necessary step before in vitro (DMPC) system exhibit the nucleation and growth of a sterol rich phase which insertion of transmembrane helices in lipid bilayers [1, 2]. Hence, the ener- increases in area fraction over a range of surface pressures. The role of sterol getics of partitioning of peptides into the bilayer interface is an important concentration, phase behavior, and droplet curvature are investigated in deter- step towards understanding the thermodynamics of protein folding. Exactly mining the viscoelastic properties of these Langmuir films. how the hydrophobic effect drives the partitioning of peptides into the mem- brane interface is unclear, because the atomic solvation parameter (s) for inter- Membrane Active Peptides and Toxins I face partitioning is one half the value observed for the bulk-phase partitioning of peptides [2, 3]. Because the hydrophobic effect is directly related to proper- 1869-Pos Board B189 ties of water, we have examined water dynamics at the POPC bilayer interface. Interaction of Aurein 1.2 and Charged Lipid Bilayers Our findings reveal that the water dynamics deviate significantly from bulk Shuo Qian, Durgesh Rai. behavior at the bilayer interface, similar to the behavior inside the translocon Oak Ridge National Laboratory, Oak Ridge, TN, USA. [4]. This suggests that the energetics of folding of helices inside the translocon Aurein 1.2 is one of more active antibacterial peptides secreted by tree frog might be similar to the energetics at the membrane interface. Litoria aurea. As a short membrane-active peptide with only 13 amino acids [1] Ladokhin AS, White SH. 1999. Folding of amphipathic a-helices on mem- in sequence, it has been found to be residing on the surface of lipid bilayer but branes: energetics of helix formation by melittin. J. Mol. Biol. 285:1363-69 it permeabilizes bacterial membranes at high concentrations. However the [2] Wimley WC, White SH. 1996. Experimentally determined hydrophobicity molecular level detail is largely unknown. In this study, we investigated the scale for proteins at membrane interfaces. Nat. Struct. Biol. 3:842-48 action of Aurein 1.2 in DMPC/DMPG lipid bilayers in the forms of unilamel- [3] Jacobs, RE. and White, SH. 1989. The nature of the hydrophobic binding of lar vesicles and multilamellar bilayers. The oriented circular dichroism (OCD) small peptides at the bilayer interface: Implications for the insertion of transbi- results show the peptide is on the surface of lipid bilayer regardless the layer helices. Biochemistry 28:3421-3437 charged lipid ratio. Only at very high peptide-to-lipid ratio (~1/10), the pep- [4] Capponi S, Heyden M, Bondar A-N, Tobias DJ, White SH. 2015. Anoma- tide is found to be inserted. To further understand how it interacts with lous behavior of water inside the SecY translocon. Proc. Natl. Acad. Sci. USA. charged lipids, we also applied Small-Angle Neutron Scattering (SANS) to 112:9016-9021 study lipid vesicles bilayer structures under the influence of Aurein 1.2.

BPJ 7847_7850 Tuesday, February 14, 2017 379a

Neutron scattering is sensitive to deuterium (D)/hydrogen (H) distribution due 1872-Pos Board B192 to significant difference in the scattering length of D/H. In the past few years, Insertion Process of Different Amphipathic a-Helical AMPS into Lipid we have developed SANS to study lipid distributions across the bilayer in Bilayers Observed in Unbiased Atomic Molecular Dynamics Simulations multi-composition umilamellar vesicles. The result with Aurein 1.2 shows Tangzhen Zhao1, Yukun Wang2, Dan Hu1, Jakob Ulmschneider1. that it interacts strongly with negatively charged DMPG lipid, causing strong 1Shanghai Jiao Tong University, Shanghai, China, 2The Johns Hopkins asymmetry in the lipid bilayer in a concentration dependent manner. At high University, Baltimore, MD, USA. concentration, it disrupts bilayer, which correlates the insertion state found in Antimicrobial peptides (AMPs) are promising candidates for the development the OCD results. We will discuss the implication of our findings in under- of novel antibiotics due to their unique mechanism of killing bacteria. They can standing the action of Aurein 1.2. directly disrupt the bacterial cell membrane rather than bind to specific recep- tors to avoid antibiotic resistance. Two main pore-forming mechanisms (labled 1870-Pos Board B190 ‘‘barrel-stave’’ and ‘‘toroidal pore’’) have been proposed to explain the anti- Molecular Basis for the Role of Cationic Residues in Antimicrobial Peptide bacterial activity of AMPs. The first necessary step is the observation of com- Interactions plex pore structures formed by transmembrane peptides in both of these two Amy Rice, Jeff Wereszczynski. models. Illinois Institute of Technology, Chicago, IL, USA. However, it’s hard to study the insertion process of amphipathic a-helices into Antimicrobial peptides (AMPs) play a vital role in the innate immune response the membrane with currently available experiments. Here, we employ unbiased and represent promising templates for developing broad-spectrum alternatives atomistic molecular dynamics simulation to explore the insertion process of to conventional antibiotics. Most AMPs are charged amino acid sequences that several a-helical AMPs in micro-second simulation timescales. We observe interact more strongly with negatively charged prokaryotic membranes than that some AMPs that have a limited amount of polar residues and carry one net neutral eukaryotic ones. Both AMPs and synthetic analogues with or two charged residues close to the N-terminus, can cross the membrane in arginine-like guanidinium groups show a greater toxic effect against bacteria relatively short simulation time. Furthermore, the presence of Histidine close than those with lysine-like amine groups, though the atomistic mechanism to the C-terminus plays an important role in the insertion process and the sta- for this increase in potency remains unclear. To examine this, we have bilization of the oligmeric structures, because of its flexible side chain and ten- conducted comparative molecular dynamics simulations of a model prokary- dency to form hydrogen bonds with other peptides. otic membrane system (POPC/POPG) interacting with two different mutants of a prototypical AMP, KR-12: one with all lysine residues mutated to arginine 1873-Pos Board B193 (R-KR12), and one with all arginine residues mutated to lysine (K-KR12). Effects of Lipid Compositions on the Entry of Cell Penetrating Peptide Simulations show that both peptides partition analogously to the bilayer and Oligoarginine into Single Vesicles display similar preferences for forming hydrogen bonds with the anionic Sabrina Sharmin1, Md Zahidul Islam1, Masahito Yamazaki1, Hideo Dohra2. POPGs. However, R-KR12 has a significantly higher propensity for hydrogen 1Bioscience, Shizuoka University, Shizuoka, Japan, 2Research Institute of bonding with the bilayer than K-KR12, as well as stronger peptide-bilayer Green Science and Technology, Shizuoka University, Shizuoka, Japan. hydrogen bonds, resulting in considerably longer interaction times. Additional To elucidate the mechanism of the entry of R9 into cells, we investigated the simulations with single and double methylated R-KR12 mutants show that the entry of carboxyfluorescein (CF) labeled R9 (CF-R9) into single giant unilamel- strength of these hydrogen bonds, rather than the number of bonds formed, is lar vesicles (GUVs) of various lipid compositions and the CF-R9-induced responsible for the extensive peptide-bilayer interaction seen in the R-KR12 leakage of a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using a system. Finally, free energy simulations reveal that both peptides are unstruc- method developed recently by us. First, we investigated the interaction of tured in solution and adopt a highly helical amphipathic structure when inserted CF-R9 with DOPG/DOPC GUVs containing AF647 and small DOPG/DOPC into the bilayer. Overall, these results help elucidate the greater toxicity of vesicles. The fluorescence intensity of the GUV membrane due to CF-R9 arginine-rich AMPs and offer potential insights for designing more potent (i.e., the rim intensity) increased with time to a steady-state value, and then analogues in the future. the fluorescence intensity of the membranes of the small vesicles in the GUV lumen increased without leakage of AF647. This result indicates that CF-R9 1871-Pos Board B191 entered the GUV lumen from the outside by translocating across the lipid mem- Daptomycin Induces Transient Membrane-Permeabilization brane without pore formation. The fraction of entry of CF-R9 at 6 min without Wei-Chin Hung1, Ming-Tao Lee2,3, Meng-Hsuan Hsie4, Hsiung Chen5, pore formation, Pentry (6 min), increased with an increase in CF-R9 concentra- Yu-Yung Chang2, Huey W. Huang6. 1 tion. Next, we investigated the entry of CF-R9 into GUVs of DLPG and DTPC Department of Physics, R.O.C. Military Academy, Fengshan, Taiwan, mixture with shorter hydrocarbon chains, which is mechanically weak. The 2National Synchrotron Radiation Research Center,, Hsinchu, Taiwan, Pentry (6 min) of CF-R9 for DLPG/DTPC (2/8)-GUVs were larger than those 3Department of Physics, National Central University, Jhongli, Taiwan, of DOPG/DOPC (2/8)-GUVs at the same CF-R9 concentrations. On the other 4Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, hand, CF-R9 could not enter DOPG/DOPC/cholesterol (2/6/4) GUVs. Analysis 51National Synchrotron Radiation Research Center, Hsinchu, Taiwan, of the rim intensity showed that CF-R9 was located only in the outer monolayer 6Department of Physics & Astronomy, Rice University, Houston, TX, USA. of the GUVs, indicating that the rate constant of the translocation of CF-R9 Daptomycin, a cyclic lipopeptide, represents a new structural class of the FDA from outer to the inner monolayer was almost zero. We proposed a hypothesis approved antibiotics. The drug interacts with the cytoplasmic membranes of that CF-R9 can translocate from the outer to the inner monolayer of the GUV by Gram-positive pathogens causing membrane permeabilization to ions and passing through prepores. On the basis of analyses of these results, we dis- cell death. The antibiotic activity is calcium ion-dependent and correlates cussed a plausible mechanism by which CF-R9 enters GUVs of various lipid with the target membrane’s content of phosphatidylglycerol (PG). Fluores- compositions.1 cence resonance energy transfer has detected daptomycin aggregation upon (1) Biochemistry, 2016, 55, 4154-4165. membrane binding and this has led to the hypothesis that daptomycin forms ion channels in the membrane. However many aspects of dayptomycin- 1874-Pos Board B194 membrane interactions are still unknown. Here we used X-ray scattering Modulating Effect of Histidine on Short Arginine- and Tryptophan-Based and CD analysis to clarify the physicochemical state of daptomycin in solu- Antimicrobial Peptides tion. With X-ray we determine that daptomycin in solution is monomeric at Lorant Janosi. concentrations relevant to its therapeutic use. A careful CD analysis of dapto- Molecular and Biomolecular Physics, Natl.Inst. for R&D of Isotopic and mycin with Ca2þ and PG-containing membranes shows that there are only two Molecular Technologies, Cluj-Napoca, Romania. states identifiable by CD, one before and one after membrane binding and the Antimicrobial peptides (AMPs) are short sequences of aminoacids that organ- stoichiometry ratio of calcium to daptomycin is 2 to 3 in their binding reac- isms use as defense mechanism against foreign bodies. The selectivity of inter- tion. To test the proposed daptomycin channels, we performed Kþ and Ca2þ action with bacterial cells over mammalian cells underlines their significant leakage experiment, comparing daptomycin with gramicidin, ionomycin, potential in developing AMP-based drugs against antibiotics resistant bacteria. valinomycin and penetratin. The results revealed the property of transient We started from a tryptophan- and arginine-rich peptide (RRWWRWWRR) ion conduction induced by daptomycin, dissimilar to gramicidin channel or which showed good antimicrobial efficacy. Seven new analogs were design ion carriers but similar to penetratin. Previous experiments have shown that by substitution of tryptophan or arginine residues with histidine residues. penetratin does not form ion channels, but both daptomycin and penetratin The peptides were tested against three bacterial strains (E. coli, B. subtilis exhibit a lipid extracting effect. The effect might create transient water pore and S. aureus). Also, their cytotoxicity on peripheral blood mononuclear cells defects that can explain the transient ion leakage induced by dayptomycin and their capacity to induce hemolysis was evaluated and used to calculate a and penetratin. therapeutic index for each peptide. Molecular dynamics simulations showed

BPJ 7847_7850 380a Tuesday, February 14, 2017 that the antimicrobial activity of these peptides can be correlated to the 3D-hy- to compare the peptide activity against synthetic membrane vesicles with the drophobic moment and to a simple structure-based packing parameter. This antiviral activities of each peptide. Taken together, our results show that inter- suggests that, in principle, one could design antimicrobial peptides based on facially active PEIs have the potential to be useful therapeutics which can such parameters. Our study shows that the nature of histidine favors its interac- possibly be applied to different enveloped viruses. tion with anionic lipid headgroups, i.e., a location at one end of an AMP, instead of the middle, and enhances the aggregation of cationic AMPs around 1877-Pos Board B197 anionic lipids, leading to transmembrane pore formation. The latter mechanism Membrane Binding and Antimicrobial Activity of a Catioinc, Porphyrin- of disruption of the membrane can be correlated with the increased antimicro- Binding Peptide bial activity of these AMPs. Hence, the position of the histidine within the pep- David Shirley, Christina L. Chrom, Gregory A. Caputo. tide sequence can be linked with AMP’s mechanism of interaction with the Chemistry and Biochemistry, Rowan University, Glassboro, NJ, USA. membrane surface. Furthermore, the presence of histidine residue reduced A designed catioinic, amphiphilic peptide was previously shown to bind the cytotoxic and hemolytic activity of the peptides, in some cases maintaining porphyrin molecules and promote the formation of excitonically coupled the same efficacy against bacteria. Some of these peptides have the potential to J-aggregate structures (Caputo et. al. 2009). Due to the catioinic and amphi- become good candidates to fight against bacteria. philic nature, this peptide was investigated for antimicrobial activity. We Acknowledgments: This work was supported by a grant of the Romanian Na- synthesized 3 variants of the peptide which differed only in the position of tional Authority for Scientific Research, CNDI-UEFISCDI, project number a single Trp residue, used as a reporter of membrane binding interactions. PNII-123/2012, PNII-98/2012, PN-II-ID-PCCE-2011-2-0027, PN 09370301 All three variants showed similar levels of antimicrobial activity against þ and PN-II-RU-TE-2014-4-2418. Gram and G- strains (low micromolar efficacy). Using these 3 versions we characterized the binding and membrane orientation of the peptides using 1875-Pos Board B195 fluorescence spectroscopy and quenching, secondary structure by CD, and Development of Cell-Wall Deficient Bacteria for the Characterization of bacterial membrane permeabilization assays to investigate mechanism of Histone-Derived Antimicrobial Peptides through Confocal Microscopy antibacterial activity. All peptides bound to anionic bilayers with higher Dania M. Figueroa1, Donald E. Elmore2. affinity compared to zwitterionic bilayers, but there was little difference 1Biochemistry Program, Wellesley College, Wellesley, MA, USA, between the three variants. Additionally, quenching experiments yielded 2Department of Chemistry and Biochemistry Program, Wellesley College, little difference between the three Trp positions in the peptides, indicating Wellesley, MA, USA. that the peptide is likely not adopting a single, uniform orientation in the Antimicrobial peptides (AMPs) are cationic, amphipathic proteins with an bilayer. The peptides adopted helical conformations when bound to the bila- innate ability to kill a wide variety of pathogens, including viruses, fungi yers. However, none of the peptides exhibited significant permeabilization of and bacteria. AMPs can be grouped into two general categories based on their the E.coli inner membrane, indicating activity may be localized to the outer mechanism of action. One group acts via permeabilization, or disruption of the membrane. Ongoing work is focused on the depth of peptide penetration cell membrane. The other group acts via translocation, or diffusion across the into the bilayer. cell membrane and disruption of an intracellular process. Confocal micro- 1878-Pos Board B198 scopy is a technique readily used to identify AMP methods of action as it Membrane Insertion of a Dinuclear Ruthenium Complex and Implications allows researchers to visualize peptide localization in bacteria by taking cross for Antibacterial Activity sections of cells. However, the small size and different orientations of bacteria Daniel K. Weber1, Marc-Antoine Sani2, Matthew T. Downton1, can produce low-resolution images. To combat this problem our lab has used J. Grant Collins3, Frances Separovic2, F. Richard Keene4. the cell-wall deficient spheroplast form of Escherichia coli to obtain higher 1Computational Biophysics, IBM Research Australia, Melbourne, Australia, quality images. Thesespheroplasts are both spherical and larger than typical 2School of Chemistry, University of Melbourne, Melbourne, Australia, E. coli, leading to improved imaging. Previously, we showed that several pre- 3School of Physical, Environmental and Mathematical Sciences, University viously characterized AMPs have the same behavior against E. coli sphero- of New South Wales, Australian Defence Force Academy, Canberra, plasts as normal cells. This project is an extension of that work aimed at Australia, 4School of Physical Sciences, University of Adelaide, Adelaide, developing protocols to consider the visualization of AMPs with cell-wall Australia. deficient forms of other bacterial strains. Characterizing AMPs against a vari- Ruthenium-based metal complexes have received considerable interest over the ety of bacteria is important as research shows that AMPs antimicrobial prop- past 30 years for their DNA/RNA-binding and anticancer properties. More erties differ against different bacteria strains. To this end, we are developing recently, however, they have been recognized as potential antibacterial agents. protocols to form protoplasts of the gram-positive bacteria Bacillus subtilis In particular, cationic dinuclear polypyridylruthenium(II) complexes, bridged and Bacillus megaterium for imaging with well-characterized control peptides, by flexible methylene linkers, have shown enhanced antibacterial activity such as buforin II and magainin. We have also investigated the membrane over their mononuclear counterparts, and generally maintain activity against integrity of cell-wall deficient bacteria using a microscopy-based permeabili- antibiotic-resistant strains. Furthermore, dinuclear complexes are known to zation assay. depolarize and increase permeability of bacterial membranes, while their activ- ity can be directly modulated by varying the length of the methylene bridge. 1876-Pos Board B196 Recently, we have applied a combination of solid-state NMR and simulation Membrane Active Peptides as a Potential Therapeutic Option for Envel- to provide an initial biophysical characterization of a dinuclear Ru(II) complex oped Viruses and a biologically-inactive Ir(III) analogue in membrane environments. We Shantanu Guha. highlight that direct membrane-permeating mechanisms and diffusion though Department of Biochemistry and Molecular Biology, Tulane University bacterial cell membranes may hinge on an exclusive ability of dinuclear School of Medicine, New Orleans, LA, USA. Ru(II) complexes to insert as transmembrane structures. Every year, influenza affects populations all across the world, and every year, new vaccines must be developed due to the intrinsically high rate of mutation 1879-Pos Board B199 and reassortment in influenza virus. Currently, there are two classes of antiviral 2H NMR Studies of Bacteria: How Does the Peptidoglycan Layer Modify drugs available to treat influenza virus infection, neuraminidase inhibitors and the Interaction between Antimicrobial Peptides and the Lipid Membrane M2 inhibitors, but both classes have limited efficacy and resistant strains of vi- Nury P. Santisteban1, Michael R. Morrow1, Valerie K. Booth2. rus are common. Due to the difficulty in treating influenza and other enveloped 1Physics and Physical Oceanography, Memorial University of viruses, there is an urgent need for new classes of antiviral agents. One poten- Newfoundland, St. John’s, NL, Canada, 2Biochemistry, Memorial University tially useful, but underdeveloped type of inhibitor is the peptide entry inhibitor of Newfoundland, St. John’s, NL, Canada. (PEI). PEIs interfere with the key early steps by which enveloped viruses bind, Antimicrobial peptides (AMPs) are a group of small peptides with antimicro- fuse, enter, and eventually infect their target cells. By testing a large number of bial effects against pathogens and have been well studied because of their membrane active peptides in an influenza infection model system, we identified promise to be part of the solution to the rising problem of antibiotic resistance. a panel of peptides which have potent inhibitory activity against the virus. We Biophysical studies with AMPs in model lipid systems are commonly used to have hypothesized that some of these peptides directly interact with viral and/or study AMPs’ permeabilizing effect on lipid bilayers. However, it is not clear if host cellular membrane lipids and disrupt viral binding and fusion. This hy- this membrane-permeabilizing characteristic is the only mechanism of cell pothesis leads to the prediction that membrane-active peptides, in general, killing. Studies suggest that at least some AMPs have additional targets, will inherently have inhibitory activity against enveloped virus due to the na- different from the lipid bilayer. This studies lead to the suggestion that mem- ture of virus-host entry mechanisms. We have used cell biology and biophysics brane permeabilization is just part of a multi-hit mechanism of AMPs, or

BPJ 7847_7850 Tuesday, February 14, 2017 381a even just a collateral effect. Additionally, AMP interactions with non-lipid cell structure and dynamics of model membrane systems. In particular, we inves- envelope components of bacteria may be important in modifying how tigated the effects of the antimicrobial peptides gramicidin and alamethecin on well AMPs are able to disrupt the lipid membrane. In order to connect studies the lipid bilayer structure using small angle x-ray and neutron scattering. The of AMPs in model lipid systems to the more complex real bacterial cell enve- structural studies were complemented by NSE experiments to probe the col- lopes, we have deuterium-labeled the membranes of the gram-positive bacteria lective bending and thickness fluctuation dynamics in these model systems. Bacillus subtilis and used 2H NMR to study how lipid acyl chain order in its Notably, the NSE results revealed enhanced thickness fluctuation dynamics membranes is affected by treatment with AMPs. We have also observed 2H in lipid bilayers containing low concentration of gramicidin that were damp- NMR spectra from Bacillus subtilis in which the peptidoglycan layer has ened with increasing peptide concentration. An enhancement in dynamics was been disrupted. This has allowed us to investigate how disruption of the pepti- not seen in bilayers containing alamethicin, suggesting that the dynamics not doglycan layer affects bacterial lipid chain order and the AMP/bacteria only depend on peptide concentration, but also peptide orientation within the interaction. membrane

1880-Pos Board B200 1883-Pos Board B203 Lipid Clustering by Antimicrobial Polymers and Lectins Mode of Action of Antimicrobial Peptides: Long and Short Amphipathic Anja Stulz1, Winfried Ro¨mer2, Karen Lienkamp3, Heiko Heerklotz1,2, Alpha-Helixes Use Different Mechanisms Maria Hoernke1,2. Erik Strandberg1, Ariadna Grau-Campistany2, Hector Zamora-Carreras3, 1Pharmaceutical Technology and Biopharmacy, Albert-Ludwigs-Universit€at, Marie-Claude Gagnon4, Philipp Muhlh€ €auser1, Parvesh Wadhwani1, Freiburg i.Br., Germany, 2BIOSS Centre for Biological Signalling Studies, Jochen Burck€ 1, Johannes Reichert1, Michele Auger4, Jean-Francois Paquin4, Albert-Ludwigs-Universit€at, Freiburg i.Br., Germany, 3IMTEK Chemistry M. Angeles Jime´nez3, Marta Bruix3, Francesc Rabanal2, Anne S. Ulrich1. and Physics of Interfaces, Albert-Ludwigs-Universit€at, Freiburg i.Br., 1Institute for Biological Interfaces, Karlsruhe Institute of Technology, Germany. Karlsruhe, Germany, 2Universitat de Barcelona, Barcelona, Spain, 3Instituto The current urge to understand the role of lipids in defense against pathogens is de Quı´mica Fı´sica ‘‘Rocasolano’’, CSIC, Madrid, Spain, 4Universite´ Laval, driven by two strategies: killing pathogens and protecting the cell from infec- Quebec, QC, Canada. tion. Antimicrobial peptides and antimicrobial polymers offer a promising We have studied the membrane structure and orientation of cationic amphi- alternative to classical antibiotics through their action on membrane integrity. pathic a-helical antimicrobial peptides (AMPs) using circular dichroism and In principle, antimicrobial peptides and polymers are able to cluster lipids solid-state NMR, combined with activity studies. For a series of model pep- through lipid selection and recruitment from a mixed membrane. We show tides, called KIA peptides, a clear length-dependent activity is found, as only that antimicrobial polymers induce leakage in lipid vesicles by transient defects peptides long enough to span the hydrophobic thickness of the membrane could rather than defined pores. The positively charged polymers efficiently cluster induce leakage in vesicles. There is also a clear threshold length for peptides negatively charged lipids from mixed model membranes. Larger domains are able to kill bacteria [1]. Using another series of KIA-like peptides of different formed (in the order of 500 lipids). The binding of the polymers to the vesicles length (from 14 up to 28 residues) but with a constant charge revealed that the is exothermic. Our findings correlate with the polymers activity against bacte- length, but not the charge, is the critical factor. In membrane systems with a ria. Similarly, lectins, carbohydrate binding proteins, can recognize certain positive spontaneous curvature, the peptides get inserted into the membrane glycolipids in mixed membranes, bind them and cluster them. By a yet un- in a transmembrane orientation. All results indicate that these peptides act by known mechanism, the membrane is locally bent. Thus bacterial lectins can forming proper oligomeric pores in the lipid bilayer. If the peptide is just initiate uptake of pathogens into host cells and are promising targets for drug long enough to span the membrane, it is aligned perfectly upright, but longer development. peptides can tilt cooperatively in the pore like an iris [1,2]. On the other hand, BP100, a highly helical peptides of only 11 amino acids, is clearly too 1881-Pos Board B201 short to form a transmembrane pore, but it is still strongly active against How Antimicrobial Peptides Permeabilize Membranes with and without bacteria. From solid-state 2H-, 15N- and 19F-NMR studies, this peptide is Pore Formation found to dip into the membrane and to show high mobility within the amphi- Jakob P. Ulmschneider. philic surface layer. This way, it most likely disturbs the lipid order and thereby Institute of Natural Sciences, Shanghai Jiao Tong University, Shanghai, induces permeability, which suggests a carpet-like mechanism of action [3]. China. The structural results on these long and short AMPs clearly demonstrate that One of the biggest enigmas of antimicrobial peptides (AMPs), which protect all peptides with similar structural characteristics can act by very different forms of life against pathogens, is why few structures of membrane pores have mechanisms. been found despite clear evidence of membrane leakage and antimicrobial ac- References: tivity. We provide a surprisingly simple explanation: For some AMPs such as [1] Grau-Campistany et al., (2015) Sci Rep 5, 9388. PGLa (charge þ5), pores are not needed to explain both leakage and peptide [2] Grau-Campistany et al., (2016) J Phys Chem Lett 7, 1116-1120. translocation. Fully converged, unbiased multi-microsecond equilibrium simu- [3] Zamora-Carreras et al., (2016). Biochim Biophys Acta 1858, 1328-1338. lations at all-atomistic level reveal that peptides spontaneously translocate across the membrane individually on a timescale of tens of microseconds, 1884-Pos Board B204 without forming pores. These findings explain why, despite vesicular leakage Simulations of Membrane Disrupting Peptides Pores Versus Surface no channel has been identified for PGLa. However, similar simulations on Binding other, lesser charged AMPs like maculatin clearly show pore formation. The B. Scott Perrin Jr.1, Riqiang Fu2, Myriam L. Cotten3, Richard W. Pastor1. 1 results suggest that for some specific antimicrobial peptides, pore formation Laboratory of Computational Biology, NHLBI/NIH, Rockville, MD, USA, 2 may not have to be invoked at all to explain both peptide translocation and National High Magnetic Field Laboratory, Tallahassee, FL, USA, 3 membrane permeabilization. Department of Applied Science, College of William & Mary, Williamsburg, VA, USA. 1882-Pos Board B202 Peptides that disrupt biological membranes are a source of new antibiotic and The Synergistic Effects of Lipids and Peptides on Membrane Dynamics antiviral therapeutics. Here, the relationships between peptide primary se- Elizabeth Kelley1, Andrea Woodka1, Paul Butler1,2, Michihiro Nagao1,3. quences, membrane bound structures, and abilities to disrupt membranes are 1Center for Neutron Research, NIST, Gaithersburg, MD, USA, 2Chemical investigated using all-atom molecular dynamics simulations. First, the arche- and Biomolecular Engineering, University of Delaware, Newark, DE, USA, type barrel-stave alamethicin (alm) pore in a 1,2-dioleoylsn-glycero-3-phos- 3Center for Exploration of Energy and Matter, Indiana University, phocholine bilayer at 313 K indicates that ~7 ms is required for equilibration Bloomington, IN, USA. of a preformed 6-peptide pore; the pore remains stable for the duration of There is a growing appreciation that the membrane physical properties are the remaining 7 ms of the trajectory, and the structure factors agree well with essential to cell and protein function. Simply altering the thickness of model experiment. A 5 ms simulation of 10 surface-bound alm peptides shows signif- membranes has been shown to influence the biological activity of several pro- icant peptide unfolding and some unbinding, but no insertion. Simulations at teins, while incorporating peptides into lipid membranes also is known to 363 and 413 K with a 0.2 V electric field yield peptide insertion in 1 ms. Inser- affect the bilayer structural properties. Clearly there is a synergy in lipid- tion is initiated by the folding of residues 3-11 into an a-helix, and mediated by protein interactions in determining the membrane properties; however, the membrane water or by previously inserted peptides. The stability of five alm nature of these interactions are not well understood. Here we use a combina- pore peptides at 413 K with a 0.2 V electric field demonstrates a significant tion of small angle scattering techniques and neutron spin echo spectroscopy preference for a transmembrane orientation. In contrast, a hypothesis that (NSE) to investigate the effects of incorporating a small peptide on both the the antimicrobial peptide piscidin 1 (p1) forms toroidal pores is tested. The

BPJ 7847_7850 382a Tuesday, February 14, 2017 primary simulation was initialized with 20 peptides in four barrel-stave pores in microbicidal activity in the presence of RBCs. Ultimately, our results suggest a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmi- that combining the power of combinatorial library screening with the nuance toyl-2-oleoyl-sn-glycero-3-phosphoglycerol bilayer. The four pores relaxed of rational sequence engineering is an effective approach to engineering novel to toroidal by 200 ns, only one pore-like structure containing two transmem- antimicrobial peptides that retain activity under physiological conditions. brane helices remained at 26 ms, and none of the 18 peptides released to the surface reinserted to form pores. The simulation was repeated at 413 K with 1887-Pos Board B207 an applied electric field and all peptides were surface-bound by 200 ns. Trajec- Development of Refined Bacterial Spheroplast Analyses to Characterize tories of surface-bound piscidin with and without applied fields at 313 and 413 Hybrid Antimicrobial Peptides Heidi M. Wade1, Louise E.O. Darling2, Donald E. Elmore1. K and totaling 6 ms show transient distortions of the bilayer/water interface 1 31 Department of Chemistry and Biochemistry Program, Wellesley College, (consistent with P NMR), but no insertion to transmembrane or pore states. 2 15N chemical shifts confirm a fully surface-bound conformation. Taken Wellesley, MA, USA, Department of Biological Sciences and Biochemistry together, the simulation and experimental results imply that transient defects Program, Wellesley College, Wellesley, MA, USA. rather than stable pores are responsible for membrane disruption by p1, and Antimicrobial peptides (AMPs) provide a promising alternative treatment for likely other AMPs. infectious bacteria resistant to antibiotics. AMPs kill bacterial cells through a variety of diverse mechanisms. While some AMPs, such as parasin, induce 1885-Pos Board B205 membrane permeabilization, others like buforin II (BF2) and DesHDAP1 enter Simulation and Database-Guided Antimicrobial Peptide Evolution the cell with little disruption to the membrane and kill bacteria by interacting Charles H. Chen1,2, Charles G. Starr3, Gregory Wiedman1, with intracellular components. Previous work in our labs demonstrated the po- William C. Wimley3, Jakob P. Ulmschneider4, Martin B. Ulmschneider1,2. tential of using bacterial spheroplasts to better visualize peptide localization via 1Whiting School of Engineering, Johns Hopkins University, Baltimore, MD, confocal microscopy. Here, we build upon our previous spheroplast work in USA, 2Institute for NanoBiotechnology, Johns Hopkins University, two key ways. First, we take advantage of the improved image quality to Baltimore, MD, USA, 3Department of Biochemistry and Molecular Biology, perform more quantitative analyses to measure the cellular entry or membrane Tulane University School of Medicine, New Orleans, LA, USA, 4Institute of localization of peptides. We then utilized these more systematic approaches to Natural Sciences, Shanghai Jiao Tong University, Shanghai, China. characterize the mechanisms of designed hybrid peptides. Recently, there has Antimicrobial peptides (AMPs) are powerful and ubiquitous components of been increased interest in designing AMPs by combining two distinct AMPs the innate immune defense in all domains of life. AMPs are amphiphilic into a single peptide. It has been shown that such hybrid AMPs are often peptides that selectively target and kill a wide variety of microbial pathogens more potent than their individual AMP components. However, to date all stud- at low micro-molar concentrations. These peptides vary widely in size, ied hybrid AMPs have combined together two AMPs that work via membrane sequence, and secondary structure, and no common activity motif has been permeabilization. Thus, we focused on characterizing hybrid peptides that discovered to date. Here, we report several powerful new synthetic AMPs combine one permeabilizing AMP (parasin) and one translocating AMP that are developed using a combination of molecular dynamics (MD) simula- (DesHDAP1 or BF2) with different orientations and linkers. Our results have tions and database screening. First, we use MD simulations to study the mech- shown that the permeabilizing peptide typically dominates the mechanism of anisms of AMP folding and pore assembly in a bacterial model membrane. action when combined with the translocating peptide. This work is an important The atomic detail information of how pores form was used to develop a novel step in determining if any trends can be generalized to other hybrids made of AMP: LDKA, which is a template sequence based on atomic detail structural engineered permeabilizing-translocating peptide pairs. information from the simulations. This simple peptide is composed a small number of amino acids and shows powerful pore-forming properties. Second, 1888-Pos Board B208 we apply statistical analysis of several thousand AMPs to optimize the LDKA Real-Time Characterization of an Antimicrobial Mechanism-of-Action template sequence. Finally, we apply a high-throughput screen using dye with Nonlinear Optical Scattering 1 2 1 leakage assays to both bacterial and mammalian membrane model vesicles. Michael J. Wilhelm , Bruk Mensa , Mohammad Sharifian Gh. , 2 1 This allows us to fine-tune the pore size and the binding selectivity for William F. DeGrado , Hai-Lung Dai . 1Department of Chemistry, Temple University, Philadelphia, PA, USA, different membrane types. From the screen, we have identified 9 different 2 LDKA analogues with different membrane selectivity and pore sizes. Our Department of Pharmaceutical Chemistry and the Cardiovascular Research work confirms that the combination of MD simulations, database optimiza- Institute, University of California, San Francisco, San Francisco, CA, USA. tion, and theory/computation guided high-throughput screening is a powerful Antimicrobial resistance is an emerging threat that demands continuous devel- strategy to design potent new antimicrobial peptides for different biomedical opment of new classes of antibiotic agents. In the interest of efficiently targets and applications. optimizing viable chemical targets, it is imperative to have experimental tech- niques capable of characterizing not only the efficacy of an antibiotic, but also 1886-Pos Board B206 the specific mechanism-of-action (MoA). We have recently demonstrated the Discovering Novel Antimicrobial Peptides using High-Throughput surface-sensitive nonlinear optical technique, second-harmonic generation Screening in the Presence of Human Erythrocytes light scattering (SHS), for quantifying transmembrane molecular transport in Charles G. Starr, Jing He, William C. Wimley. living cells(1). We now show that SHS can be applied as a sensitive probe of Department of Biochemistry and Molecular Biology, Tulane University, New membrane permeability in living bacteria. Specifically, by monitoring the up- Orleans, LA, USA. take response of an SHS-active probe molecule following administration of an Antimicrobial peptides (AMPs) have long been considered excellent candidates antibacterial agent, perturbations in the measured transport response reveal for development as antibiotic agents to combat the threat of drug resistant bac- time- and concentration-dependent changes in the permeability of the bacterial terial pathogens. Yet, the promise AMPs have displayed in the laboratory has membranes, thus permitting real-time characterization of the MoA. As an not yielded correlative clinical success. We hypothesized that a major reason initial proof-of-principle, we apply SHS for deducing the sequential MoA of for the disparity between outcomes at the bench and in the clinic is AMP inter- the antimicrobial, Bricilidin (Bn). Bn is a synthetic arylamide foldamer exhib- action with and toxicity towards host cells. Here, we validate this theory using iting amphiphilic topology similar to that of cell-penetrating peptides, and was human red blood cells as model eukaryotic cells. Indeed, the potency of several designed to disrupt bacterial membranes(2). Using malachite green (MG) as an well-studied antimicrobial peptides decreases from minimum sterilizing SHS-active probe, we characterize the MoA of Bn in Escherichia coli. In gen- concentrations (MSC) of 1-5 mMto>30 mM in the presence of RBCs at eral, for low concentrations (<1uM) and short interaction periods (%15min), 1x109 cells/ml; equivalent to 20% of the human physiological concentration. Bn is observed to primarily affect the bacterial outer membrane, resulting in To address this issue, we have developed a high-throughput method to screen enhanced permeability. However, for higher concentrations (R5uM) and a combinatorial peptide library for antimicrobial activity in the presence longer interaction periods (>15min), Bn begins to disrupt the CM, resulting of concentrated human cells. The screen consists of assays that assess the in an apparent decreased permeability, likely indicating depolarization of the effect of RBCs on antimicrobial activity as well as toxicity toward eukaryotic membrane. cells. We present data on peptides identified and characterized from the REFERENCES: high-throughput library screen and show that these new peptides were not as 1. Wilhelm, M.J., M. Sharifian Gh., and H.-L. Dai. 2015. Chemically Induced effective as they initially appeared during the screen. We demonstrate that the Changes to Membrane Permeability in Living Cells Probed by Nonlinear Light difference between observed and expected results is due to unforeseen Scattering. Biochemistry. 54: 4427–4430. proteolytic activity sequestered within the cytosol of the red blood cells. Finally, 2. Mensa, B., G.L. Howell, R. Scott, and W.F. DeGrado. 2014. Comparative we show that a library consensus sequence synthesized using all d-amino acids mechanistic studies of brilacidin, daptomycin, and the antimicrobial peptide outperforms all library isolates as well as the template sequence in terms of LL16. Antimicrob. Agents Chemother. 58: 5136–5145.

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Membrane Structure III tion profile of GUVs-NI, GUVs-HS and GUVs-2:1:2 were very similar: Triton X-100 first induced phase separation (liquid-ordered/liquid-disordered phase) 1889-Pos Board B209 followed by partial solubilization of the liquid-disordered phase. The results Liquid Membrane Fluctuations Drive Ordered Monolayer Domain Align- are discussed in terms of differences in lipid composition, as revealed from ment and Raft Stacking the fatty acid and headgroup composition analysis of the erythrocyte-lipid Timur R. Galimzyanov1,2, Veronika V. Alexandrova1, Petr I. Kuzmin2, extracts. Peter Pohl3, Sergey A. Akimov2. 1 1891-Pos Board B211 Theoretical Physics & Quantum Technologies, National University of Lipids Regulated through Membrane Topography Revealed by Single Science and Technology, Moscow, Russian Federation, Particle Tracking 2Bioelectrochemistry, A.N. Frumkin Institute of Physical Chemistry and 3 Xinxin Woodward, Abir Maarouf Kabbani, Christopher V. Kelly. Electrochemistry, Moscow, Russian Federation, Institute of Biophysics, Physics, Wayne State University, Detroit, MI, USA. Johannes Kepler University, Linz, Austria. The sorting of lipids and protein on plasma membrane are critical to the life We identify the mechanism of interbilayer and transbilayer coupling of liquid- of eukaryotic cells. For example, in the process of phagocytosis, phosphatidy- ordered domains in lipid membranes. The driving force for such coupling linositol 4,5-bisphosphate (PIP2) accumulates around phagosomal cup and has thus far remained unrevealed, yet membrane domains are always adopt leads to actin remodeling. However the mechanisms of phagocytosis initiation bilayer conformation. Liquid-ordered domains also demonstrate transbilayer is yet to be fully understood. We will be resolving these mechanisms via correlative behaviour by uniting into stacks in the multilamellar structures. understanding the regulations of phospholipids such as PIP by membrane Various hypothesis were put forward to explain coupling phenomena. Trans- 2 structure, i.e., studying the interplay between nanoscale curvature and PIP2 bilayer coupling is usually explained by some phenomenological interaction accumulation in curved supported lipid bilayers (SLBs). We hypothesize between ordered and disordered domains in opposite monolayers. While inter- that PIP2 will be sorted based on membrane topography since membrane bilayer coupling is described by hydration forces that are supposed to be curvature has been shown to induce segregation of various lipids and pro- different for ordered and disordered phases. Both hypotheses lack sufficient teins (Tian and Baumgart, Biophys. J., 2009). Since diffusion is a great indi- evidence and theoretical basis, while such a widespread cooperative behaviour cator of membrane properties, the diffusion of lipids are quantified on of rafts makes to suggest the existence of common driving force for these nanoscale curvature and flat membrane by single particle tracking (SPT) phenomena. paired with polarized localization microscopy (PLM). To create SLB with We provide the universal mechanism of such striking behaviour. Using con- nanoscale curvature sites, giant unilamellar vesicles (GUV) were burst on tinuum elasticity theory, we show that elastic deformations alone are the 48 nm diameter nanoparticles over a coverslip substrate. Fluorophorescently driving force of ordered domain coupling. The coupling is provided by two tagged phospholipids were applied to the top leaflet after the formation of independent mechanisms. The first one is due to membrane dynamic mem- SLBs to minimize the membrane-substrate interaction. Single particle brane shape fluctuations; the corresponding energy is proportional to domain tracking (SPT) indicates that DPPE-Rhodamine diffuses 4 times faster on area. This mechanism utilizes the idea that stiff regions in both monolayers flat membrane than on 48 nm curvature sites. This result implies the mem- attract each other because their registration minimizes spatial restraints on brane viscosity increase with curvature. In future experiments, TopFlour- membrane undulations, i.e. domain registration maximizes entropy. The sec- PIP2 will be introduced to SLB top leaflet with nanoscale curvature sites ond mechanism bases on the domain boundary energy minimization; the that are supported by nanoparticle of varying size. Diffusion and concentra- coupling energy is proportional to domain boundary length. In the framework tion of TopFlour-PIP2 and other phospholipids will be studied on flat mem- of thickness mismatch model we show that the minimum domain boundary brane verses nanoscale curvature sites. Understanding the molecular sorting energy is achieved in registered conformation, making monolayer domains in lipid bilayers will reveal the mechanisms of PIP2 accumulation in nanoscale and non-stacked configurations energetically unfavourable [1, 2]. Combina- curvature initiation. tion of these two mechanisms results in alignment driven by lipid deforma- tions, the membranes fundamental property, which does not require 1892-Pos Board B212 introducing any specific features, like interactions at the membrane midplane, Archaeal-Inspired Lipids Exhibit Low Membrane Permeability due to different hydration forces, etc. Fluctuation-based model is universal and Entropic Effects does not depend on particular properties of lipid membrane and can be Geoffray Leriche1, Karthikeyan Diraviyam2, Young Hun Kim1, applied to any system, composed of elastic films with non-uniform elastic Takaoki Koyanagi1, Olivia Eggenberger3, Thomas B.H. Schroeder3, characteristics. Michael Mayer3, Jerry Yang1, David Sept2. 1. Galimzyanov et al., Physical Review Letters 115.8 (2015). 1Chemistry and Biochemistry, University of California San Diego, La Jolla, 2. Galimzyanov et al., Physical Review Letters 116.7 (2016). CA, USA, 2Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA, 3Adolphe Merkle Institut, Fribourg, Switzerland. 1890-Pos Board B210 Many archaea are able to withstand extremes of temperature, pressure, pH Mechanical Properties of Model Erythrocytes Membranes from Healthy and/or levels of salt. One unique feature of these extremophiles that gives and Hereditary Spherocytosis Subjects them this capability is that their membranes are made from covalently linked, 1 1 2 3 Bruna R. Casadei , Amanda C. Carita´ , Vale´ria F. Dutra , Eneida de Paula , tetraether lipids that form a monolayer rather than a traditional bilayer. Here 4 2 1 Rumiana Dimova , Maria S. Figueiredo , Karin A. Riske . we present experimental and computational results on a series of synthetic 1Biophysics, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil, 2 archaeal-inspired lipids, looking at their biophysical properties and rates of Hematology and Hemotherapy, Federal University of Sa˜o Paulo, Sa˜o Paulo, permeation. Apart from a number of interesting mechanical and material Brazil, 3Biochemistry, State University of Campinas, Campinas, Brazil, 4 properties, we find that tethered lipids exhibit a much stronger dependence Theory & Bio-Systems, Max Planck Institute of Colloids and Interfaces, on the entropy of activation for small molecules to cross the membrane. In Potsdam, Germany. relating our measured permeation rates with properties of the membrane, Defects in the erythrocyte membrane can cause hereditary spherocytosis (HS), we find that the order parameters for the lipid tails describe the changes in a congenital hemolytic anemia caused by a deficiency in cytoskeletal or integral entropy, and the combination of area per lipid, membrane thickness and proteins. To understand the complex behavior of the composing lipids and their short-range diffusion coefficient can predict our permeation results. Interest- involvement in the stability and organization of the membrane structure, we ingly, we also see that the rates of water penetration into the core of the mem- have used optical microscopy in giant unilamellar vesicles (GUVs) and as- brane provide an excellent empirical measure that matches our experimental sessed the membrane mechanical properties and the response of GUVs to permeation rates. Triton X-100. GUVs were composed by lipid extracted from the erythrocyte membranes of both healthy individuals (GUVs-NI) and HS (GUVs-HS) pa- 1893-Pos Board B213 tients, as well as by purified lipids (palmitoyl oleoyl phosphatidylcholine, Lateral Heterogeneity of Cholesterol on Binary Lipid Mixtures of POPC/ POPC, and POPC:sphingomyelin:cholesterol, 2:1:2). The bending rigidity of CHOL: a Molecular Dynamics Study GUVs was quantified using fluctuation analysis. GUVs-HS were found to be Fernando Favela-Rosales1, Arturo Galva´n-Herna´ndez2, softer than GUVs-NI and GUVs-2:1:2, probably reflecting differences in their Jorge Herna´ndez-Cobos2, Iva´n Ortega-Blake2. acyl chain composition. The pore edge tension was measured from the dy- 1Instituto Tecnolo´gico Superior Zacatecas Occidente, Sombrerete, Mexico, namics of macropore resealing after application of electric pulses. The edge 2Universidad Nacional Auto´noma de Me´xico, Cuernavaca, Mexico. tension of erythrocyte lipids was shown to be lower than that of the biomimetic Phase diagrams on ternary and quaternary lipid mixtures showing the exis- system (2:1:2), probably due to the presence of charged lipids. The solubiliza- tence of liquid-disordered (ld), liquid-ordered (lo) and a mixed phase (ldþlo)

BPJ 7847_7850 384a Tuesday, February 14, 2017 are now well established. Although the ldþlo is generally accepted for 1896-Pos Board B216 binary mixtures of cholesterol and phospholipids with saturated acyl Bending Modulus of Bolalipids under the U-Shapes Diffusion chains, the existence of a phase separation in mixtures of cholesterol with Sergei I. Mukhin, Daniyar Gabdullin. phospholipids that have one saturated and one unsaturated acyl chain is Theoretical Physics and Quantum Technologies Department, Moscow not universally accepted. There are reports of a phase diagram on the Institute for Steel and Alloys, Moscow, Russian Federation. mixtures POPC/Chol and POPC/Erg and nystatin activity co-related to this We propose a phenomenological model to calculate frequency dispersion of the purported phase diagram; with a maximal activity appearing in the mixed bending modulus of bolalipid membrane with lateral diffusive motion of small phase. In a recent work we presented a Molecular Dynamics (MD) study of concentration of the bolalipid molecules in the U-shape conformation. A local a POPC membrane with different cholesterol concentrations along the curvature of the membrane causes an increase of the elastic energy, that could phase diagram and computed the order parameters binned in the xy- be released by dynamically induced U-shapes concentration imbalance on the plane as well as the cholesterol density. The results support the existence of opposing sides of the bolalipid layer. An effective attractive/repulsive potential a lateral heterogeneity of cholesterol, present mainly in the mixed acting upon the U-shapes on the opposite sides of the curved regions of the phase. Here we extend the previous study to consider also membrane thick- membrane are defined using the calculations of the local curvature induced ness as well as the evolution of these distributions along a ms of simulation. by a difference of the U-shapes concentrations on the opposite sides of the We observed structures that support the idea of cholesterol lateral heterogene- membrane. When local spontaneous curvature caused by the U-shapes imbal- ity is present in this binary mixture. The theoretical distributions were ance equals the local curvature induced by membrane bending the potential compared to Atomic-Force Microscopy (AFM) images from studies of the acting on the U-shapes turns to zero. Allowing for U-shapes lateral diffusion same system on supported lipid bilayers. The combined study of AFM and coefficient, the latter condition permits us to derive dynamic equations that MD simulations allowed us to advance in the understanding of the molecular describe in essence the dispersion of the bending modulus as function of the structure changes resulting from phase differences. Funding: DGAPA- bending frequency. A possible comparison of the theoretical prediction with PAPIIT-RG100416. the proper experiment is discussed.

1894-Pos Board B214 1897-Pos Board B217 Giant Vesicles Fabricated from Paper Different Static and Similar Kinetic Phase Behavior Seen for Monoolein Anand B. Subramaniam. and a Sister Compound School of Engineering, University of California, Merced, Merced, CA, USA. Andrew D. Folkerts, Paul E. Harper. Giant lipid vesicles are useful in vitro models for cells and for studying Department of Physics and Astronomy, Calvin College, Grand Rapids, biochemical processes. Here we report a straightforward and widely MI, USA. accessible technique, PAPYRUS, Paper-Abetted liPid hYdRation in aqUeous Monoglycerides form a rich variety of non-lamellar phases, including minimal Solutions, to produce cell-sized vesicles that are free of residual solvents surface based cubic phases and the inverted hexagonal phase. Monoolein is a and that are not exposed to electric fields during the growth process. We particularly popular monoglyceride that is widely utilized for drug encapsula- find that when placed in aqueous solutions, numerous giant vesicles tion and protein crystallization. We use DSC (differential scanning calorim- formed from lipid films coated on cellulose fibers of filter and chromatog- etry) to look at the static and kinetic phase behavior of a sister compound, raphy paper. Vesicles of different lipid compositions can be formed in mononoadecenoin (MNd), which differs from monoolein by the addition of a various aqueous media such as ultrapure water, physiologically relevant single methylene. Despite this small difference, the temperature of the cubic ionic buffers, and sugar solutions. Fluorescent labeling and analysis to inverted hexagonal phase transition is 30 C lower in MNd than in monoolein. demonstrated that cellulose does not incorporate into the membranes of We measure the hysteresis of this transition as function of temperature ramp the vesicles. The insolubility of cellulose in water, even at elevated tem- rate and concentration of added sucrose solution. The hysteresis exhibits a peratures, allows the formation of vesicles with lipids with high transition power law dependence on the ramp rate. Interestingly, despite the difference temperatures. The porosity of paper allows gentle fluid flow normal to the in the phase transition temperatures, the power law exponent as function of paper to free vesicles into the bulk. The extreme ease of the PAPYRUS temperature is the same for MNd and monoolein. technique, coupled with not requiring a power-source or function 1898-Pos Board B218 generator, allows massive parallelization using readily available or easily Hysteresis and the Cholesterol-Dependent Melting Transition of the improvised fluid receptacles such as Eppendorf tubes or polystyrene multiwell Martini Model plates. Alexis Webb, Clement Arnarez, Edward Lyman. University of Delaware, Newark, DE, USA. 1895-Pos Board B215 Extensive Martini simulation data, totaling 5 ms, is presented for binary mix- Manipulation of Phase-Like Domains within Intact B Cell Plasma Mem- tures of dipalmitoyl phosphatidylcholine (DPPC) and cholesterol. Using simu- branes and Visualization of their Composition using Super-Resolution lations initiated from both gel and liquid-disordered (L d ) phases, significant Microscopy and strongly cholesterol-dependent hysteresis in the enthalpy as a function of Marcos F. Nunez, Matthew B. Stone, Sarah L. Veatch. temperature is observed for cholesterol mole fractions from 0 to 20 mol% — Biophysics, University of Michigan, Ann Arbor, MI, USA. adding just a single cholesterol to each leaflet of a pure DPPC bilayer more Protein sorting based on liquid-ordered (Lo) or liquid-disordered (Ld) than doubles the width of the hysteresis loop relative to a pure DPPC bilayer. phase preference is readily observed in giant plasma membrane vesicles Although the precise phase transition temperature cannot be determined due to (GPMVs) at low temperatures but is not as easily detected in the intact cells the hysteresis, the data are consistent with a first order gel to fluid transition, from which they are derived. Here we utilize two-color super-resolution which increases in temperature with cholesterol. At 30 mol% cholesterol, no microscopy in combination with cross-correlation analysis to quantify the hysteresis is observed, and there is no evidence for a continuous transition, sorting of two minimal inner leaflet anchored-peptides proximal to clusters in either structural parameters like the area per lipid or in the heat capacity of cholera toxin B subunit (CTxB) in chemically fixed CH27 B cells. We as a function of temperature. The results are consistent with a single uniform find that the local density of a Lo-partitioning peptide is increased while phase above a critical cholesterol composition between 20 and 30 mol% in the local density of a Ld-partitioning peptide is reduced in the vicinity of Martini, while highlighting the importance and difficulty of obtaining the equi- CTxB clusters when compared to the average density of peptides on the librium averages to locate phase boundaries precisely in computational models cell surface. This in combination with additional results confirm cell plasma of lipid bilayers. membranes can support lipid domains which resemble the Lo and Ld phases in GPMVs and span leaflets of the plasma membranes. Ongoing work is 1899-Pos Board B219 aimed at detecting heterogeneity in intact cells in the absence of artificial Ultradonut Topology of the Nuclear Envelope CTxB clustering, and at distinguishing possible physical bases of the observed Mehdi Torbati1, Tanmay P. Lele2, Ashutosh Agrawal1. lipid-mediated membrane heterogeneity in intact cells. These experiments are 1University of Houston, Houston, TX, USA, 2University of Florida, characterizing the changes in enrichment and depletion of phase-partitioning Gainesville, FL, USA. peptides with respect to CTxB upon changes in temperature and composition Lipid membranes exhibit a variety of morphologies tailored to perform specific of the plasma membrane. These results will be compared to predictions of functions of cells and their organelles. A unique lipid structure is the nuclear Ising model simulations to test for consistency between that model and the envelope which houses the genome and plays a vital role in genome organiza- behavior of the phase-like domains we have observed in B cell plasma mem- tion and signaling pathways. The nuclear envelope is composed of two branes. fused membranes with thousands of toroid-shaped pores with extremely high

BPJ 7847_7850 Tuesday, February 14, 2017 385a curvatures, the origin of which remains an open question in biology. Here, we 1902-Pos Board B222 show that the architecture of this ultradonut may be generated by nanoscale Nanoscopic Lipid Domains Characterized by iSCAT Microscopy buckling instabilities triggered by membrane stresses during nuclei growth. Matthew C. Blosser1,2, Helena L.E. Coker1,2, Mark I. Wallace2. Our findings may help understand the impact of membrane mechanics on the 1Chemistry, University of Oxford, Oxford, United Kingdom, 2Chemistry, geometry and the functionality of the nucleus and more generally, other King’s College London, London, United Kingdom. double-membrane organelles in cells. Characterizing the lateral organization of lipid membranes is important for un- derstanding the function of cell membranes. Several groups have reported sub- 1900-Pos Board B220 micron heterogeneity in model membranes, using techniques such as neutron Stabilization of Cell Membrane Structures by Curvature-Inducing scattering, x-ray scattering, FRET, or AFM. Here, we study these systems using Proteins iSCAT microscopy, a recently developed technique that images the differences Morgan Chabanon, Padmini Rangamani. in light scattering from different regions of a sample. This technique is capable Mechanical and Aerospace Engineering, University of California San Diego, of label-free detection of nanometer-scale lipid domains, and is compatible La Jolla, CA, USA. with fully hydrated, micron-scale membranes. This allows us to directly mea- An astonishing variety of membrane structures can be observed in the cellular sure the membrane organization and dynamics, and to compare data from the environment, both at the plasma membrane and at the organelles. These shapes same technique across widely different length scales. are intricately related to biological function, enabling and regulating funda- mental cellular processes such as vesicle trafficking, signaling, or vectorial syn- 1903-Pos Board B223 thesis in the endoplasmic reticulum. Yet, most of the membrane structures Optimizing Conditions for the Isolation of Membrane Proteins by Styrene- observed experimentally are unstable from the stand point of lipid membrane Maleic Acid Copolymers: A Case Study on E. Coli Inner Membranes thermodynamics, unable to retain their shape without the contribution of curva- Jonas M. Doerr, Martijn C. Koorengevel, J. Antoinette Killian. ture inducing proteins. Chemistry, Utrecht University, Utrecht, Netherlands. Here, we aim to answer the following fundamental question: given a cellular Styrene-Maleic acid (SMA) copolymers have emerged as a powerful alterna- membrane structure, what is the distribution and concentration of curva- tive to detergents for the extraction of membrane proteins from cellular mem- ture inducing proteins necessary to maintain this shape? Based on Helfrich branes. This approach has been successfully demonstrated for proteins present model extended for protein surface diffusion, we solve the shape equation in the membranes of a variety of organisms including all major systems used for on fixed geometries of biological relevance, in order to find the field of spon- heterologous expression. However, for downstream characterization often high taneous curvature that stabilize the membrane structure. We further relate amounts of proteins are required, which remains a challenge in some cases. the spontaneous curvature to protein density, bending ability, and membrane Extraction of proteins from the membrane and purification of the solubilized tension. material are two major bottlenecks in this process. We first investigate the role of spontaneous curvature in minimal surfaces, Here, we report a systematic approach to optimize conditions for the use of which includes catenoids — relevant to vesicle trafficking, tubulation, and nu- SMA to extract the tetrameric potassium channel KcsA from the inner mem- clear pores — and helicoids — relevant to endoplasmic reticulum ramps. In brane of Escherichia coli, by varying different parameters such as SMA-to- these cases, the shape equation reduces to an heterogenous Helmholtz equation membrane ratio, ionic strength, pH, polymer type and polymer length. Further- for the spontaneous curvature, where the source term is proportional to the more, conditions for the purification of the channel via Ni-affinity of its poly- Gaussian curvature. We show that this latter quantity alone is responsible for His tag have been optimized. Implications of our findings will be discussed in non-uniform distribution of spontaneous curvature in minimal surfaces. We the general context of membrane protein solubilization by SMA. then expand our analysis to more general surfaces, and identify instabilities in spontaneous curvature intensity, pointing out the need for distinct proteins 1904-Pos Board B224 at different stages of membrane shaping processes. We demonstrate how het- Membrane Protein Extraction by Styrene-Maleic Acid Copolymers is erogeneities in spontaneous curvature distribution maintain energetically Independent of Subcellular Localization but is Influenced by Membrane expensive membrane shapes at lower cost. Organization Jonas M. Doerr1, Juan J. Dominguez Pardo1, 1901-Pos Board B221 Marleen H. van Coevoorden-Hameete2, Casper C. Hoogenraad2, Solubilization of Lipid Membranes by Styrene-Maleic Acid Copolymers: J. Antoinette Killian1. Importance of Polymer Composition and PH 1Chemistry, Utrecht University, Utrecht, Netherlands, 2Biology, Utrecht Stefan Scheidelaar, Juan J. Dominguez, Jonas M. Dorr, University, Utrecht, Netherlands. Martijn C. Koorengevel, Cornelis A. van Walree, J. Antoinette Killian. Styrene-Maleic acid (SMA) copolymers have emerged as a powerful alterna- Membrane Biochemistry & Biophysics, Utrecht University, Utrecht, tive to detergents for applications in membrane research. Most notably, Netherlands. these amphipathic polymers can be used to directly extract and purify The styrene-maleic acid (SMA) copolymer is rapidly gaining attention as tool membrane proteins from intact cells of different organisms in the form of in membrane research due to its ability to directly solubilize lipid membranes ‘‘native nanodiscs’’. These particles stabilize the protein in a near native envi- into nanodisc particles without the requirement of conventional detergents. ronment comprising conserved native lipids as well as other membrane compo- Although many different variants of SMA are commercially available, so far nents that allow for the study of preferential interactions by biochemical mainly those with a styrene-to-maleic acid monomer ratio of 2:1 or 3:1 have analysis. been used for model membrane studies as well as for membrane protein To evaluate the general applicability of SMA-mediated membrane protein extraction. extraction from biomembranes, we employed fluorescence microscopy to visu- Here, we present a systematic investigation of the effect of SMA composition alize SMA solubilization of HeLa cells in real time. Our results indicate that on its solution properties as well as on its interaction with membranes. A partic- SMA solubilization of (human) cells is a multi-step process that is not specific ular focus was the effect of pH, which strongly affects the charge density of the for any (sub)cellular membrane. However, we found solubilization kinetics of polymer. Using model membranes of di-14:0 PC lipids, we found that mem- plasma membrane-localized proteins to vary markedly for different proteins, brane solubilization is promoted by a low charge density and by a relatively suggesting a strong influence of membrane organization and domain formation. high fraction of maleic acid units in the polymer. To assess the influence of membrane properties on solubilization preferences of In addition, we used calorimetry and fluorescence methods to investigate the SMA in more detail, we employed an approach of partial solubilization of influence of SMA composition on the properties of lipids in nanodisc particles model membranes of different compositions. In lipid mixtures that form homo- formed of different saturated PCs. We found that all polymers affect the gel-to- geneous bilayers, we found that SMA exhibits a high degree of promiscuity liquid crystalline phase transition of the lipids but that the degree of their influ- with respect to lipid headgroups. However, in phase-separating systems of fluid ence depends on the styrene-to-maleic acid ratio. phases with either gel-phase or liquid-ordered phases, lipids in the fluid phase Altogether, the results show large differences in behavior between different were solubilized with a strong preference. SMA variants, with the one having a styrene-to-maleic acid ratio of 2:1 Our findings support the use of SMA for the study of lipid-protein interactions showing both the highest efficiency in membrane solubilization and the best of membrane proteins and suggest applications for the isolation of membrane preservation of lipid bilayer properties in nanodisc particles. proteins from ordered domains in phase-separated membranes.

BPJ 7847_7850 386a Tuesday, February 14, 2017

Protein-Lipid Interactions: Structures 1908-Pos Board B228 Direct Observation of Unidirectional Cholesterol Flipping on the Surface 1905-Pos Board B225 of P-Glycoprotein Computational Characterisation of the Modulation of Membrane Proteins Sundar Thangapandian, Emad Tajkhorshid. by Lipids Biochemistry/Beckman Institute, University of Illinois at Urbana- Carmen Domene1, Javier Iglesias Fernandez1, Richard Naftalin2, Champaign, Urbana, IL, USA. Peter J Quinn3. P-glycoprotein (Pgp) is a biomedically important member of the largest super- 1Chemistry, Kings College London, London, United Kingdom, 2Physiology, family of ABC transporters and mediates multidrug resistance in many cancer Kings College London, London, United Kingdom, 3Biochemistry, Kings types. Substrate binding and transport in Pgp are modulated by the presence of College London, London, United Kingdom. cholesterol in the membrane, while cholesterol itself is a substrate and exported Experimental evidence has shown a close correlation between the composition by Pgp. Structural information on cholesterol binding sites and mechanistic and physical state of the membrane bilayer and glucose transport activity via details of its transport pathway are largely unknown. In this study, a set of the glucose transporter GLUT1. Cooling alters the physical state of the mem- 35 independent simulations of Pgp oriented independently in cholesterol-rich brane from fluid to gel phase, and in parallel, a large decrease in net glucose lipid bilayers were performed, adding to a total of 7 ms, enabling extensive sam- transport rate is observed. How the physical state of the membrane alters pling of lipid-protein interactions. From the ensemble of cholesterol molecules glucose transport dynamics has been investigated using computational ap- (~4700) sampled in these simulations, one complete and two partial cholesterol proaches. It is found that the protein is sensitive to the membrane phase and flipping events were captured. All of the flipping events visited a region formed this in turn has implications on the glucose transport mechanism. by transmembrane (TM) helices TM1, TM2, and TM11 with Tyr 49 and Tyr 126 as key residues interacting with cholesterol. This region has been identified 1906-Pos Board B226 with two cholesterol-recognition amino acid sequence (CRAC) motifs. Choles- Molecular Dynamics Simulations of G-Protein Coupled Receptor Lipid terol flipping from the inner leaflet initially binds to this motif and assumes a Interactions 90 angle to the membrane normal burying its only hydroxyl group within George Hedger1, Hsin-Yung Yen2, Idlir Liko2, Carol V. Robinson2, the surface of the protein followed by flipping to the outer leaflet. Convergence Christian Siebold3, Mark S.P. Sansom1. and clustering analyses of cholesterol molecules around Pgp revealed that 1Department of Biochemistry, University of Oxford, Oxford, United cholesterol binds to specific regions formed between TM1-6 and TM8 helices Kingdom, 2Department of Chemistry, University of Oxford, Oxford, United interacting through its rough surface. From the generated ensemble of choles- Kingdom, 3Division of Structural Biology, Wellcome Trust Centre for terol sampling around Pgp, only a single complete cholesterol flipping from in- Human Genetics, University of Oxford, Oxford, United Kingdom. ner leaflet to outer leaflet has been directly observed, which supports the known Lipid molecules can bind selectively to specific sites on integral membrane pro- flippase activity of Pgp. Our study is the first to report that substrate transloca- teins, modulating their structure, stability, and function. We have undertaken tion by Pgp might take place through pathways on the surface of the protein. multiscale molecular dynamics simulations to investigate two distinct modes of lipid interaction with G-protein coupled receptors (GPCRs). Native mass 1909-Pos Board B229 spectrometry measurements suggest the presence of specific lipid binding sites P-gp Lipid Uptake Pathways Determined by Coarse-Grain Molecular on Neurotensin Receptor 1 (NTS1), in particular for phosphatidyl inositol phos- Dynamic Simulation phate (PIP) species. Coarse-grained molecular dynamics simulations of NTS1 Estefania Barreto Ojeda, Valentina Corradi, Ruo-Xu Gu, Peter Tieleman. embedded in a lipid bilayer indicate strong and specific interactions of PIP mol- Biological Sciences and Centre for Molecular Simulation, Univeristy of ecules with defined regions on the intracellular portion of NTS1. The predom- Calgary, Calgary, AB, Canada. inant mode of PIP binding is via interaction with basic residues located within P-glycoprotein (P-gp) belongs to the ATP-binding cassette transporters (ABC) intracellular loop regions, inaccessible to other anionic lipid species such as superfamily of membrane proteins. The transport mechanism in ABC trans- phosphatidyl serine. In silico mutagenesis of key residues within these regions porters is triggered by ATP binding and hydrolysis at cytosolic nucleotide bind- abrogates lipid binding. These simulation based predictions provide guidance ing domains (NBDs). NBDs dimerization and dissociation are coupled with for experimental mutagenesis of NTS1, toward structurally rationalising the conformational changes in the transmembrane domains (TMDs), which form proposed PIP binding sites. Molecular simulations have also been used to the translocation pathway for the substrates. Due to its ability to recognize explore interactions of cholesterol with the extracellular domain of Smooth- and pump drugs to the extracellular medium, P-gp is a key player in cancer ened (SMO), a Class F GPCR. The recent near full-length x-ray structure of multidrug resistance. Among its substrates are also lipids and lipid-like mole- SMO revealed the presence of a cholesterol molecule within a hydrophobic ste- cules, as, for example, miltelfosine, a phospholipid-based anticancer drug. For rol binding groove on the extracellular cysteine rich domain (CRD). Atomistic the development of strategies targeting P-gp inhibition, a better understanding simulations of SMO in a lipid bilayer suggest the cholesterol molecule exerts a of the steps involved in substrate recognition and uptake is needed. In this work, marked effect on the structural stability of the CRD, as well as the potential for we use coarse-grain (CG) molecular dynamics (MD) simulations to explore a degree of flexibility of extracellular domains of SMO relative to 7TM domain. possible pathways of lipid uptake in the inward-facing conformation of P-gp. Together these studies suggest how molecular simulations may be used in Five different lipid environments were considered: a pure POPC bilayer, a conjunction with experiment to guide the identification of lipid binding sites, pure POPE bilayer, a symmetrical POPC:POPE bilayer, and two additional bi- and characterise the effects of lipid binding on GPCR structure and function. layers with different ratios of POPE and POPC lipids. The simulations, each of the duration of 20ms, were carried out using the MARTINI force field. The re- 1907-Pos Board B227 sults highlight the role of specific transmembrane helices and residues in the Rational Design of New Antimicrobial Peptides Targeting Gram Negative initial steps of the uptake and for the interactions established with a given lipid Bacteria once inside the cavity. We suggest possible uptake pathways in P-gp in the Loan K. Huynh1, Goutam Gupta2. different lipid environments used in this study. 1Biology, New Mexico Consortium, Los Alamos, NM, USA, 2Life Sciences Division, New Mexico Consortium, Los Alamos National Laboratory, Los 1910-Pos Board B230 Alamos, NM, USA. Lipid Transfer Mechanism of CETP between HDL and LDL: A Coarse- Antimicrobial peptides (AMPs) have been extensively investigated as poten- Grained Simulation Study tial antibiotics because the activity of these small cationic peptides can be Venkata Reddy Chirasani, Sanjib Senapati. enhanced by rational design. Many AMPs appear to bind and subsequently Department of Biotechnology, IIT Madras, Chennai, India. disrupt the integrity of microbial membranes. However, the precise mechanisms Cholesteryl ester transfer protein (CETP) mediates the bidirectional exchange of membrane disruption remain unclear. Combining experiment and molecular of cholesteryl esters (CEs) and triglycerides between HDL and LDL (1). Ani- dynamics (MD) simulations, we investigate the mechanisms by which AMPs mal models along with early clinical studies have demonstrated that the interact with model bacterial membranes. Specifically, we define the structural impairment of CETP’s functionality is important for the efficient treatment and dynamic properties of membrane interaction for a panel of candidate of cardiovascular diseases. Despite the pharmacological interest, CETP’s lipid AMPs. Using conventional and temperature virtual replica exchange simula- transfer mechanism is poorly understood. Recent cryo-electron microscopy tions, we correlate antimicrobial activity with membrane insertion depth. studies suggested that CETP penetrates its N-and C-terminal b-barrel domains Furthermore, we show that electrostatic density and the propensity for peptide into HDL and LDL respectively to form a ternary complex (2). Further it was aggregation have competing consequences for efficacy. Overall, our studies pro- proposed that the hydrophobic tunnel of CETP can simultaneously interact vide a detailed understanding of AMP disruption of bacterial membranes, facil- with the core of HDL and LDL for continuous lipid exchange. However, the itating rational design of more effective AMPs for Gram negative bacteria. detailed lipid transfer process is still inconclusive. In this study, we employed

BPJ 7851_7854 Tuesday, February 14, 2017 387a coarse-grained molecular dynamics simulations to illuminate the lipid transfer 1913-Pos Board B233 mechanism of CETP in HDL-CETP-LDL ternary complex. The extent of pene- Alanine Charge Screening Demonstrates Cytochrome C Unfolding on tration of CETP into HDL and LDL in our ternary complex model corroborated Cardiolipin Membrane Surfaces well with the experimental findings. Further, the results demonstrated that the Margaret M. Elmer-Dixon, Bruce E. Bowler. ejection of C-terminal plug-in phospholipid (PLC) and subsequently the diffu- University of Montana, Missoula, MT, USA. sion of CETP-bound CEs into LDL droplet through PLC opening. A detailed Cytochrome c (Cytc) has been shown to play an important role in signaling analysis on interactions of PLC pocket lining residues with the discharged lipids during apoptosis. In the initial stages, positively charged regions of Cytc revealed residues with significant role in CE transfer. The detailed mechanism interact electrostatically with negatively charged, cardiolipin (CL) on the inner of lipid transfer by CETP as explored in this study might help designing future mitochondrial membrane. In the presence of reactive oxygen species, Cytc CETP research and subsequent CETP therapeutics. demonstrates increased peroxidase activity and a propensity to oxidize CL. References: Cytc dissociate from the oxidized CL freeing it to form the apoptosome in 1. Tall, A.R. Plasma cholesteryl ester transfer protein. J. Lipid Res. 34: 1255– the cytoplasm and initiate apoptosis. The exact mechanism for this initial elec- 1274 (1993). trostatic interaction and the role of structure and conformation of Cytc have 2. Zhang, L. et al., Structural basis of transfer between lipoproteins by choles- been extensively debated. Using heme Soret band circular dichroism (CD), teryl ester transfer protein. Nat. Chem. Biol. 8: 342–349 (2012). fluorescence (Trp59) and UV-visible spectroscopies, in coordination with alanine charge screening, we investigate the initial electrostatic cytochrome 1911-Pos Board B231 c/cardiolipin interaction. Role of Bound Phospholipids in Structural Stability and Functionality of To date, several cardiolipin interaction sites have been proposed on cyto- Cholesteryl Ester Transfer Protein chrome c. At pH 8, we are able to isolate and study the cytochrome c/cardio- Prasanna Diddige Revanasiddappa, Revathi Shankar, Sanjib Senapati. lipin interaction at anionic binding site A with 100% cardiolipin vesicles. Biotechnology, IITM, Chennai, India. Using alanine charge screening (Lys/Ala mutations), we investigated the Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl role of lysines 72, 73, 86 and 87 in site A during electrostatic cardiolipin bind- esters (CEs) and triglycerides (TG) between high density lipoproteins (HDL) 1 ing. Optimized titration techniques were utilized that generate reproducible and low density lipoproteins (LDL). Previous animal model studies have quantitative data that can then be fit to extract the binding contribution to car- shown that blocking the function of CETP can increase the level of HDL in diolipin for each amino acid investigated. Our data indicate that an initial bind- blood plasma and suppress the risk of cardiovascular disease (CVD). Hence, ing occurs at lipid to protein, L/P, ratios below 5. However, neither Trp59 understanding the mechanism by which CETP transfers the neutral lipids be- fluorescence nor Soret band CD is strongly sensitive to this early binding event tween lipoproteins are of immense importance. In a recent report (Chirasani making quantitative evaluation of this interaction difficult. At L/P ratios >20 a et al., J. Biol. Chem. 2016), we have shown that the bound CEs intraconvert binding event consistent with a conformational rearrangement on the surface between bent and linear conformations as a consequence of high degree of 2 of the CL vesicle occurs. Our data indicate that one lysine from either side conformational flexibility of the protein. In this study we explored the impor- of Site A (Lys72/73 versus Lys86/87) is important for both the cooperativity tance of bound phospholipids (PLs) in maintaining the structural integrity of of and L/P ratio needed for this conformational rearrangement on the liposome CETP and its role in lipid transfer. Results from all atom molecular dynamic surface. simulation show that N-terminal bound PL imparts fluctuations and C-terminal bound PL provides stability to the CETP structure. They are also found to play a 1914-Pos Board B234 crucial role in CE transfer from HDL to LDL. These observations are confirmed Exploring the Stability and Cardiolipin Affinity of Cytochrome C’s by restraining both PLs, which showed conformational rigidity in CE. The Domain Swapped Dimer Conformation detailed insights obtained here on the role of PLs in CETP could help in Harmen B. Steele1,2, Levi J. McClelland1,2, J.B. Alexander Ross1,2, devising methods to prevent the CETPs function in countering atherosclerosis. Bruce E. Bowler1,2. References 1Biochemistry and Biophysics, University of Montana, Missoula, MT, USA, 1. Mistry, Q. X., et al., Crystal structure of cholesteryl ester transfer protein 2Chemistry & Biochemistry, University of Montana, Missoula, MT, USA. reveals a long tunnel and four bound lipid molecules. Nat. Struct. Mol. Biol. Cytochrome c’s (cytc) oxidation of the lipid cardiolipin (CL) is a proposed 14, 106-13 (2007) switch involved in initiation of the intrinsic pathway of apoptosis. Recently, 2. Chirasani, V. R., et al., Structural Plasticity of Cholesteryl Ester Transfer there has been an increased number of reports in the literature of cytc Protein Assists the Lipid Transfer Activity. J. Biol. Chem. 291, 19462-19473 domain-swapped dimers (DSD). The Hirota lab has reported that the horse (2016) DSD has increased peroxidase activity compared to the monomeric form. The Bowler lab has solved DSD crystal structures with various detergents bind- 1912-Pos Board B232 ing in the hydrophobic tube of the proposed ‘‘C’’ cytc-CL binding site. We Oxidation State and PH Dependence of Cytochrome C Binding to Cardio- postulate that the DSD conformation of cytc is an evolutionary switch allowing lipin-Containing Liposomes for tighter regulation of the intrinsic pathway of apoptosis. In order to examine Bridget Milorey, Reinhard Schweitzer-Stenner. the stability of the DSD, samples were incubated temporally at fixed tem- Drexel University, Philadelphia, PA, USA. peratures and then analyzed using size-exclusion chromatography. The data, A combination of UV/visible absorption, circular dichroism and fluorescence presented here, indicate that the human DSD is more kinetically stable spectroscopies was used to probe the binding of cytochrome c to cardiolipin than horse or yeast DSD, suggesting evolution of a more kinetically stable (CL) containing liposomes and concomitant conformational changes as a conformation in primates. Additionally, fluorescence correlation spectroscopy function of cardiolipin concentration at different pH values. Our group has (FCS) data, also presented, show the binding of Zn-cytc to CL nanodiscs (ND). recently developed a model that describes oxidized cytochrome c binding to ND are discoidal lipid bilayers maintained by two alpha helical belt proteins CL-containing liposomes as a two-step process where native-like liposome bound and provide a nice model system to study protein-lipid interactions. Initial conformers convert into more unfolded conformations. We have provided evi- FCS results indicate that the Kd of cytc for CL NDs is in the micromolar range. dence that the M80 ligand in the more unfolded state has been replaced by either This poster will compare the relative CL binding affinities of monomer and a lysine or histidine as the axial ligand. A slightly modified binding model was DSD cytc. used to describe the binding of reduced cytochrome c, to account for the redox properties of the heme in the absence and presence of oxygen. In our current 1915-Pos Board B235 study, we explored how lowering the pH to values between 6.0 and 7.0 affects Selective Probing of Non-Native Cardiolipin-Bound Conformations of binding and conformation of cytochrome c in both oxidation states. It has recently Ferricytochrome C via Ferrocyanide-Mediated Photoreduction been proposed that such a protocol would facilitate the so-called L-site binding, Dmitry Malyshka, Reinhard Schweitzer-Stenner. which involves the electrostatic interaction of amino acid residues Lys22, 25, Chemistry, Drexel University, Philadelphia, PA, USA. 27 and His26 and 33 with the acidic phospholipids on the liposome surface. Upon binding to cardiolipin (CL), ferricytochrome c replaces its native Met80 Our results for binding at pH 6.5 indicate a conversion from a low spin to a hex- ligand and ultimately gains peroxidase activity, a vital step towards its dissoci- acoordinated high-spin state at moderate to high cardiolipin concentrations, ation from the inner mitochondrial membrane and its subsequent initiation of which leads to a significant blue shift of the Soret band. This very much resembles apoptosis. Multiple binding studies involving ferricytochrome c and CL-con- the behavior of denatured cytochrome c where lowering the pH leads to the pro- taining liposomes have resulted in a variety of partially conflicting binding tonation of a distal histidine ligand and its replacement by a water ligand. Thus, models, but little quantitative insight into the co-existence of native and our observations strongly suggest that histidine rather than lysine is the sixth partially unfolded conformations of ferricytochrome c has been obtained ligand in the misligated state of cytochrome c on CL-containing liposomes. thus far. While multiple groups have put forth binding models to explain this

BPJ 7851_7854 388a Tuesday, February 14, 2017 phenomenon, further validation of these models and characterization of the membranes that increase the propensity for misfolding and subsequent involved conformational states is still outstanding. Resonance Raman (RR) aggregation. spectroscopy is perfect for this task due to the high sensitivity of the heme vibrational modes to ligation, oxidation, and spin state changes of the heme 1918-Pos Board B238 iron. By utilizing ferrocyanide-mediated photoreduction, a normally undesired Structure and Dynamics of Nanopore-Confined Membrane Proteins are phenomenon that is inhibited for ferricytochrome c conformations lacking Affected by Bilayer Lipid Composition Met80 ligation, we selectively probed the non-native states of ferricytochrome Alexander A. Nevzorov, Morteza Jafarabadi, Antonin Marek, c bound to CL-containing liposomes both in the presence and absence of Alex I. Smirnov. NaCl. The thus elucidated concentrations of these non-native states at different Chemistry, North Carolina State University, Raleigh, NC, USA. lipid concentrations and ionic strengths were consistent with a previously Self-assembled nanotubular lipid bilayers confined within macroscopically reported model, where different conformations of the protein were calculated aligned high-density homogeneous nanopore array structure of anodic based on a global thermodynamic fit to a proposed [CL] dependent equi- aluminum oxide (AAO) membranes exhibit a high degree of macroscopic librium between a native-like (prone to photoreduction) and a partially alignment that facilitates their studies by oriented sample solid state (OS SS) unfolded bound state for which photoreduction is inhibited. RR spectral evi- NMR and spin-labeling EPR methods. The principal advantages of the lipid dence suggests a dominance of hexacoordinated low spin states even at high nanotube arrays are in the applicability of this method to essentially any lipid lipid content. Only trace evidence for the presence of a high spin state could composition and a tolerance of the macroscopic alignment to a wide range of be inferred from Raman spectra, but the presence of a charge transfer band environmental conditions such as, e.g., temperature, pH, and ionic strength. at 625 nm in the optical spectrum might point to the presence of a quantum- Here we describe 1) a recent progress in reconstructing small pore-forming mixed state. and transmembrane peptides as well as large membrane protein complexes, such as photosynthetic reaction center (RC) protein in inorganic nanopores 1916-Pos Board B236 and 2) utilization of this technology to study lipid-induced changes in protein Biophysical Insights into the Membrane Interaction of the Core Amyloid- conformations by magnetic resonance methods. Specifically, by using OS SS Forming Ab40 Fragment K16-K28 and its Role in the Pathogenesis of NMR we have shown that the tilt and a-helix kink angles of Pf1 coat protein Alzheimer’s Disease are affected by the bilayer composition on example of DOPC, POPC and Swapna Bera1, Kyle Korshavn2, Rajiv Kar1, Mi Hee Lim3, DMPC bilayers. Effects of unsaturated lipids on Pf1 dynamics as evidenced Ayyalusamy Ramamoorthy2, Anirban Bhunia1. by the changes in the linewidths in the Pf1 spectra are also reported. We relate 1Biophysics, Bose Institute, Kolkata, India, 2Chemistry, University of these changes to the bilayer fluidity and its effects on the uniaxial rotational Michigan-Ann Arbor, MI, USA, 3Chemistry, Ulsan National Institute of diffusion of the protein within the membrane. Further, we demonstrate the Science and Technology, Ulsan, Korea, Republic of. use of DEER spectroscopy to obtain long-range distance constraints for The aggregation of amyloid-b (Ab) on neuronal membranes is implicated in membrane protein systems incorporated into lipid nanotubes. The improved both neuronal toxicity and the progression of Alzheimer’s disease. Unfortu- nanopore alignment technique described here provides a general method for nately, the heterogeneous environment that results from peptide aggregation studying lipid-induced structural conformations of membrane proteins under in the presence of lipids makes the details of these pathways difficult to inter- physiologically relevant conditions by magnetic resonance. Supported by rogate. Hence In this study, we report an investigation of the membrane inter- U.S. DOE contract No. DE-FG02-02ER15354 to A.I.S. and NSF MRI 1229547 to A.A.N. action of an Ab fragment (K16LVFFAEDVGSNK28, KK13), which maintains the amyloidogenic nature of the full-length peptide and is implicated in 1919-Pos Board B239 membrane-mediated folding, through a combination of NMR spectroscopy Membrane Targeting of TIRAP is Negatively Regulated by Phosphoryla- and molecular dynamics simulations. Surprisingly what we have found that, tion in its Phosphoinositide-Binding Motif despite KK13’s ability to form amyloids in solution, the monomer remains un- Daniel G.S. Capelluto1, Wen Xiong1, Xiaolin Zhao1, Shuyan Xiao2, structured in the presence of lipid bilayers, unlike its full-length parent peptide. Jeffrey F. Ellena3, Geoffrey S. Armstrong4, Carla V. Finkielstein5. Additionally, NMR and molecular dynamics simulation results support that the 1Protein Signaling Domains Laboratory, Biological Sciences, Virginia Tech, presence of GM1 ganglioside, a lipid which strongly promotes binding be- Blacksburg, VA, USA, 2School of Materials and Metallurgy, Inner Mongolia tween Ab and lipid bilayers, promotes KK13 binding to but not folding on University of Science and Technology, Baotou, China, 3Biomolecular the membrane. Finally, we also show that the peptide partitions between the Magnetic Resonance Facility, University of Virginia, Charlottesville, VA, membrane and aqueous solution based on the hydrophobicity of the N-termi- USA, 4Departments of Chemistry and Biochemistry, University of Colorado nal residues, regardless of lipid composition. These results support previous at Boulder, Boulder, CO, USA, 5Integrated Cellular Responses Laboratory, discoveries suggesting the importance of GM1 ganglioside in exacerbating Biological Sciences, Virginia Tech, Blacksburg, VA, USA. membrane-driven aggregation while identifying the potential importance of Toll-like receptors (TLRs) trigger early immune system recognition and C-terminal residues in membrane binding and folding, which has previously response to infection. Pathogen-activated TLRs, such as TLR2 and TLR4, been unclear. dimerize and move laterally across the plasma membrane to phosphatidylino- sitol (4,5)-bisphosphate-enriched domains. At these sites, TLRs interact with 1917-Pos Board B237 the TIR domain-containing adaptor protein (TIRAP), triggering a signaling Structural Analysis of Tau Peptide Interactions with Lipid Membranes us- cascade that leads to innate immune responses. Membrane recruitment of ing Fourier Transform Infrared Spectroscopy TIRAP is mediated by its phosphoinositide-binding motif (PBM). We show Paige Engen, Larry Masterson. that TIRAP PBM transitions from a disordered to a helical conformation in Chemistry Department, Hamline University, St Paul, MN, USA. the presence of either zwitterionic micelles or monodispersed phosphoinositi- Alzheimer’s disease is a common form of dementia that is characterized by the des. TIRAP PBM bound phosphoinositides through basic and nonpolar residues formation of neurofibrillary tangles formed by the protein tau. The neurofibril- with high affinity, favoring a more ordered structure. TIRAP is phosphorylated lary tangles are formed when tau protein delocalizes from the microtubule at Thr28 within its PBM, which leads to its ubiquitination and degradation. binding domain and misfolds. This misfolding occurs primarily at four repeat We demonstrate that phosphorylation distorts the helical structure of TIRAP segments: R1, R2, R3, and R4. Previous studies indicate that lipid membranes PBM, reducing phosphoinositide interactions and cell membrane targeting. may be involved during this aggregation; particularly those with negative Our study provides the basis for TIRAP membrane insertion and the mecha- charge were shown to localize the aggregates of tau. In this study, we analyzed nism by which it is removed from membranes to avoid sustained innate im- the four repeat segments using FTIR spectroscopy to monitor structural mune responses. changes under various membrane conditions to investigate this phenomenon. The Amide I peak was used as a proxy for secondary structure in the presence 1920-Pos Board B240 of neutral DOPC lipid vesicles (a model of the plasma membrane) and nega- Structure, Dynamics, and Function of the Membrane Associated SRC tively charged DOPC/DOPG (80:20) lipid vesicles (a model of the Golgi Family Kinase HCK membrane). We show that the repeat segments are largely random coil in Matthew P. Pond1, Rebecca Eells2, Lydia Blachowicz1, Frank Heinrich2,3, aqueous solution and that the introduction of negatively charged vesicles in- Francisco Bezanilla1, Gianluigi Veglia4, Mathias Lo¨sche2,3, Benoıˆt Roux1. creases b-sheet and a-helix secondary structure by up to 50%. On the other 1Department of Biochemistry & Molecular Biology, University of Chicago, hand, neutral membranes induced secondary structure changes by up to Chicago, IL, USA, 2Department of Physics, Carnegie Mellon, Pittsburgh, PA, 20%. Further studies that correlate the aggregation rate of these peptides under USA, 3Center for Neutron Research, NIST, Gaithersburg, MD, USA, these conditions will be presented. Taken together, these data suggest that tau 4Department of Chemistry & Department of Biochemistry, Molecular mislocalization to different compartments may expose the repeat segments to Biology & Biophysics, University of Minnesota, Minneapolis, MN, USA.

BPJ 7851_7854 Tuesday, February 14, 2017 389a

The Src family kinases (SFKs) are a group of nine non-receptor tyrosine ki- 1923-Pos Board B243 nases known for their involvement in critical signal transduction pathways. Kinetic Off-Rates of Membrane Associated Proteins Measured in Structurally, SFKs share a common multi-domain architecture, consisting of Unroofed Cells SH3, SH2, and catalytic (SH1) domains attached to a membrane-anchoring Eric N. Senning, Nicolas Reyes, Sharona E. Gordon. SH4 domain through an intrinsically disordered ~80 residue region of low Department of Physiology and Biophysics, University of Washington, sequence conservation called the Unique (U) domain. Despite the wealth of Seattle, WA, USA. information on SFK structure and function, little is known about the nature Cellular function is orchestrated by the vast network of interacting biological of these enzymes in the membrane-associated form. Interestingly, SH4-U do- molecules in the cell, and these interactions are governed by the kinetics of mains in different species share common sequence features, despite differences association and dissociation. The biochemist’s in vitro toolkit provides a means between SFK members, arguing for their importance in differentiating the func- to measure a small part of this network with high precision, unveiling a mech- tions of SFKs and raising questions about their ability to participate in SFK anism of action. It is in this regard that careful measurements of kinetics and signaling. binding equilibria of proteins and lipids reconstituted as mimetic plasma mem- Hematopoetic cell kinase (Hck), a phagocyte specific proto-oncogene member brane has provided valuable insights into cell function (Knight and Falke, of the SFKs, is a potential drug target for HIV infections and Bcr/Abl-chronic Biophys. J. 2009). From these studies in the in vitro setting it is worthwhile myeloid leukemia. To achieve structural characterization of the membrane- asking if the same measurements may be made in the cellular context to further associated Hck via its SH4-U domains, data from solution and solid-state refine our understanding of protein - plasma membrane interactions. In order to NMR, neutron reflection, and fluorescence resonance energy transfer were frame our study of protein - plasma membrane interaction within a cellular computationally integrated using a novel maximum-entropy multi-resolution context we relied on cell ‘‘unroofing’’ for the nearly instantaneous production ‘‘restrained-ensemble molecular dynamics’’ (reMD) simulation scheme. This of a plasma membrane sheet exposed to a buffer solution. Upon unroofing, the framework has allowed us to detail features of the membrane-protein complex rapid shift in equilibrium experienced by GFP-PLCd-PH (a PIP2 binding pro- at atomic resolution and identify possible new constraints on Hck’s ability to tein, residing on the inner leaflet of the plasma membrane) led to an exponential phosphorylate downstream targets. In vivo assays are being developed to test loss of GFP fluorescence that was recorded and fit to a kinetic off-rate (1.1 5 the relevance of these findings. 0.3 s 1; N=3). These results demonstrate a practical approach to measuring the dissociation rate constant for plasma membrane associated proteins in the 1921-Pos Board B241 cellular context and potentially open the door to studying binding interactions Human Bεta Defensin Type 3 Interaction with Chemokine Receptor that have been intractable as in vitro experiments. CXCR4 1924-Pos Board B244 Liqun Zhang1, Aaron Weinberg2, Zhiming Feng2. The Use of Membrane Fusion to Understand the Role of Lung Surfactant 1Chemical Engineering, Tennessee Technological University, Cookeville, Protein B in Lipid Trafficking TN, USA, 2Biological Sciences, Case Western Reserve University, Kayla Kroning, Otonye Braide. Cleveland, OH, USA. Gordon College, Wenham, MA, USA. Defensins are cationic cysteine-rich small molecules with molecular masses Respiratory Distress Syndrome (RDS) is a disorder affecting premature babies ranging from 3 to 5 kDa. In general, human b defensins (hBD) are produced who lack or have a complete absence of the essential lung surfactant compo- by epithelial cells, and they are important to the human innate immune system. nents produced during proper fetal lung development. Composed of a mixture Among the identified hBDs, hBD type 3 (hBD-3) has 45 residues with three of lipids and proteins, Lung Surfactant lines the alveoli, providing a barrier disulfide bonds formed between six cysteine residues. It can exert chemotactic against inhaled pathogens, lowering the surface tension at the air-fluid inter- effects on T-cells and immature dendritic cells, mediated via the C C chemo- face, and facilitating oxygen absorption. Specifically, Surfactant protein B kine receptors. hBD-3 was found to interact with C C chemokine receptor (SP-B) has been identified as a major protein in surface tension reduction CXCR4 which contributes to inflammatory diseases and cancer, resulting in and lipid trafficking to the air-fluid interface of the alveoli. Previous studies antagonism of T cell migration, thus inhibition of chemotaxis. But the exact have shown selective interactions with DPPC in SP-B penetration, possibly inducing/inhibiting tumor mechanism is not yet known. suggesting the flipping of DPPC to the air-fluid interface. We will study the In this project, the interaction and binding of hBD-3 with CXCR4 embedded phase separations and trafficking abilities of SP-B to better understand how in lipid bilayer was investigated using all-atom molecular dynamics simula- it lowers surface tension. Using Fluorescence Resonance Energy Transfer tions after the initial structures predicted using docking calculations. In total, with the fluorescent probes Rhodamine-PE and NBD-PE, we will monitor three kinds of lipid membranes: POPC, POPS, POPG were set up. In order to the predicted effects of SP-B in mediating membrane fusion and inducing consider the hBD-3 structure effect on the binding, besides hBD-3 wildtype, phase separations. This method allows us to demonstrate the role of SP-B in hBD-3 analog which has three disulfide bonds removed, were also studied. lipid trafficking in a fast and cost effective manner with minimal sample Since hBD-3 can form oligomers and CXCR4 can stay in monomer and consumption. dimer forms, the binding of hBD-3 in both monomer and dimer forms with CXCR4 in both monomer and dimer forms were investigated. The structure 1925-Pos Board B245 of CXCR4 in lipid membrane and the key residues on the binding interface Delivery of Lung Surfactant SP-C Based Nanostructures to Respiratory between hBD-3 and CXCR4 agrees with experimental findings. hBD-3 Air-Liquid Interfacial Films analog shows more interaction with the lipid membrane than the wildtype. Nuria Roldan1, Antonio Cruz1, Andrea Sanz1, Marta Bruix2, Jesu´sPe´rez-Gil1, Moreover, in different lipid bilayers, the dynamics and the complex struc- Begon˜a Garcia-Alvarez1. tures of hBD-3 and CXCR4 are different. Thus, it is expected that lipids 1Biochemistry and Molecular Biology I, Complutense University of Madrid, can influence the chemotactic function of hBD-3 interacting with chemokine Madrid, Spain, 2Department of Biological Physical Chemistry, Consejo receptors. Superior de Investigaciones Cientı´ficas, Madrid, Spain. The breathing function is sustained by Lung Surfactant (LS), a macromolecular 1922-Pos Board B242 complex lining the alveolar air-liquid interface. In the context of the physiology Sphingomyelin Modulates Structure of Apolipoprotein A-I in Alzheimer’s of the LS system, the absence or dysfunction of surfactant protein SP-C is asso- Disease ciated with several lung diseases. With the main goal of developing new ther- Vanshika Singh, Vinit Sansanwal, Subhabrata Kar. apeutic tools for the treatment of patients with respiratory dysfunction, we have School of Biosciences, Apeejay Stya University, Gurgaon, India. evaluated different methodologies from the field of nanotechnology using scaf- Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder charac- folding molecules that break membranes in nanoscale particles incorporating terized by accumulation of neurotoxic b-amyloid plaques (Ab) and neurofibril- membrane proteins in their natural environment. We have explored the scaf- lary tangles in the brain. Apolipoprotein A-I (apo A-I) has been implicated in folding reversible encapsulation of lipid bilayers incorporating either palmitoy- metabolism of Amyloid Precursor Proteins/ Ab. The oligomerization of pla- lated or a non-palmitoylated recombinant version of SP-C for the study of its ques is the essence of AD, and sphingomyelin, a major class of lipids found structure-function relationships. In this work, SP-C has been solubilized using in neuronal membranes, have direct implication in the same. The role of apo nanoparticles made with different lipid composition (POPC, POPC/POPG, A-I in the inhibition of b sheet organization upon binding to Ab senile plaques POPC/POPG/DPPC or the Native Surfactant Lipid Fraction), assessing them is also known. Interaction of sphingomyelin and apo A-I has not been well char- as a potential native-like environment in order to preserve the tridimensional acterized. Our results show that sphingomyelin modulates the structure of apo structure and functional properties of the protein. The nanoparticles behave A-I. This can lead to an altered dysfunctional apo A-I with reduced physiolog- themselves as a highly adsorptive material in the air-liquid interface, where ical properties. they inhibited pulmonary surfactant adsorption. When SP-C is incorporated

BPJ 7851_7854 390a Tuesday, February 14, 2017 into the nanoparticles the interfacial adsorption capability of the complex the predominant heparan sulfate (HS) proteoglycan in the developing and adult increased slightly depending on the palmitoylation state of the protein. We human brain, has two N-linked glycans and three chains of HS. Loss-of- have also analyzed the interaction of the complexes with surfactant monolayers function mutations show that both glypican core protein and their HS chains and bilayers. Fluorescently labeled nanocomplexes seem to associate to both are important in shaping animal development. Here, to explore structure, lipid structures with no apparent lipid transfer. These nanostructures could dynamics, and interactions of Gpc1 in a membrane environment, we have per- then constitute an efficient vehicle to direct molecules such as proteins or drugs formed molecular dynamics simulation studies of Gpc1 modeled with both to the respiratory air-liquid interface. N-glycans and HS chains and anchored to a bilayer via a GPI linker. Our ana- lyses reveal that HS chains exhibit a pronounced flexibility, allowing great 1926-Pos Board B246 freedom for HS to reach out and accommodate binding to receptors and other Lipid Bilayer Association of the Alternatively Translated Region of signaling molecules. In addition, within the given simulation time, Gpc1 core PTEN-Long protein is steadily located ~50 A˚ above the membrane surface, showing a ten- Anne-Marie Bryant, Arne Gericke. dency of providing enough distance for HS assembly enzymes or a membrane Worcester Polytechnic Institute, Worcester, MA, USA. receptor to interact with the membrane-proximal region of Gpc1. PTEN-Long is a 576-amino acid translational variant of phosphatase and tension homolog on chromosome ten (PTEN), a tumor suppressor gene and antagonist to 1929-Pos Board B249 phosphoinositide-3-kinase (PI3K) signaling. PTEN-Long (PTEN-L) exhibits at Assembly of Matrix Protein 1 of Influenza a Virus and its Role in Budding the N-terminal end an alternative translation region (ATR) with additional 173 Process N-terminal amino acids to the normal PTEN open reading frame. PTEN-L is Liudmila A. Shilova1, Anna S. Lyushnyak1, Denis G. Knyazev2, secreted from cells, can exist outside the cell and shows activity towards Natalia V. Fedorova3, Liudmila A. Baratova3, Oleg V. Batishchev1. PI(3,4,5)P3 after cell entry. Therefore, PTEN-L may have therapeutic uses by 1A.N. Frumkin Institute of Physical Chemistry and Electrochemistry of RAS restoring a functional tumor suppressor protein to tumor cells. The mechanism (IPCE RAS), Moscow, Russian Federation, 2Johannes Kepler University by which PTEN-L enters cells is currently unknown. The ATR region of Linz, Institute of Biophysics, Linz, Austria, 3A. N. Belozersky Institute of PTEN-L is evolutionary conserved and contains a polyarginine stretch with ho- Physico-Chemical Biology, Moscow State University, Moscow, Russian mology to cell permeable peptides, suggesting that PTEN-L may be able to tra- Federation. verse the plasma membrane passively. It has been suggested that the N-terminal Influenza A virus is a serious pathogen belonging to the Ortomixoviridae fam- ATR part of PTEN-L is intrinsically disordered, which we confirmed using CD ily. It is an enveloped virus with outer shell represented by a lipid bilayer with and FTIR spectroscopy. Our FTIR studies did not reveal any observable struc- incorporated glycoproteins and proton channel, while the inner one is a scaffold tural change of PTEN-L ATR upon lipid binding to PC, PS, and PI(4,5)P2. We formed by matrix protein 1, M1. This protein, being the most abundant one further found that the integrity of model lipid bilayers is compromised upon among viral proteins, is known to possess a multifunctionality: along with be- interaction of PTEN-L’s ATR. We are currently further delineating the lipid ing a mechanical skeleton of a virion, it acts as a factor, which triggers crucial binding preferences of the ATR part of PTEN-L, as well as the full-length protein viral processes, such as the release of viral RNA during infection and budding using surface plasmon resonance (SPR) and FRET techniques. of newly assembled virions on a final step of viral maturation. We focused on 1927-Pos Board B247 this last stage of influenza virus life cycle: assembly and budding of new virions PTEN Binding Mechanism with Compositionally Diverse Lipid Model at the surface of infected cell. Our goal was to clarify the role of the M1 protein Membranes in formation of virus protein scaffold and budding of progeny virions. We per- Brittany M. Neumann1, Katrice E. McLoughlin1, Vanessa Pinderi1, formed experiments on giant unilamellar vesicles (GUVs) using confocal fluo- Rakesh Harishchandra1, Alonzo Ross2, Arne Gericke1. rescent microscopy. We succeed in detecting the budding process of virus-like 1Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, particles by M1 protein in vitro under special conditions. We examined the pos- MA, USA, 2Chemistry and Biochemistry, University of Massachusetts sibility of matrix protein alone to cause budding on GUVs of different lipid Medical School, Worcester, MA, USA. composition, and the role of lipid nanodomains - rafts - in this process. PTEN is one of the most frequent mutated tumor suppressor proteins in human This work was supported in part by RSF grant #15-14-00060, RFBR grant cancer. This project investigates how PTEN interacts with lipid model mem- #15-54-74002 and grant of the President of the Russian Federation (MK- branes of different composition and morphology. We hypothesize that PTEN 6058.2016.4). binds synergistically to the plasma membrane (PM) by interacting with phos- 1930-Pos Board B250 phatidylinositol-(4,5)-bisphosphate(PI(4,5)P2) through its PBM domain as well as non-specifically, electrostatically through its C2 domain with anionic Tubulin on Biomimetic Mitochondrial Membranes: Structural Features lipids. While the currently accepted model is that PTEN’s C2 domain interacts and Lipid Discrimination 1 2 3 1 with phosphatidylserine (PS), we hypothesize that the C2 domain can interact David P. Hoogerheide , Sergei Yu Noskov , Daniel Jacobs , Hirsh Nanda , 3 3 with any anionic lipid, as long as this lipid has a sufficiently high local concen- Tatiana K. Rostovtseva , Sergey M. Bezrukov . 1Center for Neutron Research, National Institute of Standards and tration. PI(4,5)P2 is present in the PM at about 1%, while PS is found at about Technology (NIST), Gaithersburg, MD, USA, 2Biochemistry Department, 30%. We observed in model membrane studies that PI(4,5)P2 and PS have only 3 a limited tendency to co-localize in a domain. In contrast, phosphatidylinositol University of Calgary, Calgary, AB, Canada, Section on Molecular Transport, Eunice Kennedy Shriver National Institute of Child Health and (PI) (about 8% of PM lipids), aids in the formation of PI(4,5)P2 enriched do- Human, National Institutes of Health, Bethesda, MD, USA. mains through headgroup hydrogen bond formation. A PI/PI(4,5)P2 domain would be an excellent platform for PTEN membrane interaction, suggesting Dimeric tubulin, an abundant cytosolic water-soluble protein, has emerged as that PI is a better second binding partner than PS. We are using stopped-flow an important regulatory factor of the permeability of the voltage-dependent fluorescence spectrophotometry as an ensemble technique that provides us anion channel (VDAC) in the mitochondrial outer membrane, with implica- with information about the association and dissociation kinetics of PTEN tions for mitochondrial energetics as well as the Warburg effect observed in when interacting with lipid model membranes of different composition. Using cancer cells. A wealth of recent in vitro and in vivo evidence points to a mech- this technique we have found that PTEN binds to pure PS and PI vesicles with a anism of this regulation, in which a charged C-terminal tail of tubulin inserts into the water-filled VDAC pore, thus blocking metabolite fluxes through similar binding constant. We see synergistic binding to 30%PS/5%PI(4,5)P2 as this passive transport channel. The membrane-binding properties of tubulin well as 30%PI 5%PI(4,5)P2. Additionally, we are utilizing single molecule TIRF microscopy to track PTEN on model membranes of different composi- have proven difficult to ascertain, as early work generated contradictory results tion. From these data, we determine the PTEN diffusion coefficient, hopping regarding the tubulin-membrane association, including whether it was integral frequency and dwell times. In the aggregate, this allows us to describe in detail or peripheral in nature. Here we report on a comprehensive biophysical study of PTEN binding mechanisms with differently composed lipid model membranes. tubulin binding to lipid membranes with compositions that mimic the mito- chondrial outer membrane. A combination of surface plasmon resonance, 1928-Pos Board B248 bilayer overtone (second harmonics) analysis, and single-channel recordings Structure, Dynamics, and Interactions of GPI-Anchored Human Glypi- show that tubulin distinguishes between lamellar and non-lamellar lipid com- can-1 having N-Glycans and Heparan Sulfates in Membranes ponents of the membrane. To obtain the structural features of the tubulin het- Hongjing Ma, Jumin Lee, Wonpil Im. erodimer on the membrane surface, we have employed neutron reflectometry Lehigh University, Bethlehem, PA, USA. (NR) of tubulin on a tethered bilayer lipid membrane platform. The NR results The glypican family of cell-surface proteoglycans, anchored to the outer leaflet definitively show that tubulin binds peripherally, and in combination with mo- of eukaryotic cell membrane through a glycosylphosphatidylinosital (GPI) an- lecular dynamics (MD) simulations suggest the binding domain to be a highly chor, is involved in important cellular signaling pathways. Glypican-1 (Gpc1), conserved amphipathic a-helix on a-tubulin. Thus tubulin joins a short but

BPJ 7851_7854 Tuesday, February 14, 2017 391a growing list of amphitropic proteins that target cell and organelle membranes General Protein-Lipid Interactions III by sensing lipid packing defects via amphipathic a-helices, suggesting a pathway by which lipid homeostasis regulates mitochondrial function. 1933-Pos Board B253 Molecular Mechanism of Lipid Scrambling by Opsin 1931-Pos Board B251 George Khelashvili, Kalpana Pandey, Harel Weinstein, Anant K. Menon. Membrane Bound Structures of Peripheral Membrane Binding Proteins Weill Cornell Medical College, Cornell University, New York, NY, USA. TIM3 and TIM1 Produced by Molecular Dynamics Informed Analysis The visual pigment rhodopsin is both a G protein-coupled signaling recep- of X-Ray Scattering Experiments tor (GPCR) and a constitutively active phospholipid scramblase capable of Daniel Kerr1, Zhiliang Gong2, Greg Tietjen3, Javier Baylon4, Luke Hwang2, translocating phospholipids rapidly across a membrane bilayer. The scramblase J. Michael Henderson5, Binhua Lin6, Mati Meron6, Wei Bu6, activity of rhodopsin - also a property of other Class-A GPCRs - is critical for Mark Schlossman7, Emad Tajkhorshid8, Erin Adams9, Ka Yee Lee10. disc membrane homeostasis because it corrects the trans-bilayer phospholipid 1Program in Biophysical Sciences, University of Chicago, Chicago, IL, USA, imbalance caused by the unidirectional lipid pumping activity of disc-localized 2Chemistry, University of Chicago, Chicago, IL, USA, 3School of ATP-driven transporters, including the ‘Stargardt’s disease transporter’ Engineering and Applied Sciences, Yale University, New Haven, CT, USA, ABCA4. Beyond its role in disc membranes, lipid scrambling is vitally impor- 4Center for Biophysics and Computational Biology, University of Illinois at tant at multiple physiological levels, from blood clotting, clearance of apoptotic Urbana-Champaign, Champaign-Urbana, IL, USA, 5PhysicoChimie Curie cells and diurnal phagocytosis of photoreceptor outer segments, to the growth (UMR CNRS 168), Institut Curie, Paris, France, 6Center for Advanced of cell membranes and protein glycosylation in the endoplasmic reticulum. The Radiation Sources, University of Chicago, Chicago, IL, USA, 7Physics, molecular mechanism of lipid scrambling is not established for any of these University of Illinois at Chicago, Chicago, IL, USA, 8Center for Biophysics systems. We analyzed large-scale ensemble atomistic molecular dynamics and Computational Biology, University of Illinois at Urbana-Champaign, (MD) simulations of opsin embedded in an explicit 9:1 POPC (1-palmitoyl- Chicago, IL, USA, 9Committee on Immunology and Department of 2-oleoyl-sn-glycero-3-phosphocholine) / POPG (1-palmitoyl-2-oleoyl-sn-glyc- Biochemistry and Molecular Biology, University of Chicago, Chicago, ero-3-phospho-(1’-rac-glycerol)) membrane and identified a pathway for lipid IL, USA, 10Chemistry and James Franck Institute, University of Chicago, translocation along the interface between specific transmembrane helices of the Chicago, IL, USA. protein. From these results we determined structural and dynamic features of Lipid binding and associating proteins are necessary components of cell the protein that are mechanistically involved in lipid translocation, and gener- signaling pathways historically overlooked for more amenably characterized ated quantitative models for the kinetics of the translocation process. The protein-protein interactions. As peripheral membrane binding proteins attract mechanistic insights and predictions emerging from these computational more attention, reliable structural methods are needed to elucidate the pro- studies are being used to guide the design of opsin mutants with defined scram- tein-lipid interactions that facilitate their function. Traditional methods such bling activity that can probe and refine the mechanistic models. as crystallography or NMR have produced structures of many peripheral mem- brane binding proteins in isolation, bound to a single lipid, or in a lipid cubic 1934-Pos Board B254 phase but not in complex with full lipid membranes. X-ray reflectivity provides An Opsin Monomer Scrambles Phospholipids structural characterization of lipid monolayer associated proteins assuming a Kalpana Pandey, Birgit Ploier, Michael Goren, George Khelashvili, known structure of the desired protein has already been obtained. Depending Anant Menon. on the experimental conditions of the given structure, it is possible this structure Weill Cornell Medical College, New York, NY, USA. is representative of the membrane associated structure. In our studies of three The G protein-coupled receptor (GPCR) opsin is a phospholipid scramblase members of the T-cell Immunoglobulin Mucin (TIM) family of proteins, that facilitates rapid transbilayer phospholipid exchange in liposomes. The involved in the recognition of the apoptotic cellular signal phosphatidylserine mechanism of opsin-mediated lipid scrambling is not known but it has been (PtdSer) in lipid membranes, the crystal structure was only representative for proposed that the lipid translocation pathway may lie within the interface TIM4 and not TIM1 or TIM3. TIM1 was crystallized without PtdSer in a closed created by the two protomers of an opsin dimer. To test this proposal we engi- conformation that cannot represent the PtdSer bound state and TIM3 has much neered QUAD opsin, in which four lipid-facing residues in transmembrane he- lower affinity resulting in a weak x-ray reflectivity signal. We developed data lix 4 are substituted with tryptophan. Herein we show that QUAD opsin has analysis methods employing molecular dynamics to refine the structures of scramblase activity similar to that of the wild type protein. However, unlike TIM1 and TIM3 to better represent their membrane bound conformations. wild type opsin, QUAD opsin reconstitutes as a monomer into the vesicles in The newly obtained structures provide much improved fits of the data and which its scrambling activity is measured. In addition to the QUAD mutant, highlight protein-lipid interactions that can explain the differences in binding we have found that some unclassified Retinitis Pigmentosa-linked mutations affinity between the TIM protein family members. (V209M and F220C in TM5 and F45L in TM1), which are otherwise functional proteins, also scramble as monomers. These results indicate that opsin dimer- 1932-Pos Board B252 ization is not a prerequisite for lipid scrambling. Curvature Mediated Interactions in Highly Curved Membranes Afshin Vahid Belarghou1,AnCela Sari c2, Timon Idema1. 1935-Pos Board B255 1Bionanoscience, Tu Delft, Delft, Netherlands, 2University of College Role of Soft Matter in G-Protein-Coupled Receptor Signaling London, London, United Kingdom. Udeep Chawla1, Suchithranga M.D.C Perera1, Andrey V. Struts1,2, Cellular membranes exhibit a variety of morphologies, from a simple spherical Michael C. Pitman1,3, Michael F. Brown1,4. liposome to bewildering complex structures like interconnected tubular net- 1Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA, works found in the Endoplasmic Reticulum, or connected stacks of perforated 2Laboratory of Biomolecular NMR, St. Petersburg University, St. Petersburg, membrane sheets as in the Golgi apparatus. Membranes form these structures Russian Federation, 3Raytheon Company, Tucson, AZ, USA, 4Department of through different mechanisms, the most important of which is through the inter- Physics, University of Arizona, Tucson, AZ, USA. play between membrane lipids and inclusions like proteins. Spatial organiza- Current understanding of GPCR function is based on protein structures ob- tion of such proteins in biological membranes is essential for stabilizing the tained from X-ray crystallography yet the role of membrane lipids and water membrane and for the dynamic behavior of cellular organelles. Despite making (soft matter) needs further investigation. Lack of understanding of the role of significant strides in understanding membrane mediated interactions, it is not soft matter acts as an impediment for development of effective drugs against completely understood how inclusions interact in a highly curved regime. It GPCRs. Here we discovered that hydration plays a key role in the binding is, therefore, warranted to investigate how proteins in strongly bent membranes and dissociation of the G-protein transducin that accounts for rapid amplifica- self-organize. We do so using both analytical calculations based on the course- tion and high fidelity in rhodopsin signaling. We conducted osmotic stress grained Helfrich model, and with Monte Carlo simulations. First, we find that studies with varying molecular weight osmolytes and used UV-visible spectros- the forces between identical inclusions, which are known to be always repul- copy to analyze our results [1, 2]. Osmolytes with larger molecular weight shift sive on a flat membrane, become attractive on curved and closed membranes. the equilibrium to the inactive Meta-I state, whereas small molecular weight Second, we show how many point-like inclusions embedded in a tubular mem- osmolytes shift the equilibrium to the ‘‘extra’’ Meta-II state. We also discov- brane spontaneously self-assemble into rings and lines. Finally, we quantita- ered there is an influx of about 60 water molecules upon rhodopsin activation tively show how inclusions adhered to an ellipsoidal membrane likewise consistent with earlier molecular dynamics simulations [3]. This size reversal is self-assemble into a ring perpendicular to its semi-major axis. The structures surprising because small osmolytes exert higher osmotic pressure and are ex- we find are highly relevant in biology, and are found for instance in endocytosis pected to expel more water from the protein. We suggest that small osmolytes and cell division. Our results therefore reveal that purely physical interactions interact with the transducin binding cleft and the membranes to form ‘‘extra’’ underlie these biological phenomena. Meta-II, whereas large osmolytes are completely excluded from the cleft.

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Binding assays of osmolytes with a high-affinity C-terminal transducin peptide lowed by neurotransmitters diffusion through the synaptic cleft to the opposite suggest that small osmolytes do not compete with peptide for the transducin neuron, and is finally completed by the binding of neurotransmitters to their cleft. Small osmolytes go along with water in the transducin binding site and receptors, thus initializing the activation of a postsynaptic neuron. This descrip- replace hydrated volume. Based on our studies, we propose a wet-dry cycling tion, however, may be too simplified given that there are numerous macromol- model for transducin binding which explains the rapid amplification and high ecules in the synaptic cleft, which interact with neurotransmitters water-soluble fidelity in rhodopsin signaling. domains in a non-specific way. This suggests that the binding of certain neuro- [1] A.V. Struts et al. (2011) PNAS 108, 8263-8268. transmitters to their receptor’s binding site does not take place directly from [2] A.V. Struts et al. (2015) Meth. Mol. Biol.1271, 133-158. the water phase but is instead membrane mediated. This possibility is largely [3] N. Leioatts et al. (2013) Biochemistry 53, 376-385. overlooked in the literature. Our recent study of 13 neurotransmitters showed that neurotransmitters can be categorized into two groups: membrane- 1936-Pos Board B256 binding (e.g., dopamine, serotonin, epinephrine, norepinephrine, melatonin, Automated Identification of Cholesterol Interaction Sites on G-Protein and adenosine) and membrane-nonbinding molecules [1]. This categorization Coupled Receptors by Coarse-Grained Simulation corresponds to the receptor types of their neurotransmitters: receptors of Eric Rouviere,Ce´ment Arnarez, Edward Lyman. membrane-binding neurotransmitters are predominately G-protein coupled University of Delaware, Newark, DE, USA. receptors with the entry to the binding pocket located close to the lipid mem- Extensive experimental evidence suggests that cholesterol influences the func- brane surface, while receptors of membrane-nonbinding neurotransmitters tion of G-protein coupled receptors (GPCRs), perhaps by direct and specific are ligand-gated ion channels with a binding pocket located far from the lipid interactions with the protein surface. Interaction motifs (cholesterol recognition membrane surface. These observations allowed us to postulate the existence of amino acid consensus motif and cholesterol consensus motif) have been iden- a membrane-mediated mechanism for neurotransmission where neurotransmit- tified by bioinformatic analysis and inspection of crystal structures. Here we ters first attach to the membrane surface and then through lateral diffusion find report an approach based on analysis of molecular simulation data to identify and enter their receptors binding site. Following this idea, we performed studies other putative interaction sites. In unbiased simulations of GPCRs in choles- of dopamine and adenosine entry pathways to the binding pockets in their terol containing membranes, cholesterol samples the protein surface. This receptors. Using accelerated molecular dynamics simulations, free energy data is then analyzed to identify localized, specific, and reproducible interaction calculations, and atomistic molecular dynamics simulations we confirmed sites. The result of the calculation is a ranking of membrane-facing residues in that the most probable path of dopamine and adenosine entry into their receptor terms of a cholesterol interaction ‘‘score,’’ which may then be used to guide takes place via the membrane-water interface. [1] P. Postila, I. Vattulainen, T. experimental tests based on mutagenesis. Results based on coarse-grained Ro´g. 2016. Selective effect of synaptic membrane on neurotransmission. Sci. ‘‘Martini’’ simulations of the A2A adenosine receptor are presented, which pre- Rep. 6:19345. dict that cholesterol is most likely to bind in three distinct areas on the surface of the protein. 1939-Pos Board B259 1937-Pos Board B257 Role of Hydrophilic Groove of the TMEM16 Protein in Lipid Scrambling Single Molecule Studies of a Membrane-Bound, Master Kinase Circuit... George Khelashvili, Byoung Cheol Lee, Maria Falzone, Anant K. Menon, Detecting Interactions of Two Master Kinase Pairs on the Membrane, Alessio Accardi, Harel Weinstein. Weill Cornell Medical College, Cornell University, New York, NY, USA. PDK1-AKT1 and PDK1-PKC Alpha 2þ Brian P. Ziemba, Joseph J. Falke. The TMEM16 proteins are Ca -dependent ion channels and/or phospho- Biochemistry, University of Colorado, Boulder, CO, USA. lipid scramblases that play key roles in a variety of physiological functions. Mutations in TMEM16F cause Scott syndrome, a bleeding disorder caused In chemotaxing ameboid cells, a leading edge signaling network formed on 2þ the cytoplasmic leaflet of the plasma membrane directs actin and membrane re- by impaired Ca -dependent externalization of phosphatidylserine (PS) in activated platelets, suggesting that this homologue functions as a scramblase. modeling to propel the cell up an attractant gradient. AKT1 (PKB) and PKCa 2þ kinases are central to the regulation of this chemotactic and other signaling Explicit demonstration that some TMEM16 proteins are Ca -dependent pathways. To achieve full catalytic competence, PKCa and AKT1 are phos- phospholipid scramblases comes from reconstitution and assay of purified phorylated at key, C-terminal residues by PDK1, a master regulator of several fungal TMEM16 homologs (afTMEM16 and nhTMEM16). The recently AGC-family kinase members that control cell growth, proliferation, survival determined crystal structure of nhTMEM16, revealed a dimeric arrangement regulate metabolism. Interactions of AKT1, PDK1 and PKCa with the mem- of the protein and the presence of hydrophilic groove on the opposite sides brane are controlled by regulated generation of the signaling phosphoinositide of the dimer interface exposed to the lipid membrane. Extensive mutagenesis studies have established the importance of the groove in the lipid scrambling lipid PIP3 (AKT1 and PDK1) and the influx of intracellular calcium (PKCa). Both AKT1 and PKCa possess a C-terminal PIF (PDK1 Interacting Frag- by showing that mutations of specific residues in the groove region to either ment) motif thought to interact with a ‘‘PIF pocket’’ at the PDK1 catalytic Trp or Ala significantly reduce the kinetics of lipid translocation. We have domain. Productive collisions between membrane-bound AKT1:PDK1 and used a combination of unbiased and enhanced sampling atomistic molecular PKCa:PDK1 likely accelerate formation of the PIF:PIP pocket complex such dynamics (MD) simulations of nhTMEM16 in lipid membrane and water that activating phosphorylation events by PDK1 at PKCa and AKT1 more environments to evaluate the structural context and detailed mechanisms efficient. Despite convincing work describing complexes between these regula- for the measured changes in scrambling efficiency by mutant nhTMEM16 tory kinases in solution, there has not been any evidence of AKT1:PDK1 or constructs. The findings center on the formation and mechanistic role of a PKCa:PDK1 complex formation on a lipid membrane. The present work inves- continuous water pathway along the membrane-facing surface of the groove tigates the complexes between AKT1, PKCa and PDK1 using single molecule in the wild type protein that enables lipid translocation. The manner in which TIRF microscopy. The poster will present the findings of our work that will test the mutations cause a significant reduction in the level of the hydration in the the hypothesis that AKT1 and PKCa are each able to form a stable complex groove region points to a key role of the hydrophilic groove of TMEM16 in with PDK1, and the complex can be dissociated by adding a PIF peptide mimic. lipid scrambling. Moreover, by monitoring complex formation via increased protein:membrane friction and diffusional slowing, the affinity of each interaction can be derived. 1940-Pos Board B260 These findings are expected to yield new insight into the PDK1-dependent Regulation of Membrane Binding by the C2-Domain of Cytoplasmic maturation of AKT1 and PKCa and may also assist with the development Phospholipase A2 by Ceramide-1-Phosphate and Calcium 1 1 2 1 and testing of new therapeutics directed towards the kinases and kinase Xiuhong Zhai , Yong-Guang Gao , Ivan A. Boldyrev , Lucy Malinina , 3 2 4 complexes. Dinshaw J. Patel , Julian G. Molotkovsky , Charles A. Chalfant , Rhoderick E. Brown1. 1938-Pos Board B258 1Hormel Institute, University of Minnesota, Austin, MN, USA, Lipids, Neurotransmitters and their Receptors 2Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Hanna Juhola1, Fabio Lolicato1, Pekka Postila2, Ilpo Vattulainen3, Academy of Sciences, Moscow, Russian Federation, 3Structural Biology Tomasz Rog3. Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA, 1Department of Physics, Tampere University of Technology, Tampere, 4Department of Biochemistry and Cell Biology, Virginia Commonwealth Finland, 2Faculty of Science and Engineering, A˚ bo Akademi University, University, Richmond, VA, USA. 3 Turku, Finland, Department of Physics, University of Helsinki, Helsinki, Cytoplasmic phospholipase A2a (cPLA2a) is a key generator of arachidonic Finland. acid that is used for downstream production of pro-inflammatory eicosanoids. Neurotransmission by chemical synapses is often described as a simple process, The N-terminal C2-domain of cPLA2a helps drive calcium-dependent which starts from neurotransmitters release from presynaptic vesicles, is fol- interaction with phosphatidylcholine (PC) membranes. The sphingolipid,

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ceramide-1-phosphate (C1P), also enhances cPLA2a C2-domain partitioning to molecular basis for protein-lipid interactions of ACAP1 proteins during mem- PC membranes. Here we show the usefulness of a new Fo¨rster resonance en- brane remodeling process. ergy transfer (FRET) approach for gaining insights into the C1P and calcium 1943-Pos Board B263 interdependence of membrane partitioning by cPLA2a C2-domain. Our assay relies on energy transfer between intrinsic tryptophan (Trp71 in C2-domain) Activation of PI3K by Ras: Single Molecule Studies of the Activation and anthryl-vinyl fluorophore that is omega-linked to the sn2 acyl chain of Mechanism PC. A wide range of Caþþ concentration has been studied with PC membranes Thomas Buckles1, Brian Ziemba1, John E. Burke2, Glenn Masson3, containing or lacking C1P. Enhancement of C2-domain partitioning to the Roger Williams3, Joseph J. Falke1. 1 2 membrane by Caþþ and C1P is not simply additive because of their interaction Biochemistry, Univ. Colorado Boulder, Boulder, CO, USA, University of 3 with each other. Depending on conditions, binding of calcium by the C1P head Victoria, Saanich, BC, Canada, Cambridge University, Cambridge, United group may mask the phosphate moiety and mitigate interaction with cationic Kingdom. residues that comprise the C1P head group binding site in the C2-domain. The lipid-anchored GTPase Ras is well known for its role in oncogenesis – about Thus, the C1P/Caþþ ratio appears to regulate membrane partitioning by the one quarter of all human tumors show mutations in a Ras family member. One of the best characterized Ras targets is the lipid kinase Phosphatidylinositol 3-Ki- C2-domain of cPLA2a. Our findings shed light on the regulatory role played by bioactive sphingo-lipids, such as C1P, and changing calcium levels that nase (PI3K) which, like Ras, is highly oncogenic. PI3K phosphorylates the occur during inflammatory events associated with diseases such as sepsis. [Sup- constitutive plasma membrane lipid phosphatidyl-inositol-(4,5)-bisphosphate port: NHLBI HL125353 & NIGMS GM45928; Russian Foundation for Basic (PIP2) yielding the essential signaling lipid phosphatidylinositol-(3,4,5)-bi- Research 012-04-00168; Hormel Foundation]. sphosphate (PIP3). The resulting PIP3 lipid signal regulates an array of cell pro- cesses including migration and growth. 1941-Pos Board B261 The underlying mechanism by which Ras activates PI3K is unclear - this proj- Non-Native Metal Ions Reveal the Role of Electrostatics in Synaptotagmin ect aims to better elucidate the activation mechanism using single molecule 1-Membrane Interaction TIRF microscopy to probe the interactions and activities of single proteins Sachin Katti1, Atul Srivastava1, Bin Her2, Alexander B. Taylor2, on a target membrane surface. One hypothesis is that the lipid-anchored, mem- Tatyana Igumenova1. brane-bound Ras protein increases the surface density of PI3K on the mem- 1 Biochemistry & Biophysics, Texas A&M, College Station, TX, USA, brane by increasing kon and/or decreasing koff. An alternative hypothesis 2 UTHSCSA, San Antonio, TX, USA. proposes that Ras binding increases the PI3K kcat and/or affinity for substrate Synaptotagmin 1 (Syt1) belongs to the family of evolutionarily conserved C2 lipid PI(4,5)P2 (PIP2) and/or ATP. Single-molecule fluorescence methods domain proteins that are named after the second conserved domain of protein will be used to distinguish between these possibilities by observing the number kinase C (PKC). The two C2 domains of Syt1 (designated as C2A and C2B) of single particle diffusion tracks, track length, diffusion speed, specific activ- þ act as Ca2 sensors for the evoked synchronous neurotransmitter release. ity, k , and K of PI3K in the presence and absence of Ras. Our poster will þ cat m The binding of Ca2 ions to the tip of Syt1 C2 domains changes the local elec- present the latest findings of these studies. trostatic potential from positive to negative. This ‘‘electrostatic switch’’ is believed to be sufficient to allow the protein to interact with anionic phospho- 1944-Pos Board B264 lipids in the presynaptic membranes, a function essential for synaptic vesicle Quantitation of Cardiolipin Binding to Human Autophagy (Mitophagy)- fusion. Non-native toxic metal ions such as Cd2þ and Pb2þ can potentially Related Atg-8 Proteins bind to the C2 domains as Ca2þ mimics and interfere with this critical function. Zurin˜e Anto´n1, Ane Landajuela1, Javier H. Herva´s1, L. Ruth Montes1, The goal of our research is to determine how the binding of such toxic metals Sonia Herna´ndez-Tiedra1,2, Felix M. Goni1, Guillermo Velasco1,3, affects the structure and function of Syt1. Using solution NMR spectroscopy Alicia Alonso1. 1 2 and the lanthanide (Tb3þ) luminescence experiments, we determined that Instituto Biofisika (CSIC-UPV/EHU), Leioa, Spain, Department of C2 domains have a higher affinity for Cd2þ and Pb2þ than the native ligand, Biochemistry and Molecular Biology I, Universidad Complutense, Madrid, 3 Ca2þ. We were able to obtain high-resolution crystal structures of both C2 Spain, Universidad Complutense, Madrid, Spain. domains of Syt1 complexed to Cd2þ and Pb2þ and identify the high-affinity The phospholipid cardiolipin (CL) has been proposed to play a role in selective sites. We also demonstrate that although Cd2þ binds to Syt1 with high affinity, mitochondrial autophagy, or mitophagy. CL externalization to the outer mito- it fails to support the protein-membrane interactions. In contrast, Pb2þ acts chondrial membrane would act as a signal for the human Atg8 ortholog sub- as a functional surrogate of Ca2þ in driving Syt1-membrane association. family, MAP1LC3 (LC3). The latter would mediate both mitochondrial This seemingly contradictory behavior suggests that the simple ‘‘electrostatic recognition and autophagosome formation, ultimately leading to removal of switch’’ model does not accurately describe Syt1-membrane interactions. damaged mitochondria. We have applied quantitative biophysical techniques The absence of membrane binding with Cd2þ bound C2 domains could be to the study of CL interaction with various Atg8 human orthologs, namely due to the inability of Cd2þ to effectively coordinate the oxygen atoms of LC3B, GABARAPL2 and GABARAP. We have found that LC3B interacts anionic phospholipid head groups. Our data supports the notion that the direct preferentially with CL over other di-anionic lipids, that CL-LC3B binding coordination of the protein-bound metal ion by anionic lipid head group is occurs with positive cooperativity, and that the CL-LC3B interaction relies essential for the membrane binding of Syt1. only partially on electrostatic forces. CL-induced increased membrane fluidity Acknowledgement: This research is supported by the funds from Welch Foun- appears also as an important factor helping LC3B to bind CL. The LC3B C dation grant A-1784, NSF CAREER award CHE-1151435, and NIH grant R01 terminus remains exposed to the hydrophilic environment after protein binding GM108998. to CL-enriched membranes. In intact U87MG human glioblastoma cells rotenone-induced autophagy leads to LC3B translocation to mitochondria 1942-Pos Board B262 and subsequent delivery of mitochondria to lysosomes. We have also observed Molecular Details of the PH Domain of ACAP1 Protein Binding to PIP2- that GABARAP, but not GABARAPL2, interacts with CL in vitro. However Containing Membranes neither GABARAP nor GABARAPL2 were translocated to mitochondria in Chun Chan1, Lanyuan Lu2, Fei Sun3, Jun Fan1. rotenone-treated U87MG cells. Thus the various human Atg8 orthologs might 1Department of Physics and Materials Science, City University of Hong play specific roles in different autophagic processes. Kong, Hong Kong, Hong Kong, 2Nanyang Technological University, Singapore, Singapore, 3Chinese Academy of Sciences, Beijing, China. Exocytosis and Endocytosis II ACAP1 protein dimers were previously reported to specifically bind to PIP2- containing cell membranes and form well-structured protein lattices in order 1945-Pos Board B265 to conduct membrane tubulation. We first carried out unrestrained molecular Membrane Defects Recruit C-Terminus of Complexin to Synaptic Vesicles dynamics (MD) simulations to characterize orientation of the PH domains Jiajie Diao. with respect to the BAR domains of the protein dimer. Owing to the atomic pre- University of Cincinnati College of Medicine, Cincinnati, OH, USA. cision, we present a comprehensive orientation analysis of PH domain under Single vesicle-vesicle fusion assays based on FRET of lipid or content mixing different lipid-bound states. Furthermore, we sought to investigate nature of [1-3] are widely used to investigate complexin on vesicle docking [4] and fast the two binding pockets on the PH domain revealed by our MD simulations. fusion process [5]. Most recently, we discovered the important role of its N-ter- We performed additional restrained MD simulations and multiple PMF profiles minal domain in promoting fast fusion via membrane interaction [6] and the of the two pockets were presented in order to account for their preference to association of its C-terminus to synaptic vesicles for suppressing spontaneous PIP2 over other charged lipids, e.g. POPS lipids. Combining orientation fusion [7]. For the membrane curvature sensing mechanism of complexin, analysis and studies of binding pockets, our simulations results reveal valuable through molecular dynamics simulations, we found that a high density of

BPJ 7851_7854 394a Tuesday, February 14, 2017 membrane defects on synaptic vesicles is responsible for recruiting amphiphilic mammalian cell culture. Using this methodology, we conclude that one otofer- regions in C-terminal domain of complexin. lin molecule may bind both up to 4 Cav1.3 and 4 SNARE proteins simulta- 1. Diao, J. et al. Nat. Protoc. 7, 921 (2012). neously, and that physiologically relevant calcium concentrations alter the 2. Diao, J. et al. Bioessays 35, 658 (2013). stoichiometry and affinity. Our results motivate a model in which otoferlin 3. Diao, J. et al. Langmuir 25, 7177 (2009). acts as a scaffolding/priming protein that localizes SNARE proteins and the 4. Diao, J. et al. J. Am. Chem. Soc. 135, 15274 (2013). calcium channel in close proximity to synchronize calcium influx and 5. Lai, Y. et al. Elife 3, e03756 (2014). exocytosis. 6. Lai, Y. et al. Proc. Natl. Acad. Sci. USA, 113, E4698 (2016). 7. Gong, J., Lai, Y., / Yang, X., Brunger, A.T., Diao, J. The C-terminal 1948-Pos Board B268 domain of mammalian complexin-1 localizes to highly curved membranes. Snare Proteins Entropically Expand Membrane Fusion Pores 1 2 3 3 Proc. Natl. Acad. Sci. USA 113, (2016). Sathish Thiyagarajan , Zhenyong Wu , Oscar D. Bello , Sarah M. Auclair , Wensi Vennekate2, Shyam S. Krishnakumar3, Erdem Karatekin2, Ben O’Shaughnessy4. 1946-Pos Board B266 1 2 Soluble Beta-Amyloid Modifies Presynaptic Membrane Cholesterol and Physics, Columbia University, New York, NY, USA, Cellular and Molecular Physiology, Yale University, New Haven, CT, USA, 3Cell Rigidity 4 Qi Zhang. Biology, Yale University, New Haven, CT, USA, Chemical Engineering, Pharmacology, Vanderbilt University, Nashville, TN, USA. Columbia University, New York, NY, USA. Neurotransmitters and hormones are released through fusion pores whose dy- Cholesterol is influential for the physicochemical properties of lipid namics are a critical determinant of the release amount and rate. Fusion pores membranes in mammalian cells. In neurons, synaptic vesicles that mediate neurotransmitter release have the highest membrane cholesterol content may flicker repeatedly, before resealing (‘‘kiss & run’’ fusion) or dilating irre- (~40mol%). Cellular and physiological analyses have shown that vesicular versibly (full fusion). These dynamics are physiologically regulated, but the cholesterol is essential for vesicle release and retrieval. However, little is mechanisms are not understood. Prior studies either monitored single exocy- known about its distribution, trafficking and regulation during neurotransmis- totic pores in live cells with uncontrolled biochemistry, or used in vitro recon- sion and how that affects presynaptic membrane architecture. Using new fluo- stitutions lacking single pore sensitivity. The unavailability of key parameters has hampered development of meaningful models to describe pore dynamics. rescent ratiometric reporters for cholesterol concentration and membrane Here we combine mathematical modeling and single pore measurements to fluidity, we uncovered the correlation between vesicle turnover and choles- terol trafficking, illustrated resulted changes in presynaptic membrane fluidity, investigate regulation of pore dilation. Experimentally, we measured individual and elucidated the homeostatic adjustment of membrane cholesterol under pore conductances with controlled SNARE copy number for the first time. Re- variable neuronal activities. Structural studies have suggested that beta- constituting v-SNAREs into ~23 nm diameter nanolipoprotein particles (discs), amyloid peptides (Abeta) pathologically linked to Alzheimer’s disease (AD) we fused discs with patch-clamped cells expressing complementary t-SNAREs and measured currents. can interact with membrane-embedded cholesterol. Furthermore, three major We found that SNARE numbers dramatically affected pore size. Just ~2 secretases that cleave amyloid precursor protein (APP) to produce beta- amyloid peptides (Abeta) exhibit distinct cholesterol-sensitivity. To test if SNAREs nucleated pores, but with increasing numbers of SNAREs pores APP and/or Abeta modify synaptic cholesterol and synaptic vesicle turnover, became progressively more dilated. We determined pore size distributions and to understand the cause of synaptic dysfunction found in early stage of and free energy versus pore size. AD, we applied soluble Abeta1-40 (Ab40) and measured neurotransmitter We developed a coarse-grained mathematical model that reproduced the exper- release, vesicle recycling, cholesterol distribution, membrane dynamics during imental free energy profiles. Free energies were averaged over fusion pore shapes and configurations of SNARE complexes which could zipper at the and after electric stimuli. We have found that extracellular Ab40 binds to and pore waist or roam unzippered. Protein-free pores resisted expansion with force sequesters synaptic membrane cholesterol, disrupts its homeostasis and im- pairs synaptic vesicle release and retrieval. Consequently, a gradual decrease ~19 pN due to membrane tension and bending, close to the experimental value. in the sustainability of neurotransmission emerged after the chronic treat- With SNAREs, zippering drove SNARE assembly at the pore, where SNARE ment of near pathological concentration of Ab40. In conclusion, synaptic crowding generated entropic pore expansion forces, since bigger pores gave vesicle cholesterol is homeostatically regulated despite activity variance, zippered SNAREs more entropy. With 15 SNAREs the entropic expansion force of 14 pN was close to the 19 pN resistance. and APP or Abeta may be the physiological modulator for synaptic choles- Our results suggest that SNAREs critically regulate pores post-fusion by terol homeostasis. entropic expansion forces. Several SNAREs are required to fully dilate pores for efficient contents release. 1947-Pos Board B267 Characterizing Three Component Heteromeric Complexes Mediated by 1949-Pos Board B269 Otoferlin, a Multivalent Membrane Fusion Protein, Using a Novel Examination of Antigen-Induced Endocytic Structure Formation in Single-Molecule Assay B Lymphocytes Colin Johnson, Nicole Hams. Thaddeus M. Davenport, Andrea M. Dickey, Kem A. Sochacki, Oregon State University, Corvallis, OR, USA. Justin W. Taraska. Multidomain proteins reside at the heart of cellular signaling, where they can National Heart Lung and Blood Institute, National Institutes of Health, act as multivalent scaffolds for the assembly of protein complexes and as Bethesda, MD, USA. ligand-dependent effectors of signal transduction. Despite their prevalence, it B lymphocytes are an essential component of the adaptive immune response is unclear how multidomain proteins simultaneously interact with multiple cli- against pathogens. The ability of a B cell lineage to adapt to better recognize ents, and how each domain contributes to the overall activity of the protein. a given antigen depends critically upon the ability of each B cell to bind and Challenges, including the large size and low solubility have largely limited internalize antigen through its B cell receptor (BCR). The mechanism of studies to smaller truncated forms of these proteins. Although more tractable, antigen internalization by B cells appears to require cooperation between the relevance of studies on truncated proteins to our understanding of the BCR signaling, the actomyosin network and clathrin-mediated endocytic pro- full-length protein is questionable. Otoferlin, which consists of 6 consecutive cesses. However, it remains unclear how these actors are coordinated to C2 domains, serves an example. Expressed almost exclusively in vertebrate achieve efficient and controlled uptake of structurally heterogeneous anti- sensory hair cells, otoferlin is a 240 kDa transmembrane protein hypothesized gens. To better understand this process, we set out to directly observe to act as the calcium sensor for neurotransmitter release at hair cell ribbon syn- antigen-induced endocytic structures in the human DG-75 B cell line using apses during the encoding of sound. While studies have suggested that some C2 a combination of conventional and super-resolution microscopy techniques domains of otoferlin may associate with SNARE proteins and the voltage gated including confocal laser scanning microscopy (CLSM), total-internal re- calcium channel CaV1.3, no study has quantitatively characterized multivalent flected fluorescence microscopy (TIRF-M), and correlative super-resolution and concomitant binding properties of multi-domain or full-length otoferlin light and electron microscopy (CLEM). Cells expressing GFP-tagged BCR owing to its size and solubility. Thus, it remains unclear whether otoferlin and red fluorescent protein-tagged endocytic proteins were stimulated with can bind these proteins simultaneously and more generally. To address this lim- anti-human IgM Fab’2 to model BCR clustering and internalization. We itation, we have developed a generally applicable real-time single-molecule co- observed recruitment of early adaptors of clathrin mediated endocytosis – localization binding titration assay which in combination with single-molecule FCHO1, Eps15, Epsin, CALM, and clathrin itself – to Fab’2-induced photobleaching can resolve the binding multivalencies, stoichiometries, and BCR clusters by TIRF-M and CLSM. CLEM revealed that BCR clusters binding affinities of large full-length multidomain proteins enriched from are heterogeneous in size and frequently partition with clathrin on larger

BPJ 7851_7854 Tuesday, February 14, 2017 395a membrane invaginations. This work reveals novel structural features of 1952-Pos Board B272 antigen-induced endocytic structures and expands our understanding of the A C1-C2 Module in MUNC13 Inhibits Calcium-Dependent Neuro- mechanism by which B cells may accommodate antigen of varying size dur- transmitter Release ing internalization. Jeremy Dittman1, Francesco Michelassi1, Haowen Liu2, Zhitao Hu2. 1Biochemistry, Weill Cornell Medical College, New York, NY, USA, 1950-Pos Board B270 2Queensland Brain Institute, The University of Queensland, Brisbane, Bulky Ligands as Entropic Inhibitors of Receptor Uptake by Endocytosis Australia. Andre C.M. DeGroot1, David J. Busch1, Carl C. Hayden1, Aaron T. Alpar1, Almost all known forms of fast chemical synaptic transmission require the syn- Jeanne C. Stachowiak1,2. aptic hub protein Munc13, but the mechanisms by which it controls vesicle 1Department of Biomedical Engineering, University of Texas at Austin, 2 fusion are not well understood. Using the C. elegans Munc13 ortholog UNC- Austin, TX, USA, Institute for Cellular and Molecular Biology, University 13, we show that deletion of the C2B domain, the most highly conserved of Texas at Austin, Austin, TX, USA. domain of Munc13, enhances calcium-dependent exocytosis downstream of Receptor internalization by endocytosis controls diverse cellular processes vesicle priming, revealing a novel autoinhibitory role for the C2B. Further- from the rate of nutrient uptake to the timescale of essential signaling events. more, C2B inhibition is relieved by calcium-mediated binding of C2B to the While many of the biochemical motifs that enable uptake have been thoroughly plasma membrane. Finally, DAG binding to the neighboring C1 domain or investigated, the physical factors that influence receptor internalization remain its complete deletion also disengages C2B inhibition. Inhibition by the poorly understood. Specifically, recent work from several groups has demon- C1C2B module requires a conserved linker sequence leading into the MUN strated that the steric bulk of receptors inhibits their uptake by multiple types domain, and disruption of this linker structure relieves autoinhibition. There- of trafficking vesicles including caveolae, COPII vesicles, and clathrin coated fore, C1-C2B exerts a basal inhibitory effect on Munc13 in the primed state, pits. However, the mechanism by which endocytic structures differentiate while elevated calcium and DAG release this inhibition. Calcium-dependent re- among receptors of different sizes to control their uptake remains unclear. Us- lief of C1-C2B inhibition may contribute to superpriming, short-term plasticity, ing bulky ligands to acutely increase receptor size, here we show that receptor and modulation by lipid signaling pathways. uptake is governed by a thermodynamic balance between receptor-vesicle bind- ing energy and the entropic cost of confining receptors within nascent vesicles. 1953-Pos Board B273 Specifically, we used quantitative fluorescence imaging to precisely map the Homologous C2A Domains of Myoferlin and Dysferlin have Distinct Lipid distribution of receptors among clathrin-coated pits at the plasma membrane Binding Specificities surface. In agreement with a simple thermodynamic model, these data show Faraz Harsini1, Anne Rice2, Sukanya Chebrolu1, Kerry Fuson1, that the increased entropic cost of internalizing bulky ligands inhibits receptor Andrei Turtoi3, R. Bryan Sutton1,4. uptake, driving accumulation of ligated receptors at the plasma membrane. 1Cell Physiology and Molecular Biophysics, Texas Tech University Health Further, by examining the differential uptake of two receptors competing for Sciences Center, Lubbock, TX, USA, 2Johns Hopkins University, Baltimore, space within the same population of endocytic structures, our results reveal MD, USA, 3Metastasis Research Laboratory, Department of Chemistry, the relative importance of receptor size and receptor affinity for endocytic sites. University of Lie`ge, Lie`ge, Belgium, 4Center for Membrane Protein From a biological perspective, these results can predict how processes such as Research, Texas Tech University Health Sciences Center, Lubbock, ligand binding and dimerization, which alter both the effective size of receptors TX, USA. and their biochemical affinity for endocytic structures, will impact receptor up- Myoferlin and dysferlin are members of the ferlin protein family, and both are take rate. From an applied perspective, this work guides the design of bulky li- known to play an essential role in membrane fusion events and membrane gands that can drive the accumulation of specific receptors at the plasma repair. They are homologous multi-C2 domain proteins that are highly ex- membrane surface, a tool that may be useful for modulating diverse pathways pressed in skeletal and cardiac muscle cells, and to a lesser degree, in brain, in cell physiology and signaling. kidney, lung, and placenta cells. These proteins share a high degree of overall sequence identity and a similar overall architecture. Despite similarities be- 1951-Pos Board B271 tween myoferlin and dysferlin, only mutations in dysferlin have been shown Molecular Mechanisms of V-SNARE Function in Secretory Granule to be associated with muscular dystrophies. Specifically, DYSF is the only Exocytosis gene in which mutations are associated with Limb Girdle Muscular Dystrophy Misty Marshall, Per-Eric Lund, Sebastian Barg. Type 2B and Myoshi Myopathy; two phenotypes of dysferlinopathies which Department of Medical Cell Biology, Uppsala university, Uppsala, Sweden. are an autosomal recessive subgroup of muscular dystrophies. Myoferlin Insulin is released by regulated exocytosis and docking of insulin granules at mRNA and protein are upregulated in damaged myofibers, especially in the plasma membrane, followed by the assembly of the secretory machinery Duchenne muscular dystrophy. This suggests that myoferlin plays a role in is required for insulin release. Newly formed granules require a complex matu- muscle repair mechanisms. To date, this mechanism is unknown. Moreover, ration process to become release-ready and the formation of these fusion it has been shown previously that myoferlin is unable to compensate for a competent mature secretory granules involves a series of discrete and unique lack of functional dysferlin in dysferlinopathies. These observations raise ques- events comprised of granule sorting, maturation, docking, priming, and finally 2þ tions about the differences between myoferlin and dysferlin. Answering these Ca -dependent fusion. Soluble NSF attachment receptor (SNARE) proteins questions will lead us to a better understanding of the myoferlin-associated are the conserved core machinery required for all intracellular membrane membrane repair mechanism and its role as a protein which is expressed in fusion events. While SNAREs specifically control fusion, vesicular SNAREs many different tissue types. The first C2 domain of dysferlin, C2A, is the (v-SNAREs) perform additional roles that are still elusive. In secretory cell best studied domain of this protein. C2A serves as the Ca2þ sensor of dysferlin types, v-SNAREs are pivotal for targeting vesicles to different locations, medi- and it binds to lipids with high affinity in a Ca2þ-dependent manner. In this ating both sorting and membrane protein distribution and reductions in study, we will assess the Ca2þ and lipid binding properties of myoferlin C2A v-SNAREs result in defective regulated exocytosis. Using total internal reflec- domain, and will discuss the different structural and biophysical characteristics tion fluorescence (TIRF) microscopy, we studied the role of VAMP8 in secre- between the C2A domains of myoferlin and dysferlin. tory granule exocytosis in pancreatic beta cells. We found that VAMP8 associates with early, late and recycling endosome compartment, rather than in- 1954-Pos Board B274 sulin containing granules. However, VAMP8 traffics and fuses at the plasma Calcium-Mediated Docking and Fusion of Purified Dense Core Vesicles membrane specifically with the recycling endosomal compartment. VAMP8 with Reconstituted Membranes expression compromised secretory granule fusion, whereas VAMP2 expression Alex Kreutzberger, Volker Kiessling, Binyong Liang, Patrick Seelheim, facilitated granule fusion, delineating a VAMP8-dependent fusion step be- J. David Castle, Lukas K. Tamm. tween recycling endosomes and the plasma membrane. Truncation of the University of Virginia, Charlottesville, VA, USA. VAMP8 C-terminal region resulted in attenuated exocytosis and a change in Regulated exocytosis is a process by which neurotransmitters, hormones, and secretion kinetics. Moreover, secreting insulin measurements are negatively secretory proteins are released from the cell in response to elevated levels of correlated with increased VAMP8 copy number in human islets. We conclude calcium. This is mediated by SNARE proteins which catalyze membrane fusion that VAMP8 vesicles play an important role in granule genesis and sorting of of the secretory vesicle to the plasma membrane. While SNAREs are sufficient essential membrane proteins regulating insulin secretion. Additionally, the to catalyze fusion in vitro, they do not convey calcium sensitivity to the pro- TMD of VAMP8 may be necessary for initiating fusion upon stimulation and cess. The calcium sensor is thought to be the protein synaptotagmin, which appears to have some direct interaction on insulin secretion. This work should has two calcium binding C2 domains and resides on the secretory vesicle. To fill critical gaps in our understanding of regulated secretion and the treatment of investigate the calcium response of native secretory vesicles, we developed type-2 diabetes. a novel iso-osmotic fractionation procedure to purify dense core vesicles

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(DCVs) from PC12 cells, an immortalized neuroendocrine cell line. Fusion as- 1957-Pos Board B277 says based on bulk lipid mixing of DCVs with proteoliposomes containing a Simulation-Guided Optimization of Electrode Arrays for Electrochemical highly reactive SNARE acceptor complex were used to determine the overall Imaging of Quantal Exocytosis calcium response of fusion. A fluorescently tagged secretory protein, neuropep- Seyedmehdi Orouji1, Kevin D. Gillis2. tide Y (NPY), was used to specifically label the contents of the DCVs to 1Dalton Cardiovascular Research Center, University of Missouri - Columbia, monitor single vesicle docking and fusion events to a planar supported bilayer Columbia, MO, USA, 2Dept. Bioengineering, University of Missouri - containing reconstituted acceptor SNARE complexes. The results show that Columbia, Columbia, MO, USA. docking and fusion both depend on calcium, but in different fashions, indi- Fluorescence imaging can provide important information about the mecha- cating that different molecular players contribute to each sub-reaction. Knock nisms of exocytosis at the single-vesicle level, e.g., conformational changes downs using shRNA were used to determine which resident DCV proteins are in SNARE proteins reported via FRET. FRET signals are very small and there- responsible for the calcium response of docking and fusion. Soluble accessory fore the time and location of the fusion events are not readily apparent to allow proteins known to modulate SNARE proteins were added to fusion assays to signal analyses. The Lindau lab has pioneered the use of ‘‘electrochemical determine the molecular requirements to have a fully calcium dependent imaging’’ to identify the time and location of quantal exocytosis by comparing SNARE mediated fusion process. the amperometric signal between 4 electrodes arranged around a cell. Since it is necessary to obtain a measurable signal in at least three electrodes in order to 1955-Pos Board B275 ‘‘triangulate’’ the position, a limitation of this method is that it can only localize Fusion Pore Selectivity in Chromaffin Cells exocytosis events that originate near the center of the electrode arrays. We Joannalyn Delacruz1, Meng Huang2, Joan Lenz2, Manfred Lindau2, therefore carried out finite-element-array (COMSOL) reaction-diffusion simu- Shailendra Rathore2. 1 2 lations to guide the design of electrode arrays that can maximize the area on the Field of Pharmacology, Cornell University, Ithaca, NY, USA, Applied cell surface where a release event can be localized with high precision. The Engineering Physics, Cornell University, Ithaca, NY, USA. number and placement of electrodes around the cell were varied and the SD Healthy physiological conditions depend on dynamic release of neurotransmit- of localization precision was simulated. Our results indicate that as the number ters and hormones by exocytosis, which is initiated by the fusion pore. Previous of electrodes arranged circumferentially around the cell increases, the detect- studies show that reduction of extracellular [NaCl] decreases the amperometric able area further from the center of the array increases, however, the precision foot current, indicating no co-release of anions with transmitter. It is unknown of localization in the center decreases because the amperometric charge is if this is due to cation selectivity of the fusion pore or due to negatively charged divided between more electrodes. Thus the optimal number of electrodes de- ions, such as ATP, being bound to the granular matrix and unavailable for pends on the signal-to-noise ratio of the amperometric recordings and the release. Such selectivity might be expected if the fusion pore is lined by nega- acceptable location precision. Under our simulation conditions, if a localization tive charges from lipids and protein. If the initial fusion pore is selective for precision (SD) less than 0.23 mm is needed, an array of 4 electrodes will give cations, then its selectivity will affect the rate of transmitter release via the the largest detectable area. However, if slightly less precision is acceptable, 5 or reversal potential across the fusion pore. If the fusion pore is non-selective, 6 electrodes allows a larger detectable area than 4 or 3 electrodes. Supported by then the flux of anions from extracellular space into the vesicle is expected NIH R44MH096650. to reduce the rate of release of positively charged catecholamines by making the vesicle lumen more negative. Using amperometry we investigate in chro- 1958-Pos Board B278 maffin cells if the transmitter release rate through the fusion pore changes Development of Biophysical Markers That Quantify Metastatic Potentials when extracellular chloride is exchanged by the less mobile D-glutamate anion. of Prostate Cancer Cells using Tsunami Microscope Electrodiffusion calculations predict that for a non-selective anion permeable Yen-Liang Liu1, Aaron M. Horning2, Evan P. Perillo1, Cong Liu1, fusion pore the average foot current amplitude should increase by ~30% Mirae Kim1, Rohan Vasisht1, Hannah Horng3, Andrew K. Dunn1, when chloride is exchanged by D-glutamate with a mobility of 0.25 relative Chun-Liang Chen2, Hsin-Chih Yeh1. to chloride, whereas no change in foot current is expected for a cation selective 1Department of Biomedical Engineering, The University of Texas at Austin, fusion pore. Preliminary analysis of all amperometric events in chromaffin Austin, TX, USA, 2Department of Molecular Medicine, University of Texas cells, results in a mean amperometric foot current of 1.1450.139 pA in the Health Science Center at San Antonio, San Antonio, TX, USA, 3Department presence of chloride, and 1.0450.07 pA for D-glutamate. Further sample col- of Bioengineering, University of Maryland, College Park, College Park, MD, lections and analysis will reveal if there is a significant difference in foot cur- USA. rent amplitude depending on anion mobility and reveal if the early narrow Dysregulated trafficking of receptor tyrosine kinases has been linked to onco- fusion pore is cation selective. genesis. Here we study the trafficking patterns and dynamics of epidermal growth factor receptor (EGFR) trafficking of benign (BPH1), non-invasive ma- 1956-Pos Board B276 lignant (LNCaP), and highly invasive malignant (PC-3) prostate cells using an Dissecting the Biomechanical Feedback Between Plasma Membrane Cur- advanced 3D single-particle tracking technique termed TSUNAMI (Tracking vature and Endocytic Proteins in Mammalian Cells using Nanostructured of Single particles Using Nonlinear And Multiplexed Illumination). As a Substrates feedback-control tracking system, TSUNAMI is capable of tracking fluorescent Jessica R. Marks1, Guiseppe Calafiore2, Stefano Cabrini2, David Drubin1. 1 2 nanoparticle-tagged EGFR for up to 10 minutes and in the z-direction for University of California Berkeley, Berkeley, CA, USA, Lawrence Berkeley up to 550 microns. To analyze the long 3D trajectories generated by the National Laboratory, Berkeley, CA, USA. TSUNAMI microscope, a trajectory analysis algorithm is developed to classify Plasma membrane curvature is the hallmark of endocytosis. A mechanistic trajectories and extract the dynamic parameters, such as diffusivity, inward feedback loop between endocytic proteins and the plasma membrane drives movement, and internalization duration. These parameters can be used to quan- clathrin-mediated endocytosis from pit to vesicle; however, the macro-scale tify the metastatic potentials of prostate cancer cell lines. For instance, the dif- relationship between the endocytic protein complex and membrane curvature fusivities of EGFRs on the highly invasive malignant PC-3 cells (0.010 5 is still not understood. We previously used a novel quartz ‘‘nanostructured’’ 0.014 mm2/s) are around one-quarter of those estimated from the benign substrate to induce stable plasma membrane curvature, showing that endocytic BPH1 cells (0.036 5 0.058 mm2/s), possibly due to the abnormally high expres- < proteins are preferentially recruited to curvatures 200 nm. Here we use a sion of EGFRs on the PC-3 cells. The highly invasive PC-3 cells also exhibit polymer molded on to a glass substrate and live genome edited cells to define longer (2.83 5 0.23 mm vs. 1.45 5 0.16 mm) and faster (0.021 5 0.016 the relationship between curvature-sensing endocytic proteins and the curved mm/s vs. 0.005 5 0.002 mm/s) inward movement as compared with the non- plasma membrane. MDA-MB231 and human fibroblasts, endogenously ex- invasive LNCaP prostate cancer cells, which could be due to the high endocy- pressing the fluorescent fusion proteins AP2, clathrin, and/or dynamin2 were totic activity associated with the invasive PC-3 cells. In addition, the dynamics used to monitor endocytosis by time-lapse fluorescence microscopy. Cells seri- parameters extracted from the EGFR trajectories are correlated with the expres- ally depleted for the late stage endocytic BAR domain proteins SNX9, Bin1, sion levels of a number of epithelial-mesenchymal-transition (EMT)-related and EndophilinA2 were imaged to determine the molecular requirements for genes. The high EGFR expression is related to the decrease of EGFR diffu- clathrin-mediated endocytosis in cells with a pre-curved plasma membrane. sivity, and the increase of dynamins coheres with more active inward move- We also monitored how actin destabilization, and the depletion of N-WASP ment. After EMT induction, the non-invasive LNCaP prostate cancer cells or the Arp2/3 complex affected the outcome of clathrin-mediated endocy- also exhibit gene expression profiles and EGFR trafficking patterns similar to tosis in the context of induced membrane curvature. Finally, the size of the those shown in invasive PC-3 cells. Our work demonstrates that EGFR curvature-generating substrates was varied to determine the relevant range trajectory-derived dynamics parameters are linked to metastatic potentials. A of membrane curvatures for clathrin-mediated endocytosis under depletion new class of biophysical markers can be established based on the analysis of conditions. membrane protein trajectories.

BPJ 7851_7854 Tuesday, February 14, 2017 397a

1959-Pos Board B279 islet. Elevated or dysregulated secretion of glucagon severely impairs the glyce- Involvement of Actin Dynamics in Endocytotic Process Revealed by mic control of diabetic patients. Thus it is important to clarify how all these stim- Fast-Scanning Atomic Force Microscope uli co-operate and interact to stimulate and or inhibit glucagon secretion. Using Aiko Yoshida1, Nobuaki Sakai2, Yoshitsugu Uekusa2, Yanshu Zhang1, hyperspectral fluorescence microscopy, we measure the effect of glucagon mod- Masahiro Kumeta1, Shige H. Yoshimura1. ulators, insulin, somatostatin, and ephrins simultaneously on the activity of two 1Graduate School of Biostudies, Kyoto University, Kyoto, Japan, 2R&D, second messengers, calcium and cyclic adenosine monophosphate (cAMP), in Olympus Corporation, Hachioji, Japan. islet cells. We target genetically encoded biosensors to the cytoplasm, to the The endocytosis proceeds with a series of morphological changes of the plasma plasma membrane or to the nucleus. We hypothesize that in response to different membrane, which is mediated by a successive assembly of a number of stimuli, second messengers’ activities change differently in various cell com- membrane-associated proteins. In addition to a local binding of those proteins, partments. Individual stimuli or their combination result in various patterns of a mechanical tension of the membrane, as well as a dynamics of cortical cyto- activities, which we can use to improve our current mathematical model of skeletal network is also involved in this process. Here, we utilized a fast- glucose-inhibited glucagon secretion. These studies utilize both cell line models scanning atomic force microscope (AFM) which is specially designed for and primary cultured islet cells. This combination of specimens allows us to live-cell imaging (BIXAM), to directly visualize the dynamic morphological determine the conditions under which we can rely on cell lines to mimic intact changes of the plasma membrane and cortical actin network, together with islets hormone output. Having a reliable cell model for glucagon secretion localizations of fluorescently-labeled proteins. COS-7 cells expressing EGFP- form a-cells will be very beneficial for drug screening studies. fused clathrin were observed by BIXAM. Overlaying confocal fluorescence 1962-Pos Board B282 images and AFM images clearly identified clathrin-coated membrane pits on 2D the cell surface. The diameter of the pit was ~300 nm, which is similar to Novel Approximation of a Stationary Single-Channel Ca Nanodomain that observed by an electron microscope, and the life time was 300 seconds. Victor Matveev. The membrane started to invaginate 10-20 seconds after the initial accumula- Department of Mathematical Sciences, New Jersey Institute of Technology, tion of clathrin. The clathrin signal kept slowly increasing, and suddenly disap- Newark, NJ, USA. We consider the stationary solution for the Ca2þ concentration near a point peared 20-30 seconds after the pit closes. Interestingly, when the pit closes, a 2þ 2þ brief swelling of the adjacent membrane (~50 seconds, ~50 nm in height) ap- Ca source describing a single-channel Ca nanodomain, in the presence of a single mobile buffer with one-to-one Ca2þ binding. Previously, a number peared and covered the pit. Knock-down of dynamin did not affect this closing 2þ motion, indicating that dynamin is not necessary for the pit closure in COS-7 of Ca nanodomains approximations have been developed, such as the Excess cells. Scanning of cortical actin with increased loading force of AFM, we found Buffer approximation, the Rapid Buffering approximation, and the Linear approximation, each valid for appropriate buffering conditions. Apart from that the membrane swelling accompanied with local formation of densed 2þ cortical actin network. Inhibitors for actin polymerization/depolymerization providing a simple method of estimating Ca and buffer concentrations (cytochalasin B and Jasplakinolide), as well as inhibitor for Arp2/3 complex without resorting to computationally expensive numerical solution of (CK-666), inhibited the formation of the membrane swelling and caused revers- reaction-diffusion equations, such approximations proved useful in revealing the dependence of nanodomain Ca2þ distribution on crucial parameters such ible open-close motions of the pit, suggesting that actin dynamics is a major 2þ driving force for irreversible closing of the clathrin-dependent membrane pit. as buffer mobility and its Ca binding properties. Here we present a novel form of analytic approximation, which is based on matching the short-range 1960-Pos Board B280 Taylor series of the nanodomain concentration with the long-range asymptotic Effects of Membrane Tension on Snare-Mediated Single Fusion Pores series expressed in inverse powers of distance from channel location. Namely, Natasha Dudzinski1, Erdem Karatekin2. we use a ‘‘dual’’ Pade´ rational function approximation to simultaneously match 1Interdepartmental Neuroscience Program, Yale University, New Haven, CT, terms in the short- and the long-range series, and show that this provides an USA, 2Department of Cellular and Molecular Physiology, Yale University, accurate approximation to the nanodomain Ca2þ and buffer concentrations. New Haven, CT, USA. We compare this approximation with the previously obtained approximations, Fast and temporally controlled release is imperative for neurotransmitter release and show that it yields a better estimate of the free buffer concentration for a via evoked synaptic vesicle exocytosis. This process is driven by the interaction wide range of buffering conditions. The drawback of the presented method is between vesicle v-SNAREs with target t-SNAREs on the plasma membrane. The that it has a complex algebraic form above the lowest, bilinear order, and initial, nanometer-sized connection between two fusing lipid bilayers is called the cannot be readily extended to multiple Ca2þ channels. However, it may be fusion pore. This structure only lasts for a short time before it either dilates possible to extend at least some of the features of this method to the case of completely (resultingin full fusion), or reseals again (kiss-and-run fusion). Fusion cooperative Ca2þ buffers with two Ca2þ binding sites (e.g. calretinin), the pore dynamics determine the amount and size of cargo released, as well as vesicle case which existing analytic methods do not address. recycling. Thus they constitute a fundamental mechanism that controls the extent This work was supported in part by the National Science Foundation grant of postsynaptic neuronal activity. Due to its small size and transient nature, the DMS-1517085. fusion pore has been an elusive object to study. We have recently developed an assay using biochemically well-defined components to look at current flow 1963-Pos Board B283 through single SNARE-mediated fusion pores. The components of the assay Calcium Microdomains and Modulation in Mechanosensory Hair Cells include HeLa cells with ‘‘flipped’’ t-SNARE proteins expressed on the outside Holly A. Holman. of the cell (t-cells), and nanodiscs with v-SNARE proteins embedded within Bioengineering, University of Utah, Salt Lake City, UT, USA. them (v-discs). When we fill a patch pipette with v-discs and establish a cell- Hair cells from the cochlea and vestibular system contain highly ordered rows attached patch onto a t-cell, the v- and t-SNAREs interact, leading to the forma- of actin-based stereocilia that translate nanoscale physical force from sound, tion of a fusion pore connecting the cytosol to the pipette solution. Currents head movement or gravity, into an electrical neural code, through mecha- through these fusion pores report single fusion pore dynamics. noelectrical transduction (MET). Here we report on calcium signaling in hair Membrane tension has been shown to affect fusion pore dynamics in neuroen- cells and neurons of the inner ear using a transgenic mouse line Gad2- Cre::GCaMP5G-tdTomato. Swept field confocal microscopy was used to docrine cells. In our assay, we can quickly and accurately change the pressure 2þ in the patch pipette, thereby modulate membrane tension, using a high-speed image [Ca ] modulation in the inner ear of transgenic mice, in response to pressure clamp. We will show how an increase in membrane tension causes physiological hair bundle deflection from either micromechanical movement extended pore lifetimes, as well as increased pore conductance and charge or chemical stimuli. Images were collected through the intact membranous influx through the pore. labyrinth or, in some cases, through a fenestration in the membrane. In semicir- cular canals, spatially localized [Ca2þ]i transients were observed and quantified Calcium Signaling II at the extreme tips of stereocilia, the apex of hair cells, in microdomains at the base of hair cells, and in postsynaptic terminals. Hair bundles were either 1961-Pos Board B281 stimulated with a fluid jet directly attached to a picospritzer or by piezoelectric Studying the Effects of Glucagon-Modulators on the Activity of Second micromechanical indentation applied to mimic physiological head rotation. Messengers in Subcellular Compartments of Pancreatic Islet Cells Fluorescence were imaged using a custom upright Scientifica microscope Alessandro Ustione, Jing Hughes, Subhadra Gunawardana, David W. Piston. configured with Olympus optics and a fast swept field confocal imaging system. Cell Biology and Physiology, Washington University in St. Louis, Saint In the semicircular canals, ~1mm deflection of the intact cupula revealed punc- Louis, MO, USA. tate increases in GCaMP5G fluorescence (DF/F) at the tips of stereocilia. þ The convergence of multiple paracrine, juxtacrine, and intracellular signals is [Ca2 ]i transients were restricted to the tips of stereocilia where MET channels necessary to achieve proper glucagon secretion from the a-cells of the pancreatic are located and were not present in kinocilia. Cupula deflection also evoked

BPJ 7851_7854 398a Tuesday, February 14, 2017 large [Ca2þ]i transients just below the cuticular plate, putatively colocalizing we demonstrate that Pb2þ binds to EF-hands in DREAM with an equilibrium with the striated organelle. Depolarization with application of KCl revealed affinity higher than that determined for Ca2þ.Pb2þ association triggers changes [Ca2þ] transients in microdomains at the base of hair cells, likely reflecting in the secondary and tertiary structure of the protein that are analogous to those voltage gated calcium channels modulating synaptic release. Preliminary re- observed in Ca2þ bound DREAM based on the Trp 169 emission data and CD sults using adult vestibular organs of Gad2-Cre::GCaMP5G-tdTomato trans- spectra. Namely, Pb2þ binding to DREAM leads to a decrease of Trp169 emis- genic mice reveal highly localized time-resolved [Ca2þ] modulation at the sion intensity and decrease in the CD signal at 220 nm. The hydrophobic cavity tips of stereocilia, in microdomains of hair cells, and at synaptic terminals. in the C- terminal domain of DREAM is solvent exposed in the presence of 2þ Pb as determined using a hydrophobic probe 1, 8-ANS. The Kd values for 1964-Pos Board B284 1, 8-ANS binding to Ca2þ and Pb2þ bound DREAM were found to be similar; Targeted Optogenetic Activation of Calcium Transients in Developing 73510 mM and 103510 mM, respectively. Pb2þ binding to DREAM also mod- Skeletal Muscle Cells ulates DREAM interactions with intracellular partners. For example, titrations 1 1 1 1 Stephane Sebille , Oualid Ayad , Christian Cognard , Patrick Bois , of presenilin-1 with DREAM in the presence of Ca2þ or Pb2þ showed that Pb2þ Aurelien Chatelier2. 5 1 bound DREAM has a similar affinity for presenilin-1 (Kd = 2.44 0.19 mM) as Universite´ de Poitiers, Signalisation et Transports Ioniques Membranaires, 2þ 5 2þ 2 Ca bound DREAM (Kd = 6.23 1.60 mM). The impact of Pb association poitiers, France, Universite´ de Poitiers/CNRS, Signalisation et Transports on DREAM conformational dynamics was proved in time-resolved fluores- Ioniques Membranaires, poitiers, France. cence studies and the thermodynamic parameters for Pb2þ association to EF- Activation of calcium transients through the electrical stimulation of myotubes is hands were obtained using isothermal titration calorimetry (ITC). Those results a pre-requisite for advanced differentiation in developing skeletal muscle. This indicate that DREAM and likely other neuronal calcium sensors bind Pb2þ with stimulation occurs early in the muscle development with the appearance of the a high affinity and Pb2þ association to neuronal calcium sensors may contribute neuromuscular junction (NMJ) that allows to depolarize cell membrane and to to lead-induced neurotoxicity. induce post-synaptic potentials. Consequent generated action potentials activate excitation-calcium release coupling mechanism also known to participate in the 1967-Pos Board B287 calcium-dependent differentiation pathways for the maturation of striated Rewiring Calcium Signaling for Genome Editing and Transcriptional muscle cells. To date, depolarization-evoked intracellular calcium increases Reprogramming are mainly investigated in vitro using electrical field stimulation or high potas- Nhung T. Nguyen1, Lian He1, Yi Liang1, Yun Huang2, Yubin Zhou1,3. sium solution perfusion. However, these methods are not representative of a 1Center for Translational Cancer Research, Institute of Bioscience and spatiotemporal NMJ stimulation. Moreover, calcium signaling kinetics and local Technology, Texas A&M University, Houston, TX, USA, 2Center for calcium increases depend on the stimulation approaches. Here we have devised a Epigenetics and Disease Prevention, Institute of Bioscience and Technology, non-invasive experimental approach to enable genetically targeted photostimu- Texas A&M University, Houston, TX, USA, 3Department of Medical lation of developing skeletal muscle cells with fine temporal and spatial resolu- Physiology, College of Medicine, Texas A&M University, Temple, tion. We applied optical stimulation to C2C12 myotubes, genetically engineered TX, USA. to express ChR2-GFP to investigate depolarization-dependent calcium increases Tools capable of modulating gene transcription in living organisms is very initiated from a very small and localized area of light stimulation. We found that useful for interrogating the gene regulatory network and controlling biological the optical stimulation of membrane area smaller than 1 mm2 allowed the depo- processes. Recently, the CRISPR/Cas9 of Streptococcus pyogenes was re- larization of the whole cell membrane and activated depolarization-induced cal- engineered to be catalytically inactive, which functions as a general platform cium increases in ChR2-expressing myotubes. Optical stimulation conditions for RNA-dependent DNA binding while leaving the genome intact. This (area, surface and light power), associated with the use of pharmacological target-gene recognition complex upon fusing to either repressive or activating blockers, were investigated to understand the required conditions for obtaining effectors is able to interfere with transcriptional initiation, transcriptional voltage-dependent calcium transients. Moreover, the analysis of localized cal- elongation, RNA polymerase recruitment and transcription factor binding. cium increases demonstrates very different kinetics and amplitude of increases However, without temporal and/or spatial control, the application of these re- in different subcellular compartments. These results suggest that local optical programming tools will likely cause off-target effects and lack strict revers- stimulation, that mimics motor neurons inputs, allows to investigate finely the ibility. To overcome this limitation, we report herein the development of a consequent calcium increases and provides new results about calcium homeosta- chemical- or light-inducible transcriptional reprogramming device that can sis in subcellular compartments. control gene expression and cell fate at high spatiotemporal precision. We 1965-Pos Board B285 demonstrated the use of a photo-switchable calcium channel to remotely con- Plasma Membrane Orai1 and Septin Organization during Calcium trol calcium and NFAT-dependent transcriptional outputs. By fusing engi- Signalling neered NFAT fragments with Cas9 or dCas9 and transcriptional coactivators, Zachary Katz1, Chan Zhang2, Ariel Quintana2, Bjo¨rn Lillemeier1, we can harness the power of light to achieve photo-inducible gene editing Patrick Hogan2. and transcriptional reprogramming in mammalian cells. This system can also 1Salk Institute for Biological Studies, La Jolla, CA, USA, 2La Jolla Institute, be used to document calcium-dependent activity in mammalian cells after La Jolla, CA, USA. exposure to ligands or chemicals that would elicit calcium response inside cells. The STIM-ORAI-mediated calcium release-activated calcium channel, or [This work is supported by the Welch Foundation and CPRIT]. CRAC channel, is a key source of calcium influx that maintains cellular cal- cium balance and mediates calcium signaling. STIM in the ER membrane 1968-Pos Board B288 senses depletion of ER calcium stores and moves to ER-plasma membrane Calmodulin Interacts and Regulates Enzyme Activity of the Mammalian junctions, then recruits plasma membrane ORAI channels to the junctions Sperm Phospholipase C 1 2 3 and directly gates the channels. We have shown previously that septins are Michail Nomikos , Angelos Thanassoulas , Brian L. Calver , 3 3 2 2 crucial for efficient STIM1-ORAI1 cluster formation following store depletion. Maria Theodoridou , Luke Buntwal , Zili Sideratou , George Nounesis , 3 Septins are known to specify diffusive barriers in the plasma membrane and to F. Anthony Lai . 1College of Medicine, Qatar University, Doha, Qatar, 2‘‘Demokritos’’, serve as scaffolds to recruit signalling proteins, but their detailed role in cal- 3 cium signaling remains to be characterized. Here we utilize live-cell super-res- National Centre for Scientific Research, Aghia Paraskevi, Greece, College olution microscopy and single-molecule tracking to map ORAI1 relative to of Biomedical and Life Sciences, Cardiff University, Cardiff, United STIM1 and membrane-localized septins. These observations at high spatial Kingdom. Sperm-specific Phospholipase C zeta (PLCz) is widely considered to be the and temporal resolution enable precise analyses of ORAI1 movements before 2þ and after store depletion, and of the relation of septins to the membrane nano- sole, physiological stimulus responsible for the generation of Ca oscillations domains where STIM and ORAI interact. that induce egg activation and early embryo development during mammalian fertilization. PLCz, which is delivered from the fertilizing sperm into the egg 1966-Pos Board B286 cytoplasm, catalyzes the hydrolysis of its membrane-bound phospholipid Lead Tightly Associates to Neuronal Calcium Sensor (NCS) Protein Dream substrate phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], triggering the 2þ and Promotes Structural Changes Analogous to Calcium Bound Dream cytoplasmic Ca oscillations through the inositol 1,4,5-trisphosphate (InsP3) Samiol Azam, Jaroslava Miksovska. signaling pathway. Despite the recent advances the detailed regulatory mecha- Chemistry and Biochemistry, Florida Int University, Miami, FL, USA. nism of PLCz is still unclear, as binding partners of this protein within the Downstream regulatory element antagonist modulator (DREAM), is a 29 kDa sperm or the fertilizing egg have not yet been identified. Calmodulin (CaM) Ca2þ binding protein that controls several neurological processes including is a ubiquitous Ca2þ sensor in eukaryotic cells. A previous study has reported gene expression, apoptosis, and modulation of Kv4 voltage channels. Here that CaM directly interacts and regulates the activity of PLC delta 1 protein, a

BPJ 7851_7854 Tuesday, February 14, 2017 399a somatic PLC isoform with structural similarities to sperm PLCz. Bioinformat- 1971-Pos Board B291 ics analysis revealed putative CaM-binding sites on PLCz sequence. In the pre- Novel Approach to Study SERCA Function In Situ sent study, we have used co-immunoprecipitation analysis and we show that in Elisa Bovo, Siddharth Bhayani, Olga Raguimova, Seth L. Robia, the presence of Ca2þ, human PLCz directly interacts with CaM. Isothermal Aleksey V. Zima. titration calorimetry (ITC) experiments were performed to map the interaction. Cell and Molecular Physiology, Loyola University Chicago, Maywood, Three different peptides corresponding to disparate sequences within human IL, USA. PLCz were used and it was shown that PLCz interacts with CaM via one region Previously used techniques to measure the sarcoplasmic/endoplasmic reticu- of the molecule. In addition, recombinant proteins corresponding to the N- and lum Ca-ATPase (SERCA) function either are cell destructive or lack sensi- C-lobe of human CaM were used for ITC experiments, which revealed that tivity. We designed a new approach that allows studying SERCA function in CaM interacts with PLCz in the presence of Ca2þ, only through one of its the cellular environment under well-controlled conditions. We used the genet- lobe domains. In vitro PIP2 hydrolysis assays revealed that CaM alters PLCz ically encoded Ca sensor CEPIA-1er to directly measure ER [Ca] ([Ca]ER)in 2þ PIP2 hydrolytic activity at high Ca concentrations and, as suggested by lipo- HEK293 cells expressing SERCA2a. These cells were also expressing ryano- some binding assays, this appears to be due to CaM binding to PLCz affecting dine receptor (RyR2) for pharmacological control of ER Ca content. The proper access of the enzyme active site to its substrate PI(4,5)P2. plasma membrane was permeabilized with saponin to control cytosolic [Ca] and energy supply. HEK293 cells expressing wt-SERCA2a manifested periodic 1969-Pos Board B289 [Ca]ER depletions (Ca waves) due to spontaneous activation of RyR2. Ca waves Amino Acid Contributions to Binding across Paired EF-Hands in were rarely seen in cells expressing only RyR2, because endogenous SERCA Calmodulin was unable to increase [Ca]ER to a critical level that triggers spontaneous Suzanna Bennett1, Margaux Miller2, Thomas Middendorf1, Ca-induced Ca release (CICR). Application of 10 mM caffeine completely Richard Aldrich1. depleted [Ca]ER. Once caffeine was removed, RyR2 inhibitors were applied 1Neuroscience, University of Texas at Austin, Austin, TX, USA, 2School of to measure the rate of [Ca]ER recovery. Thus, ER Ca uptake can be analyzed Medicine, University of Utah, Salt Lake City, UT, USA. throughout the whole physiological range of [Ca]ER. ER Ca uptake was pro- The ubiquitous calcium-binding protein calmodulin participates in a network of gressively decreased with increasing cytosolic [ADP] and can be blocked by interactions influencing cellular processes like gene regulation and ion channel thapsigargin. At the end of each experiment, the CEPIA-1er signal was modulation. The immediate function of calmodulin is to bind calcium to its four calibrated with ionomycin. Using this approach, we characterized properties EF-hand calcium binding domains grouped into two pairs, each pair residing in of wt- and AAA-SERCA2a (partial loss-of-function SERCA2a mutant). ER either lobe of the dumbbell-shaped protein. Ligand binding involves a constel- Ca uptake as a function of [Ca]ER was analyzed to estimate maximum ER lation of amino acid interactions localized within the binding site as well as Ca uptake rate and maximum ER Ca load generated by wt and the SERCA across paired sites. Our previous work has shown that each of the four binding mutant. We found that AAA-SERCA2a had slower Ca uptake rate than sites has a unique apparent affinity that appears to have been selected for during wt-SERCA2a, as a result HEK293 cells expressing AAA-SERCA2a were not evolution. We have investigated how the differences in binding loop amino effective to increase ER Ca load to the level that triggers Ca waves. Thus, acid sequences cause differences in binding affinity of the individual EF- this new approach can be used as a sensitive screening tool to study different hands, as well as how the amino acid sequence of one site influences binding drugs and mutations that affect SERCA function. at its paired site through cooperative mechanisms. Between the two paired N-lobe EF-hands, all but five positions within twelve residue binding loops 1972-Pos Board B292 are identical. Each of these five loop positions were individually mutated to Stimulatory and Inhibitory Effects of PKC Isozymes are Mediated by its counterpart from the paired site and site-specific binding measurements Serine/Threonine PKC Sites of the Cav2.3a1 Subunits were performed on N-lobe fragments to explore the two roles each position Ganesan L. Kamatchi1, Senthilkumar Rajagopal2, Brittney K. Burton1. plays to determine binding in the paired EF-hand system: locally within their 1Biology, Norfolk State University, Norfolk, VA, USA, 2Biotechnology, own site (cis-effects) and at the paired site (trans-effects). Several mutations Rayalseema University, Kurnool, India. had both cis- and trans- effects whereas others had predominantly cis-ortrans- Protein kinase C (PKC) isozymes are known to potentiate voltage-gated effects. The complexity of the mutant effects on binding of both sites suggests calcium (Cav) currents through Cav2.2 and Cav2.3 channels by targeting the that the N-lobe has been conserved to act as a complex multiple site calcium serine/threonine (Ser/Thr) phosphorylation sites of Cava1 subunits. While binding unit, rather than two independent EF-hands. some of these Ser/Thr sites of Cav2.2a1 subunits mediated the stimulatory ac- tion (Thr-422, Ser-2108 and Ser-2132), others facilitated the inhibitory action 1970-Pos Board B290 (Ser-425) of PKCs bII and ε. In the current study, it was investigated if the Automated Characterization of Dynamic Parameters of Intracellular homologous sites of Cav2.3a1 subunits (stimulatory: Thr-365, Ser-1995 and Calcium Signals Ser-2011; inhibitory: Ser-369) behaved in a similar manner. In this regard, Laurent MacKay. several Ala and Asp mutants were constructed in Cav2.3a1 subunits in such Physiology, McGill University, Montreal, QC, Canada. a way that the Ser/Thr sites can be examined in isolation or in selected combi- Dynamic processes, such as intracellular calcium signaling, are hallmark of nation. These mutants or WT Cav2.3a1 along with auxiliary b1b and a 2/d sub- cellular biology. As real-time imaging modalities become widespread, a need units were expressed in Xenopus oocytes and the effects of PKCs bII and ε for analytical tools to reliably characterize time-series data without prior studied on the barium current (IBa). The results showed that Thr-365 and knowledge of the nature of the recordings becomes more pressing. We develop Ser-1995 mediated the stimulatory and Ser-369, the inhibitory effects of a signal-processing algorithm that autonomously computes the parameters PKCs bII and ε. While these sites behaved similar to their homologs, Ser- characterizing prominent single transient responses (TR) and/or multi-peaks 2011 differed from its homolog Ser-2132 as it failed to mediate the stimulatory responses (MPR). The algorithm corrects for signal contamination and decom- effects of these isozymes. Furthermore PKCs produced neither stimulation nor poses experimental recordings into contributions from drift, TRs and MPRs. inhibition when stimulatory Thr-365 or Ser-1995 and inhibitory Ser-369 were This is achieved by sequentially decomposing the signal into its individual com- present together. However, the PKCs potentiated the IBa when two stimulatory ponents, characterizing them individually, and finally combining the distinct sites, Thr-365 and Ser-1995 were present together, thus overcoming the inhib- components to yield a coherent representation of the entire recording. It subse- itory effect of Ser-369. Taken together net PKC effect may be the difference quently provides numerical estimates for the following parameters: time of onset between the responses of the stimulatory and inhibitory sites. after stimulus application, activation time (time for signal to increase from 10% to 90% of peak), and amplitude of response. It also provides characterization of 1973-Pos Board B293 the (i) TRs by quantifying their area under the curve, response duration (time Dynamic Substrate Gating in CaMKII by Autophosphorylation between œ amplitude on ascent and descent of the transient), and decay constant Derrick E. Johnson, Swarna S. Ramaswamy, Andy Hudmon. of the exponential decay region of the deactivation phase of the response, and (ii) Biochemistry and Molecular Biol, Indiana Univeristy, Indianapolis, IN, USA. MPRs by quantifying their number of peaks, mean peak magnitude, periodicity, Calcium/calmodulin (CaM)-dependent protein kinase II (CaMKII) regulates oscillatory persistence (time between first and last discernable peak) and duty diverse substrates in cellular processes ranging from metabolism and cell cycle cycle (fraction of period during which system is active). We validate the outputs control to calcium homoeostasis, excitable cell activity and plasticity. CaMKII of the algorithm through comparison with user-mediated measurements of the is a multimeric holoenzyme composed of 8–14 subunits from four closely recordings, and demonstrate that parameter estimates obtained manually and related isoforms (a, b, g, d). The calcium sensor CaM binds to a target algorithmically are correlated. This opens the possibility for such an algorithm sequence within the autoregulatory domain (ARD) of each CaMKII subunit. to be used as a general framework for comprehensively analyzing dynamic Coincident CaM binding within the holoenzyme supports intersubunit time-series with transient or multi-peaked responses. autophosphorylation (T286 a-isoform and T287 b-, g-, and d-isoforms) within

BPJ 7851_7854 400a Tuesday, February 14, 2017 the ARD; a process enabling calcium-spike frequency encoding in excitable reticulum calcium load in the Pitx2þ/- mice (23.855.8 vs 14.451.9 amol/ cells. We now show that T287 autophosphorylation tunes substrate phosphory- pF, p<0.05). Moreover, only myocytes from Pitx2þ/- had spontaneous action lation to couple the activation state of CaMKII to substrate selectivity. Specif- potentials at a resting potential of 80 mV (0.550.4/min). At 60 mV the ically, unlike high-affinity substrates, weak substrates require T286 frequency of spontaneous action potentials was 5.351.8/min for Pitx2þ/- and autophosphorylation. Substrate switching may allow many of CaMKII sub- 0.451.2/min for Pitx2þ/þ mice. Importantly, spontaneous after-potentials strates with weak non-consensus sites to be coupled to T287 autophosphoryla- were also recorded in myocytes subjected to field stimulation, but only in tion. The mechanism of this substrate filtering appears linked to the ARD as a myocytes from Pitx2þ/- mice. These results were not unique to right atrial catalytic fragment devoid of the ARD (and T286) exhibits broad substrate spec- myocytes as the calcium spark frequency was also 3.9 fold higher in left ificity as seen with T287 autophosphorylation (and phosphomimetic substitu- atrial myocytes from Pitx2þ/- mice (p<0.001). tions). Consistently, we find that substrates differential compete for access Together, these results demonstrate that Pitx2 insufficiency promote both to the catalytic domain as indicated by differential ARD displacement as spontaneous calcium release and spontaneous action potentials. Both of these measured by associative changes in CaM binding. Graded CaMKII activity/au- features are hallmarks of myocytes from patients with atrial fibrillation, sug- tophosphorylation within the holoenzyme produces differential substrate phos- gesting that Pitx2-mediated modulation of intracellular calcium handling plays phorylation profiles consistent with calcium-spike frequency encoding. We an important role in electrophysiological processes associated with atrial provide a structural model to explain T287 autophosphorylation-associated fibrillation. regulation of substrate selectivity and hypothesize that calcium-spike fre- quency encoding into T286 autophosphorylation tunes CaMKII’s substrate 1976-Pos Board B296 selectivity to yield diverse cellular responses. Contribution of Mitochondrial Calcium Uptake to Intracellular Calcium Homeostasis Studied with Organelle-Targetted Probes in Skeletal Muscle 1974-Pos Board B294 Fibers Single-Cell Investigation of the Role of Calcium Bursts in Human Immune Carlo Reggiani1, Marta Canato1, Paola Capitanio1, Lina Cancellara1, Cells Feliciano Protasi2. Emmet A. Francis, Volkmar Heinrich. 1University of Padova, Padova, Italy, 2University of Chieti, Chieti, Italy. Biomedical Engineering, University of California at Davis, Davis, CA, USA. Organelle-targetted Cameleons provide a useful tool to quantify variations in The intracellular calcium concentration of immune cells is known to exhibit local free calcium concentration. We studied murine FDB single fibers with a dramatic bursts, during which the calcium level peaks throughout the whole probe targetted to sarcoplasmic reticulum (SR) and a probe targetted to mito- cell on a timescale of seconds. Despite the well-established importance of cal- chondria and assuming organelle volumes available in the literature (Luff & cium ions in numerous cellular processes, the physiological role of these bursts Atwood, J Cell Biol 1971). In wild type C57 mice, SR free calcium concentration remains unclear. What causes calcium bursts? What do they cause in turn? was found to be 570 uM and, assuming a bound/free calcium ratio of 60/1 (Mur- When do they occur, and why do they occur at those particular times? By ad- phy et al, J Physiol 2009) and a volume of 5%, this corresponded to 1.7 mmole/ dressing questions like these, our ongoing work aims to contribute to a deeper fiber liter. Following similar approach the amount released was 0.4 and 0.6 mechanistic understanding of the behavior of immune cells, in particular hu- mmole/fiber liter in a 2 s - 60Hz tetanus and, respectively, after 20 mM caffeine man neutrophils. Our single-cell experiments combine micropipette manipula- administration. Resting mitochondrial calcium concentration was 140 nM and, tion with fluorescence imaging of the intracellular calcium concentration. The assuming a ratio of bound to free of 100/1 and a volume of 5%, this corresponded use of micropipettes enables us to separately inspect chemotaxis, adhesion, and to a 1.2 umol/fiber liter. According to this line of reasoning, the increase in mito- phagocytosis during one-on-one encounters between neutrophils and patho- chondrial calcium content was 1.32 and 1.4 umole/fiber liter in a 2 s - 60Hz genic particles, while simultaneously monitoring the cortical tension of the tetanus and, respectively, after 20 mM caffeine administration. The results cells. Surprisingly, we find that cell polarization and actin remodeling during showed that the fraction of calcium released by SR and taken up by mitochondria complement-mediated pure (i.e., adhesion-free) chemotaxis neither require is below 1 % of the calcium release, with minimal impact on cytosolic calcium nor cause calcium bursts. On the other hand, our experiments confirm that cal- level, but likely sufficient to activate metabolic mitochondrial response. Sup- cium bursts consistently accompany phagocytosis and cell spreading. Further- ported by Telethon Grant GGP 13 213. more, the occurrence of calcium bursts during phagocytosis appears to roughly correlate with a drastic increase of the cortical tension. Although treatment of 1977-Pos Board B297 the cells with 100 mM caffeine, or removal of calcium from the extracellular Necroptosis Execution is Mediated by Plasma Membrane Nanopores that medium, modulates the strength and dynamics of calcium bursts, the bursts still are Independent of Calcium occur during all phagocytosis experiments. These findings show that adhesion, Uris L. Ros Quincoces, Ana J. Garcia-Saez. but not extracellular calcium, is essential to stimulate calcium bursts. More- Interfaculty Institute of Biochemistry, Membrane Biophysics laboratory, over, the concurrent rise of the cortical tension indicates that in addition to Tubingen, Germany. adhesion, other mechanical processes may be closely related to these bursts. Necroptosis is a form of regulated necrosis that results in cell death and con- tent release after plasma membrane permeabilization. However, little is 1975-Pos Board B295 known about the molecular events that mediate the disruption of the plasma Atrial Spceific Pitx2 Insufficiencyincreases the Frequency of Calcium membrane. Here, we have quantified the kinetics of calcium and propidium Sparks, Waves, and After-Depolarizations in Mouse Atrial Myocytes iodide influx during necroptosis using live cell imaging in multiple cell lines Carmen Tarifa1, Adela Herraiz-Martinez1, Alexander Vallmitjana2, used as standard models in necroptosis research. In addition to the commonly Selma A. Serra1, Diego Franco3, Raul Benitez2, Leif Hove-Madsen1. used treatment to induce necroptosis via the TNF receptor, we examined other 1Cardiovascular Research Centre, CSIC, Barcelona, Spain, 2Automatic pathophysiologically relevant pathways, such as those involving Toll-like Control Department, Universidad Politecnica de Catalunya, Barcelona, receptors (TLR) 3/4, with relevance during bacterial and viral invasion. We Spain, 3Experimental Biology Dept., University of Jaen, Barcelona, Spain. found that early calcium influx upon TNF-induced necroptosis in the presence The transcription factor Pitx2 has been proposed as a molecular link between of a Smac mimetic is mediated by ion channels and is cell-type dependent. single nucleotide polymorphisms on chromosome 4q25 and increased risk of Importantly, this early event is mediated by Smac treatment, does not require atrial fibrillation in carriers of the risk variant. Since atrial fibrillation has the activation of the necrosome machinery and is dispensable for necroptosis. been associated with calcium handling disturbances in isolated atrial myocytes, Indeed, necroptosis induced by the activation of TLR3/4 pathways does not we here tested the hypothesis that Pitx2 insufficiency alters the calcium homeo- trigger early calcium influx. Moreover, using osmoprotectants of different stasis in atrial myocytes. sizes, we show that it is membrane pores of less than 4nm in diameter that To test this hypothesis, we used right atrial myocytes from a transgenic mouse mediate necroptosis execution in a cell- and treatment-independent manner. model with inducible atrial specific Pitx2 deletion. Spontaneous calcium Strikingly, extracellular calcium is not required for pore formation. To our release was detected with confocal calcium imaging and resulting ion currents best knowledge, this represents the first evidence obtained in living cells or membrane depolarizations were measured with patch-clamp technique in regarding the role of membrane pores in necroptosis. Those pores allow the myocytes from wild-type (Pitx2þ/þ) and heterozygous Pitx2þ/- mice. passage of small molecules and ions. Therefore, we settle the debate about Calcium imaging revealed that the frequency of calcium sparks (2.150.7 vs ion influx in necroptosis signaling. In fact, our findings reconcile apparent 0.250.1 events/cell/s, p<0.05) and waves (3.251.2 vs. 0 events/min, contradictions about the essential role of different ions (i.e calcium or sodium) p<0.05) were significantly higher in Pitx2þ/- mice. This was also true for the in necroptosis taking into account that different ions could pass through frequency of transient inward currants activated by calcium waves (2.850.5 punched membranes. Collectively, our data demonstrate that early calcium vs 1.250.5 events/min, p<0.05). The higher frequency was of spontaneous influx does not modulate the size of the later pores formed in the plasma calcium release was likely due to a higher caffeine releasable sarcoplasmic membrane.

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Cardiac, Smooth, and Skeletal Muscle classic Ca-dependent pathway. In isolated Langendorff perfused mouse hearts, adding ATP (30 mM) to the perfusate triggered both atrial and ventricular pre- Electrophysiology II mature beats. We conclude that low micromolar extracellular ATP activates an inward current that is sufficient to generate DADs and spontaneous action po- 1978-Pos Board B298 tentials in the absence of spontaneous SR Ca release. Although the molecular Attenuation of Conduction Slowing during Global Ischemia in Guinea Pig identity of ATP-activated inward current remains to be determined, the current Hearts through Increased Extracellular Calcium study demonstrates that ATP release by acute ischemic stress could facilitate Gregory S. Hoeker, Steven Poelzing. arrhythmia susceptibility via a Ca-independent mechanism. Virginia Tech Carilion Research Institute, Roanoke, VA, USA. Cardiac ischemia is associated with decreased conduction velocity (CV) which 1980-Pos Board B300 can lead to conduction block and reentrant arrhythmias. Previously we have The Comparison of Voluntary and Forced Exercises on Cardiac Function demonstrated that physiologically relevant changes in extracellular [Kþ] and of DCM Model Mice [Ca2þ] can modulate CV. The aim of the current study was to test whether Masami Sugihara1, Ryo Kakigi2, Takashi Murayama3, Takashi Sakurai3, changes in perfusate [Kþ] or [Ca2þ] could alter loss of conduction during Takashi Miida1, Sachio Morimoto4, Nagomi Kurebayashi3. global ischemia and reperfusion. Guinea pig Langendorff hearts were perfused 1Clinical Laboraotry of Medicine, Juntendo University, Tokyo, Japan, with a modified Tyrode’s solution containing 153mM Naþ, 4.6 or 6.9mM Kþ, 2Physiology(2), Juntendo University, Tokyo, Japan, 3Pharmacology, and 1.25 or 2.0mM Ca2þ, and loaded with the voltage-sensitive dye di-4- Juntendo University, Tokyo, Japan, 4Clinical Pharmacology, Kyusyu ANEPPS. Hearts were paced at a cycle length of 300ms at baseline and every University, Fukuoka, Japan. 2 minutes during no flow ischemia to assess changes in transverse CV (CVT) Background:Recent studies have demonstrated that exercise has beneficial ef- and the time to conduction block. In separate studies, hearts were perfused fects as therapy for heart failure (HF). However, effects of exercise on patients with Tyrode’s solution containing 146mM Naþ, 4.6mM Kþ, and 1.25mM with inherited dilated cardiomyopathy (DCM), one of major causes of HF, has 2þ Ca , then subjected to 15 minutes of no flow ischemia before reperfusion not been established because DCM is associated with high risk of sudden death with the test solutions for 15 minutes. At baseline (preischemia), there were (SD) by lethal arrhythmia. Recently, a knock-in mouse model of inherited þ þ no significant differences in CVT among the test solutions (153Na /4.6K / DCM was created. They show similar characteristics to DCM patients and 1.25Ca2þ: 19.351.7 cm/s, n=6; 153Naþ/4.6Kþ/2.0Ca2þ: 16.451.9 cm/s, are useful model for evaluating therapeutic effects. We compared effects of n=7; 153Naþ/6.9Kþ/1.25Ca2þ: 20.851.1 cm/s, n=6; p=NS). By 4 minutes of voluntary exercise and forced exercise on this model mouse. þ þ 2þ ischemia, CVT decreased significantly in the 153Na /4.6K /1.25Ca (95% Methods: We used a knock-in mouse model having one of human inherited þ of baseline, p<0.05) and 153Naþ/6.9Kþ/1.25Ca2þ (84% of baseline, DCM mutation, TNNT2 DK210, which have decreased Ca2 sensitivity of 2þ þ p<0.05) solutions. In contrast, increasing [Ca ] preserved CVT (153Na / myofilaments and show enlarged heart and frequent SD with t1/2 of ~70 þ 2þ 4.6K /2.0Ca : 111% of baseline, p=NS). This preservation of CVT persisted days. Homozygous DK210 mice (DCM mice) at 1 month-old were housed through 12 minutes of ischemia (98% of baseline, p=NS), whereas at the same with a running wheel (diameter = 12 cm), and daily voluntary running distance þ þ 2þ time point CVT in the 153Na /4.6K /1.25Ca solution had decreased by 40% was recorded. Other group of DCM mice underwent a forced treadmill exercise (p<0.01) and all hearts with 153Naþ/6.9Kþ/1.25Ca2þ exhibited conduction every 1-2 days (4 times a week) from 1 month of age. Running period and block by 8 minutes. Despite the Ca2þ-mediated differences in ischemia- speed were gradually increased from 11m/min of speed for 15 min to 20m/ induced changes in CV, the median time to conduction block was not signifi- min for 60 min in initial 2 weeks and kept constant for the rest of the weeks. cantly different between the 153Naþ/4.6Kþ/1.25Ca2þ (16 minutes) and Following each exercise until 2 month-old, cardiac function was assessed by 153Naþ/4.6Kþ/2.0Ca2þ (12 minutes, p=NS) solutions. However, increasing echocardiography and ECG. Heart, lung and body weights were excised and [Kþ] significantly shortened the median time to block (153Naþ/6.9Kþ/ weighed. Gene expressions of heart failure-related genes were quantified by 1.25Ca2þ: 7 minutes) relative to either solution containing 4.6mM Kþ q-PCR analysis. (p<0.01). Upon reperfusion, there were no significant differences in CVT Results and discussion: Voluntary exercise significantly prolonged t1/2 of among the test solutions, with a restoration of preischemia values within 3 mi- DCM mice (from 77 days to 128 days) and attenuated deterioration of left ven- nutes for each solution. In conclusion, increased extracellular Ca2þ improved tricular systolic function. Forced exercise also similarly attenuated the deterio- conduction during global no flow ischemia, whereas decreased extracellular ration of systolic function, though the left ventricular wall was significantly Kþ prolonged the time to conduction block. Therefore, altering plasma ionic thicker than that of voluntary exercise group. Gene expression of aMHC was milieu during acute ischemia, in order to preserve cardiac conduction, may consistently higher in forced exercise group. We will also discuss other differ- be a novel mechanism for preventing reentrant arrhythmias. ences between two exercise groups. 1979-Pos Board B299 1981-Pos Board B301 Extracellular ATP Triggers Action Potentials in Ventricular Cardio- Alteration of Action Potential during Early Ischemia in the Heart myocytes Rimantas Treinys, Regina Macianskiene,_ Mante Almanaityte, Kyungsoo Kim1, Frank Raucci1, Sabine Huke2, Bjorn C. Knollmann1. Jonas Jurevicius. 1Vanderbilt University Medical Center, Nashville, TN, USA, 2School of Institute of Cardiology, Lithuanian University of Health Sciences, Kaunas, Medicine, University of Alabama at Birmingham, Birmingham, AL, USA. Lithuania. Myocardial ischemia causes a high frequency of ventricular arrhythmias. A Ischemic heart disease still is the most common cause of death in the world. long established metabolic response to hypoxia in the heart is the release of Metabolic inhibition is typical for ischemic heart disease and heart failure ATP. Here we tested the hypothesis that ATP released from cardiomyocytes and we investigated the effect of induced early ischemia and the changes of during acute myocardial injury can contribute to ventricular ectopy by causing the action potential parameters in the isolated rabbit heart. In cardiac myocytes, delayed afterdepolarizations and triggered action potentials. Isolated mouse L-type calcium current provides calcium for the activation of the contractile ventricular cardiomyocytes were loaded with Fura2-AM to measure intracel- apparatus and is a crucial determinant of cardiac contractile activity. The sup- lular Ca. Spontaneous Ca waves (SCW) and triggered beats (TB) were quanti- pression of L-type calcium current during ischemia is usually reported effect fied after a train of field-stimulated Ca transients (for 20s with 3 Hz) in presence but our experiments revealed a dual respond of calcium current to the uncou- of inotropic stimulation with isoproterenol (1 mM) and partial inhibition of the pling of oxidative phosphorylation. When isoprenaline stimulated cardiac cells inward rectifier with Ba (5 mM) to facilitate TBs. Application of extracellular were exposed to FCCP, the uncoupler induced a biphasic effect on L-type cal- ATP (3-50 mM) significantly increased the incidence and frequency of TBs, cium current. The exposure of the myocytes to FCCP evoked a rapid initial whereas the rate of SCWs was not significantly changed. Notably, extracellular stimulation that was followed by a strong inhibition of L-type calcium current. ATP application generated a different type of TBs that was not preceded by We tested how this phenomenon is reflected in the action potential of isolated SCW, which were termed ATP-triggered beats (TBATP). Higher concentrations rabbit heart. Results showed that in the early phase of ischemia the action po- of ATP (> 50 mM) reduced the rates of TBATP. Simultaneous [Ca]i and mem- tential duration (APD) was briefly increased. At 20% repolarization APD brane potential measurements using perforated patch method demonstrated that increased up to 10% after 0.5 min of ischemia. This increase of APD was fol- both regular TBs and TBATP were preceded by a delayed afterdepolarization lowed by a typical gradual decrease of APD when ischemia progresses. We (DAD) of similar shape. However, in presence of ATP, 34% of DAD that trig- attribute this dual change of APD to the biphasic changes of L-type calcium gered beats occurred WITHOUT concomitant elevations of cytosolic [Ca]. In current and calcium transient increase in the early stage of ischemia in the other words, general DADs and TBs were mediated by the classic Ca- heart. dependent mechanism (i.e., Na-Ca exchange current in response to spontaneous This research is funded by a grant (No. MIP-58/2015) from the Research Coun- SR Ca release), whereas ATP generated DADs occurred in the absence of the cil of Lithuania.

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1982-Pos Board B302 The role of Naþ homeostasis in cardiac pacemaking is not well established. Beta-Adrenergic and ATP Sensitive Potassium Channel Effects on Blocking of the Naþ-Kþ ATPase (NKA) to raise intracellular Naþ concentra- þ 2þ Ventricular Action Potential Duration: Alterations with Exercise Training tion ([Na ]i) in ventricular myocytes, thereby increasing Ca load, is widely Xinrui Wang, Robert H. Fitts. used to improve cardiac inotropy in congestive heart failure patients. How- Department of Biological Sciences, Marquette University, Milwaukee, ever, NKA-blocking agents have a narrow therapeutic window, as excessive WI, USA. Ca2þ accumulation is pro-arrhythmic and decreases lusitropy. Here, we opti- Regular exercise-training is known to reduce the incidences and severity of mize an existing mathematical model of the mouse sinoatrial node (SAN) ischemic heart disease, but the mechanistic nature is unknown. Recently, we cell based on our extensive dataset of electrophysiological experiments showed that regular exercise prolongs the left ventricular action potential dura- (including cell-to-cell variability). Using an established population-based þ tion (APD) at resting heart rate (HR) while shortening it at high HR. These ad- approach, we investigate the consequences of increased [Na ]i on pacemaker þ aptations enhance cardiac contractility at rest while preventing Ca2 overload function. Model parameter sensitivity analysis reveals that NKA modulation and/or inadequate filling during exercise. Here we test the hypothesis that impacts Naþ and Ca2þ homeostasis, as well as several action potential exercise-training induced changes in the APD are due to a reduced responsive- characteristics. NKA dynamically modulates cell automaticity: upon partial ness to b-adrenergic regulation, and increased ATP sensitive potassium channel NKA inhibition SAN firing rate instantaneously increases, due to the direct þ (KATP) function. Female and male Sprague-Dawley rats were randomly as- effect on the membrane potential dynamics. Over time, reduced Na extrusion þ 2þ signed to voluntary wheel running or control groups. After 6-8 weeks training, causes slow [Na ]i accumulation, paralleled by an increase in Ca load, and cardiac myocytes were isolated from the apex and base regions of the left SAN cell firing rate. We show that this positive chronotropic effect can be ventricle with collagenase–protease dispersion technique. APD were measured drastically reversed. In fact, when simulating stronger NKA inhibition, exces- þ 2þ with glass micro-electrode at 1 or 10 Hz stimulation. At 1 Hz, the non-selective b sive [Na ]i and thus Ca overload cause loss of automaticity. Logistic agonist Isoproterenol shortened the APD an effect largely reversed by selective regression analysis highlights that perturbations in conductances of L-type 2þ þ þ b1-AR blocker Atenolol (0.1 mM), but not selective b2-AR antagonist ICI Ca current, hERG K current, and background Na current, and NKA 118,551 (0.1 mM). Wheel running shifted the Isoproterenol dose-response curve maximal transport rate induce the most pronounced effects on the probability (0.01-100 nM) rightward compared to controls, with LogIC50 decreased by that the cell will stop firing. These parameters are also those maximally influ- þ exercise-training in both male and female rats by ~ one log unit. The addition encing [Na ]i regulation as shown in the sensitivity analysis. Hence, by con- þ of KATP inhibitor glibenclamide (2 mM) prolonged APD at 10 Hz, with a greater firming that [Na ]i has a fundamental role in the regulation of cell prolongation observed in myocytes from exercise-trained rats. In conclusion, the automaticity, our study suggests that a detailed characterization of Naþ ho- exercise program down regulated the b1-AR to isoproterenol an adaptation that meostasis in isolated SAN myocytes is essential to fully understand cardiac would contribute to prolonged action potential in non-stressed hearts where the pacemaker function. activation of the sympathetic nervous system is low, while the exercise-training induced decline in the APD under energy demanding conditions (e.g. high HR) 1985-Pos Board B305 Loss of Caveolin-3 Causes Heart Rhythm Abnormalities by Affecting seems caused at least in part by increased KATP repolarizing current. Ca2D-Voltage Coupling in the Mouse Sinoatrial Node 1983-Pos Board B303 Di Lang, Aleah Warden, Timothy Kamp, Alexey Glukhov. Ionic Current Changes during Action Potentials in Porcine Post-MI Heart Medicine, University of Wisconsin Madison, Madison, WI, USA. Failure Model Caveolin-3 (Cav3) is a myocyte-specific scaffolding protein within caveolar Bence Hegyi1, Julie Bossuyt1, Leigh G. Griffiths2,3, Rafael Shimkunas1,4, membranes that compartmentalize a number of ion channels and transporters Zana Coulibaly1, Kenneth S. Ginsburg1, Leighton T. Izu1, Tama´s Ba´nya´sz1,5, involved in sinoatrial node (SAN) pacemaking. These channels and trans- Donald M. Bers1, Ye Chen-Izu1,4. porters are coupled with intracellular Ca2þ cycling (Ca2þ-voltage coupling) 1Department of Pharmacology, University of California, Davis, Davis, CA, through rhythmic local Ca2þ releases (LCRs) from ryanodine receptors to USA, 2Department of Veterinary Medicine and Epidemiology, University of initiate an action potential. We hypothesize that disruption of caveolae- California, Davis, Davis, CA, USA, 3Department of Cardiovascular Diseases, associated macromolecular signaling complexes alters SAN automaticity Mayo Clinic, Rochester, MN, USA, 4Department of Biomedical Engineering, leading to SAN dysfunction. In vivo ECG telemetry, in vitro high-resolution University of California, Davis, Davis, CA, USA, 5Department of optical mapping, and confocal imaging of intracellular Ca2þ cycling were Physiology, University of Debrecen, Debrecen, Hungary. performed in wild type (WT, n=8) and conditional tamoxifen-induced RATIONALE: Heart failure (HF) following chronic myocardial infarction (MI) a-MHC-controlled Cav3 knockout (Cav3-/-, n=8) mice and isolated SAN my- is associated with increased susceptibility for cardiac arrhythmias. However, the ocytes. Both in vivo and in vitro,Cav3-/- mice exhibited SAN pacemaking complex electrophysiological changes causing cardiac arrhythmias remain abnormalities characterized by alternating periods of tachycardia-bradycardia incompletely understood, especially in large animal models. METHOD: In a rhythm (cycle length varied from 123.652.6 ms to 464.45130 ms), signifi- porcine model of ischemic HF we used action potential (AP)-clamp and sequen- cant beat-to-beat cycle length variability (3.851.1 ms in WT vs. tial dissection of the major ionic currents during the cardiac AP in ventricular 19.553.6 ms in Cav3-/-,p<0.01) and shift of the leading pacemaker from myocytes (from infarct border and remote zones 5 months after MI induction). the SAN to ectopic foci. In SAN myocytes, Cav3 deficiency led to irregular RESULTS: (1) Compared to healthy controls, remote zone myocytes exhibited spontaneous rate with significant cycle length variability (30.955.2 ms in increased late sodium current, calcium-activated chloride current and calcium- WT vs. 644.75256.4 ms in Cav3-/-, p<0.01). Because LCRs are critically activated potassium current, decreased rapid delayed rectifier potassium current important for the Naþ/Ca2þ exchanger mediated component of slow diastolic and also altered sodium/calcium exchange current dynamics. These changes depolarization of transmembrane potential, we measured the period from combine to lengthen the AP duration (APD) in the remote zone. (2) In the border LCRs to Ca2þ transient initiation in order to evaluate Ca2þ-voltage coupling. zone, the above changes also occurred, but with additional pronounced de- Cav3-/- SAN myocytes showed significant prolongation (73.956.7 ms in WT creases in L-type calcium current and inward rectifier potassium current, and vs. 130.6515.0 ms in Cav3-/-, p<0.01) of this period with large beat-to-beat calcium-activated potassium current was further enhanced to become a major variability (15.552.6 ms in WT vs. 71.6517.4 ms in Cav3-/-, p<0.01). Our repolarizing current. These additional changes shortened the APD in the border findings demonstrate that Cav3 plays a crucial role in supporting functional zone. CONCLUSIONS: Ischemic HF involves complex changes in many ionic integrity of the SAN by synchronizing Ca2þ-voltage coupling and regulating currents. The changes in each individual current are modest; however, the com- rhythm of LCRs. bined changes in multiple currents integrate to cause APD shortening in the border zone but APD lengthening in the remote zone. Changes in many ionic 1986-Pos Board B306 currents significantly increase the APD dispersion surrounding the infarct to pro- Electrically Dormant Sinoatrial Nodal Cells (SANC) are Awakened by vide a substrate for cardiac arrhythmias. Increased Camp-Dependent Phosphorylation of Coupled-Clock Proteins Kenta Tsutsui1, Mary S. Kim1, Ashley N. Wirth1, Oliver J. Monfredi1,2, 1984-Pos Board B304 Bruce D. Ziman1, Rostislav Byshkov1, Alexander A. Maltsev1, Sodium Homeostasis Dynamically Regulates Sinoatrial Node Pacemaker Victor A. Maltsev1, Edward G. Lakatta1. Activity 1Laboratory of Cardiovascular Science, National Institute on Aging, Stefano Morotti1, Christian Rickert2, Joshua St Clair2, Nicholas Ellinwood1, Baltimore, MD, USA, 2Institute of Cardiovascular Sciences, University of Catherine Proenza2, Eleonora Grandi1. Manchester, Manchester, United Kingdom. 1Department of Pharmacology, University of California Davis, Davis, CA, SANC firing rate and rhythm is controlled via the degree of coupling between USA, 2Department of Physiology and Biophysics, University of Colorado rhythmic local Ca2þ releases (LCRs) emerging beneath the cell membrane Denver-Anschutz Medical Campus, Aurora, CO, USA. (‘Ca2þclock’) and sarcolemmal electrogenic mechanisms (‘membrane clock’).

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Dormant SANC (ones that don’t fire spontaneous action potentials (APs)) may of the L-type Calcium current (ICaL) to improve its AP shape and to best fit therefore represent the extreme of clock uncoupling. We hypothesized that the model to experimental results. increased cAMP-mediated, PKA-dependent phosphorylation of the coupled- Methods: Both the time constants and the steady-state value of the ICaL inac- clock system proteins would restore rhythmic AP firing in dormant SANC. tivation gates were modified, with validation performed at two different levels: 2þ We recorded membrane potential (Vm) and intracellular Ca kinetics in enzy- 1)The results for the new ICaL formulation were compared against three matically isolated single, dormant guinea pig SANC prior to and during b-adre- different experimental datasets using in silico simulations of the experimental noceptor stimulation (BARs, isoproterenol 0.1-1 mM) or during exposure to protocols. 2)A set of arrhythmic risk markers were calculated using the cell-permeant cAMP (CPT-cAMP, 300 mM). Dormant SANC that did not modified AP model, including: systolic and diastolic intracellular calcium con- 2þ fire still maintained a resting membrane potential of 35þ/-3mV (n=17), centration ([Ca ]i), AP duration, AP triangulation at different pacing fre- and exhibited small, random LCRs. ~40% responded to BARs/CPT-cAMP. quencies, and APD adaptation to abrupt cycle length changes. This initial response involved increases in LCR size and rhythmicity, and hy- Results: The mean square error of steady-state ICaL inactivation was 25% lower perpolarization of Vm, accompanied by small amplitude, spontaneous APs than in the original CRLP model and satisfactorily reproduced the available 2þ (rate ~1Hz) triggering whole-cell Ca transients. During the subsequent tran- experimental data. The modified time constants of the ICaL inactivation gates sition to steady-state AP firing, average LCR size increased while average LCR led to physiological AP peak and dome shapes. All the computed arrhythmic period shortened, partially synchronizing LCRs to late diastole, (rate ~2Hz); risk markers remained in the physiological range or were very similar to the this was paralleled by MDP hyperpolarization to ~-60 mV. AP amplitude ones reported for the original CRLP model. increased, and average AP cycle length shortened similar to LCR period. Conclusions: The proposed modification in the CRLP model improves the When BARs/CPT-cAMP was washed out, all changes reversed in order, and behavior of the ICaL current and the AP shape, providing a model suitable for SANC again exhibited small random LCRs, with Vm ~-40 mV. In conclusion, the investigation of ventricular arrhythmias. The strategy proposed in this at least in some SANC, dormancy represents membrane & Ca2þ clock uncou- work can be extended to other human ventricular cell models available in the pling that is rescuable through increased cAMP and PKA-dependent phosphor- literature. ylation of both membrane and Ca2þ clock proteins. Specifically, rescuing impaired clock coupling occurs through: (1) increased spatial LCR synchroni- 1989-Pos Board B309 zation, yielding larger LCRs; (2) increased temporal LCR synchronization, Memory and Stability of the Cardiac Action Potential Repolarization in decreasing average LCR period and shifting larger LCR occurrence to late the Space of its Allowed States: A Single Cell Simulation Study diastole. Massimiliano Zaniboni. Life Sciences, University of Parma, Parma, Italy. 1987-Pos Board B307 The rate-dependent partition of the cardiac cycle into diastole and systole is A Sexy Approach to Pacemaking measured at the cellular level as rate-dependence (RD) and electrical restitu- Ursula Doris, Sunil Logantha, Maria Petkova, Yu Zhang, Sanjay Kharche, tion (ER) of the action potential (AP) duration (APD), the first describing Halina Dobrzynski, Joseph Yanni. changes in APD after steady state changes in cycle length (CL), and the sec- Institute of Cardiovascular Sciences, Manchester, United Kingdom. ond APD changes after a sudden switch from constant to a variably delayed Introduction: The sinoatrial node (SAN) is the primary pacemaker of the heart. CL, as a function of the pre-switch CL (or DI). A dynamic ER curve Clinical evidence shows that SAN function differs between sexes. Males have (DER) can also be measured under beat-to-beat changes in pacing rate, and slower resting (extrinsic and intrinsic) heart rates, and longer corrected SAN the increase in its slope is a recognized predictor of electrical alternance recovery time compared with females. Hypothesis: Sex based differences and ventricular fibrillation. An hysteretic behavior has been described previ- may be due to SAN ion channel differences. Therefore, we investigated func- ously in DER curve and associated with cardiac memory and repolarization tional behaviour and molecular makeup of SANs in male and female rats. stability. In the present study, by means of simulations with three numerical Methods: Intracellular action potential recordings were obtained using sharp models of the human ventricular AP, I measure DER as APD vs DI and microelectrode technique (n=5 each). Gene expression was measured with APD vs CL plots from high frequency paced AP series, and find that both quantitative PCR (n=6 each). Results: The amplitude of the action potential show hysteresis with remarkably different phase shifts. Based on both repre- was significantly higher in male SANs (45.3 5 2.4) compared with female sentations, I define and measure a space of states (SPoS) for APs elicited, in (31.7 5 2.5). The upstroke velocity (dV/dtmax) was significantly faster in fe- turn, at constant, periodically changing, and randomly changing (1, 2, and 3 male SANs (16.4 5 1.6) compared with male (9.1 5 1.5). Out of 96 transcripts dimensional SPoS) pacing rates, and use them to study perturbations (a single investigated, two were significantly different between the sexes. L-Type Ca2þ missing beat) of the pacing protocol. I show how the maximum conductance channel, Cav1.3, and acetylcholine activated Kþ channel, Kir3.1, mRNA and gating kinetics of three plateau ion currents (IKs,IKr, and ICaL), change levels were significantly higher in female SANs (66.6% and 45.6% respec- shape and size of SPoS. I show how geometrical parameters of SPoS, for a tively) compared with male. Immunolabelling for Cav1.3 showed levels were given AP type and a given pacing law, can be used to numerically quantify significantly higher in female SANs compared with male (18.6%, n=6 each). memory and stability of its dynamics. Inhibition of Cav1.3 with nifedipine (5mM) showed greater effect on the beating rate of male SANs compared with female (43%). A non-coding 1990-Pos Board B310 RNA, miR-139-3p (predicted by Ingenuity IPA software to affect Cav1.3) An Integrated Model of Human Beta-Adrenergic Signaling and Ventricu- showed greater effect on the beating rate of male SAN preparations compared lar Electrophysiology Reveals Contributors to Positive Inotropy 1 2 2 1 with female. Mathematical modelling showed that a higher density of Cav1.3 Jingqi Gong , W. Clayton Thompson , Cynthia J. Musante , Eric A. Sobie . 1Department of Pharmacological Sciences, Icahn School of Medicine at (as in the case of females) has a protective role from sinoatrial node arrhyth- 2 mias. Conclusion: This study identified functional and molecular differences Mount Sinai, New York, NY, USA, Cardiovascular and Metabolic Diseases in key pacemaker ion channels in SAN between sexes. These differences Research Unit, Pfizer Inc., Cambridge, MA, USA. advance our understanding of gender differences in cardiac electrophysiology Adrenergic regulation is well recognized as an important player in cardiac and may have implications for gender specific design of anti-arrhythmic drugs functions and contributes to pathology underlying arrhythmia and heart failure. and biological pacemakers. Mathematical models of beta-adrenergic signaling have been developed and adrenergic effects on cardiac electrophysiology have been analyzed in animal 1988-Pos Board B308 cells. However, key contributors of these effects in human myocytes remain A Methodology to Improve Human Ventricular Models for the Investiga- largely unknown. We have integrated detailed beta-adrenergic signaling tion of Cardiac Arrhythmias cascade with human ventricular electrophysiology based on published models Jesu´s Carro1,2, Jose´ F Rodrı´guez-Matas2,3, Esther Pueyo2,4. of these two systems. PKA targets in human ventricular electrophysiology: fast 1Universidad San Jorge, Villanueva de Ga´llego, Spain, 2Instituto de sodium channel, slow delayed rectifier potassium channel, sodium potassium Investigacio´n en Ingenierı´a de Arago´n, Universidad de Zaragoza, Zaragoza, ATPase, L-type calcium channel, ryanodine receptor, phospholamban and Spain, 3Polite´cnico di Milano, Milano, Italy, 4Centro de Investigacio´n troponin-I, are simulated to incorporate both baseline and phosphorylation- Biome´dica en Red en Bioingenierı´a, Biomateriales y Nanomedicina, Madrid, modified behaviors. Responses to different concentrations of adrenergic Spain. agonist isoproteronol are also monitored and adjusted. The model was validated Introduction: A number of human action potential (AP) models can be found by ensuring these behaviors matched published voltage-clamp data before and in the literature for the study of ventricular arrhythmias. One of the latest pro- after phosphorylation. Simulations to analyze sensitivity of each PKA target posed models is the Carro-Rodrı´guez-Laguna-Pueyo (CRLP). Although this were performed with blocking the phosphorylation of a single target one at a model reproduces many human ventricular physiological features, it lacks time. These simulations showed that phosphorylation of L-type calcium chan- the characteristic AP peak and dome. In this work we propose a modification nel and phospholamban were by far the largest contributors to the positive

BPJ 7855_7859 404a Tuesday, February 14, 2017 inotropic response observed at 1 Hz with beta-adrenergic stimulation. Further Voltage-gated K Channels and Mechanisms of analysis of this integrated model would dissect contribution from parameters in the adrenergic signaling cascade to human ventricular electrophysiology. These Voltage Sensing and Gating III results will greatly facilitate understanding of human adrenergic regulation on cardiac functions and diseases. 1993-Pos Board B313 KV2.1/KV6.4 Heterotetramers are Functional in Two Stoichiometric 1991-Pos Board B311 Configurations Capacitive Memory Suppresses Alternans and Promotes Spontaneous Glenn Regnier, Dirk J. Snyders. Activity in a Fractional-Order Minimal Cardiomyocyte Model Biomedical Sciences, University of Antwerp, Antwerpen, Belgium. Tien Comlekoglu, Seth H. Weinberg. Members of the voltage-gated Kþ (Kv) subfamilies Kv5, Kv6, Kv8, and Kv9, Biomedical Engineering, Virginia Commonwealth University, Richmond, which are collectively designated as electrically silent (KvS) subunits, selec- VA, USA. tively modulate the biophysical properties of Kv2 channels by forming heter- Electrical activity in cardiomyocytes is typically modeled using an ideal par- otetrameric Kv2/KvS channel complexes: e.g. Kv2.1/Kv6.4 heterotetramers allel resistor-capacitor circuit. However, studies have suggested that the pas- display a 40 mV hyperpolarizing shift in the voltage-dependence of C-type sive properties of cell membranes may be more appropriately modeled with inactivation. Fo¨ster resonance energy transfer (FRET) analysis has previously a non-ideal capacitor, in which the current-voltage relationship is given by a demonstrated that Kv2.1/Kv9.3 channels are composed of three Kv2.1 fractional-order derivative. Fractional-order membrane potential dynamics in- subunits and one Kv9.3 subunit. However, it remains unknown whether troduces capacitive memory effects, i.e., dynamics are influenced by the prior Kv2.1/KvS channels can have other stoichiometries. We investigated this spe- membrane potential history. We recently showed that fractional-order mem- cifically for the Kv2.1/Kv6.4 combination by comparing the biophysical prop- brane dynamics alters ionic currents and spiking rates in a neuronal model. erties of different concatemeric constructs to these of the corresponding Here, we investigate the effects of fractional-order membrane dynamics in a monomers. Kv2.1-Kv2.1 (2_2) dimers yielded delayed rectifier currents cardiomyocyte model using the minimal 3-variable Fenton-Karma (FK), cho- with biophysical properties similar to those of channels composed of individ- sen because the FK model, with first-order derivative membrane dynamics, ual Kv2.1 subunits. Kv2.1-Kv6.4 (2_6) or Kv6.4-Kv2.1 (6_2) dimers dis- does not have short-term memory. We performed simulations for fractional- played a midpoint of inactivation of 5953 and 5851 mV (n=6), orders between 0.5 and 1 and variable cycle lengths. We found that the action respectively, similar to that of Kv2.1/Kv6.4 channels obtained from co- potential duration (APD) was shortened as the fractional-order decreased, for transfection the individual subunits (6252 mV). This suggested that these all cycle lengths. As a consequence, the minimum cycle length (MCL) for loss channels are functional with a 2:2 stoichiometry. Analysis of tetrameric con- of 1:1 capture decreased as fractional-order decreased. Further, at short cycle catemers revealed that the positional arrangement of the Kv6.4 subunits was lengths at which APD alternans was present in the first-order model, alternans crucial. Indeed, we could not detect any functional channels when two was suppressed, such that the cycle length for alternans onset decreased for Kv6.4 subunits were positioned side by side (2_6_6_2), but the tetramer decreasing fractional-order. For fractional-order less than ~0.82, alternans with alternating subunits (2_6_2_6) displayed a midpoint of inactivation of was not present at any cycle length. Finally, for fractional-order less than 62 52 mV (n=6). Additionally, tetramers that represented a 3:1 stoichiom- ~0.75, we found that the model produced spontaneous action potentials etry (2_6_2_2) also displayed the hyperpolarizing shift of the inactivation. following pacing. Short-term memory effects were represented by a Interestingly, the voltage-dependence of activation became shallower with hypothetical memory ‘‘current,’’ which we found was primarily outward for incorporation of more Kv6.4 subunits: slope factors were 9 mV for fractional-order closer to 1, shortening APD, while it was primarily inward 2_2_2_2, 15 mV for 2_6_2_2 and 22 mV for 2_6_2_6. Taken together, our for fractional-order closer to 0.5, generating spontaneous action potentials. data suggest that the Kv2.1:Kv6.4 stoichiometry in heteroteramers can be Collectively, our results suggest the capacitive memory, reproduced by a either 3:1 or 2:2 with the restriction that Kv2.1 and Kv6.4 have to alternate fractional-order model, may play a role in both alternans formation and sup- in the channel complex in the 2:2 configuration. pression and pacemaking. 1992-Pos Board B312 1994-Pos Board B314 Activation of TRPM3 in Perivascular Sensory Nerves Induces Dilation of Lipid-Dependent Gating of Kv Channels and Excitability Change of Cere- Mouse Resistance Arteries bellar Purkinje Neurons in an NPC1 Model Mouse Lucia Alonso-Carbajo1,2, Yeranddy A. Alpizar1, Justyna Startek1, Qiu-Xing Jiang1, Hui Zheng Zheng2, Hong Xing3, Gaya Yadav1, Yuqing Li3. Jose Ramo´n Lo´pez-Lo´pez2, Maria Teresa Pe´rez-Garcı´a2, Karel Talavera1. 1Department of Microbiology and Cell Science, University of Florida, 1Lab of Ion Channel Research, KU Leuven, Leuven, Belgium, 2Dpto de Gainesville, FL, USA, 2Department of Physiology, UT Southwestern Bioquı´mica, Biologı´a Molecular y Fisiologı´a, IBGM, Universidad de Medical Center, Dallas, TX, USA, 3Department of Neurology, University of Valladolid, CSIC, Universidad de Valladolid, Valladolid, Spain. Florida, Gainesville, FL, USA. The vascular tone is determined by a complex interplay of vasodilator and Recent studies of Kv channels in different membrane systems with altered ra- vasoconstrictor stimuli that modulate the contractile state of vascular smooth tios between phospholipids and nonphospholipids support our lipid-dependent muscle cells (VSMCs). Activation of the cation channel Transient Receptor gating hypothesis that the nonphospholipids favor the conformational switch Potential Melastatin 3 (TRPM3) has been shown to induce contraction of of the voltage sensor domains of a Kv channel to a resting state. To enable VSMCs in aorta. However, the contribution of this channel to the vascular more precise control of lipid composition in an artificial membrane and avoid tone in resistance arteries remains unknown. Real-time qPCR and immuno- possible solvent contamination or phase separation of lipids in bilayer mem- chemistry experiments showed Trpm3 expression in mesenteric arteries iso- branes, we constructed a bead-supported unilamellar membrane (bSUM) and lated from wild type (WT) C57BL/6J mice. Myography experiments carried used it to define a more quantitative relationship between nonphospholipid out in intact pressurized mesenteric arteries from WT mice showed that the content and change in the gating property of a voltage-gated potassium chan- TRPM3 agonist PS induces vasodilation at concentrations above ~5 mM, nel. In the bSUMs, we were able to show for the first time that a small amount with a concentration-dependency featuring two distinct increasing phases. In of cholesterol (~10 molar %) in a phospholipid membrane in a fluidic phase contrast, PS only induced vasodilation above 10 mM following a single Hill- exerts a strong inhibitory effect on a KvAP channel, suggesting that the Kv type behavior in preparations from Trpm3 knockout (KO) mice. Recordings channels might be highly sensitive to change in membrane cholesterol con- in WT arteries in the presence of the CGRP receptor antagonist BIBN 4096 tent. To test this prediction in a physiological system where lipid metabolic recapitulated the results of Trpm3 KO preparations, indicating that the defects cause significant neurological disorders, we analyzed the neuronal TRPM3-mediated effect of PS entails CGRP release from perivascular nerve excitability in a mouse model for the Niemann-Pick disease type C, an endings. The effect of 10 mM PS was inhibited to about 50% by the combina- NPC1-I1061T knockin mice. Systematic comparison of five different groups tion of potassium channel blockers (500 nM paxilline, 10 mM correolide and of cerebellar Purkinje neurons from both knockin mice and control animals 50 nM stromatoxin). Electrophysiological recordings in freshly isolated mesen- revealed a significant decrease in the firing frequency of action potentials in teric VSMCs, revealed that basal currents are not sensitive to PS (10 mM and the tonic-burst firing Purkinje neurons of the NPC1 mice, which appears to 30 mM) and that PS has no effect on potassium currents. Our data indicates be consistent with the predicted cholesterol effects on Kv channels. Detailed that activation of TRPM3 channels in perivascular sensory nerves induces studies of the gating properties in these Purkinje neurons are being conducted CGRP release, which leads to activation of potassium channels in smooth mus- to understand the underlying mechanism. Our data support a potential connec- cle cells, resulting in dilation of mesenteric arteries. These findings reveal a po- tion among cholesterol content in cell membranes, cholesterol-dependent tential role of TRPM3 in vascular tone regulation, and support the recent notion gating of voltage-gated K channels and change in neuronal excitability in that this channel may play roles in neurogenic inflammation. CNS neurons.

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1995-Pos Board B315 rate of inactivation of Kv3.3 currents, while TBK1 overexpression slowed their Trabectedin Re-Educates Resting Peritoneal Macrophages into M1 inactivation rate. In order to explore the role of TBK1 in cell death caused by Subtype G592R Kv3.3, we measured levels of cleaved caspase 7 in different conditions, Diego A. Peraza1, Marina Mojena2, Alicia de la Cruz1, Teresa Gonzalez1, and found that the level of cleaved caspase 7 is increased in cells expressing Lisardo Bosca2, Carlos M. Galmarini3, Carmen Valenzuela1. mutant Kv3.3 channels compared with that in cells expressing wild type chan- 1Modelos Experimentales de Enfermedades Humanas, Institute of nels, particularly under condition of depolarization with high Kþ. Pretreatment Biomedical Research CSIC-UAM, Madrid, Spain, 2Metabolismo y with the TBK1 inhibitor, however, reduced expression levels of cleaved cas- Sen˜alizacio´n Celular, Institute of Biomedical Research CSIC-UAM, Madrid, pase 7 under high Kþ stimulation in both types of cells, but not in untransfected Spain, 3Departamento de Investigacio´n y Desarrollo, PharmaMar S.A., cells. Our results suggest that the interactions of the Kv3.3 potassium channel Colmenar Viejo, Madrid, Spain. with TBK1 and Hax-1 regulate channel gating and may link the channel to Macrophages play an important role in the inflammatory response, acting as signaling pathways that protect cerebellar neurons from apoptosis. antigen-presenting cells and modifying the cytokine milieu and the intensity 1998-Pos Board B318 of T-cell signaling. The electrophysiological properties of macrophages depend via on their state of functional activation. Trabectedin induces rapid apoptosis exclu- HIV-Tat Induces a Decrease in IKr and IKs Reduction in Phosphatidy- sively in mononuclear phagocytes and this effect contributes directly to its anti- linositol-(4,5)-Bisphosphate Availability 1 1 1 tumor activity. The aim of this study was to evaluate the effects of trabectedin on Zeineb Es-Salah-Lamoureux , Mariam Jouni , Olfat A. Malak , 1 1 1 K and K channels in resident peritoneal and bone marrow-derived macro- Nadjet Belbachir , Zeina Reda Al Sayed , Marine Gandon-Renard , V ir 1 1 1 phages (BMDM) under both short- and long-term incubation with the drug. Ex- Guillaume Lamirault , Chantal Gauthier , Isabelle Baro´ , 1 2 1 periments were performed using the whole-cell configuration of the patch-clamp Flavien Charpentier , Kazem Zibara , Patricia Lemarchand , 3 1 1 technique. Following exposure (ca. 10 min) of peritoneal macrophages or Bruno Beaumelle , Nathalie Gaborit , Gildas Loussouarn . 1Institut du Thorax, Nantes, France, 2Lebanese University, Beirut, Lebanon, BMDM to 100 nM trabectedin neither KV nor Kir were modified. The electro- 3 physiological effects of prolonged treatment with trabectedin were studied after CNRS, Montpellier, France. 16 h of incubation with the drug. Under these conditions, trabectedin produced a Patients with HIV present with a higher prevalence of QT prolongation, of concentration-dependent effect that was qualitatively similar to the response which molecular bases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulates viral genes expression and is required for effi- observed after LPS challenge. Moreover, the increase in KV observed appeared in parallel with a use-dependent decline, typical of macrophages stimulated with cient HIV replication. Tat is actively secreted into the blood by infected T-cells LPS. Similarly, the degree of inactivation of the current was greater and faster at and affects organs such as the heart. Tat has been shown to alter cardiac repo- higher trabectedin concentrations. All these results suggest that: 1) Trabectedin larization in animal models but how this is mediated and whether this is also the case in human cells is unknown. In the present study, we show that Tat trans- does not interact with Kir or KV channel. 2) Trabectedin acts by re-educating mouse resting peritoneal macrophages into M1-subtype. Supported by Pharma- fection in heterologous expression systems led to a decrease in hERG (under- Mar, SAF2013-45800-R and FIS RD12/0042/0019 Grants. lying cardiac IKr) and human KCNE1-KCNQ1 (underlying cardiac IKs) currents and to an acceleration of their deactivation. This is consistent with a 1996-Pos Board B316 decrease in available phosphatidylinositol-(4,5)-bisphosphate (PIP2). A mutant Slo3 Channel is Essential for Fertilized Egg Development by Controlling Tat, unable to bind PIP2, did not reproduce the observed effects. In addition, Critical Molecules for Mitosis WT-Tat had no effect on a mutant KCNQ1 which is PIP2-insensitive, further Qiong-Yao Tang1, Ye Liu1, Zheng Xu1, Fang-Fang Zhang1, Fei-Fei Zhang1, confirming the hypothesis. Twenty-four-hour incubation of human induced Yan Li1, Jose M. Eltit2, Diomedes E. Logothetis2, Zhe Zhang1. pluripotent stem cells-derived cardiomyocytes with Wild-type Tat reduced 1Xuzhou Medical University, Xuzhou, China, 2Physiology and Biophysics, IKr and accelerated its deactivation. Concordantly, this Tat incubation led to Virginia Commonwealth University, Richmond, VA, USA. a prolongation of the action potential (AP) duration. Events of AP alternans were also recorded in the presence of Tat, and were exacerbated at a low pacing Slo3 channels are specially expressed in testis and sperms. Slo3 KO mice are þ male infertile but the mechanism remains elusive. In this abstract, we first cycle length. Altogether, these data obtained on human K channels both in use Ca2þ imaging method measured Ca2þ entry of wild type sperms and heterologous expression systems and in human cardiomyocytes suggest that Slo3 KO mice sperms. We found that Ca2þ entry can be normally induced Tat sequesters PIP2, leading to a reduction of IKr and IKs, and provide a molec- by high Kþ, cGMP and cAMP in all tested conditions in both capacitated or ular mechanism for QT prolongation in HIV-infected patients. uncapacitated sperms. Thus, the male infertile phenotype cannot be explained 1999-Pos Board B319 2þ by abnormal Ca entry in fertilization process. Furthermore, in vitro fertiliza- Flavonoids as Natural Modulators of Mitochondrial Potassium Channel tion indicated fertilized eggs with slo3 KO sperms can develop into two cell Piotr Bednarczyk1, Anna Kicinska2, Wieslawa Jarmuszkiewicz2, stage but cannot pass through this developing stage. In order to investigate Renata Debowska3, Adam Szewczyk4. the reason of stopping development, we performed micro-array analysis using 1Department of Biophysics, Warsaw University of Life Sciences – SGGW, testis tissues both from wild type mice and Slo3 KO mice. The results indicate Warsaw, Poland, 2Laboratory of Bioenergetics, Adam Mickiewicz that expression level of large numbers genes had been altered in Slo3 KO mice University, Poznan, Poland, 3Dr Irena Eris Cosmetic Laboratories, Piaseczno, testis, not only in spermtocytes but also in steroli cells. Further analysis re- Poland, 4Laboratory of Intracellular Ion Channels, Nencki Institute of viewed that critical gene is required for mitosis had been missed. In the mean- Experimental Biology, Warsaw, Poland. time, we confirmed that fertilized eggs stayed in mitosis stage and cannot Flavonoids belong to a vast group of polyphenolic compounds that are widely develop further. Thus, we concluded that Slo3 channels are essential for fertil- distributed in all foods of plant origin. They exhibit a large variety of biological ized eggs development by controlling critical molecules in mitosis. properties. Interest in health benefits of flavonoids has increased due to their potent antioxidant and free-radical scavenging activities. The biological activ- 1997-Pos Board B317 ity, bioavailability and low toxicity set broad prospects of the usage of some of Role of Tank-Binding Kinase in Neurodegeneration Produced by Spino- these substances as potential therapeutics for a number of human diseases. cerebellar Ataxia Type 13 Some flavonoids have also been shown to be cardioprotective. Although the Yalan Zhang, Luis Varela, Tamas Horvath, Leonard Kaczmarek. antioxidant effect of flavonoids has been long thought to be a crucial factor ac- Yale, New Haven, CT, USA. counting for cellular cardioprotection, mitochondrial pathways (including Inherited mutations in the Kv3.3 potassium channel, such as the G952R Kv3.3 mitochondrial ion channels) are presently emerging potential targets for a spe- mutation, Kv3.3 mutations result in Spinocerebellar Ataxia 13 (SCA13). In cific pharmacological action of some flavonoids in the anti-ischemic strategies. contrast to wild type Kv3.3 channels, G592R mutant channels have slowed The detailed research describing a direct interaction of flavonoids with mito- inactivation and fail to trigger the formation of a Hax-1- and Arp2/3- dependent chondrial potassium channels is lacking. Therefore, the scientific aim of the actin cytoskeleton under the plasma membrane, but significantly increase the these studies is the description of the regulation of the mitoBKCa and mitoKATP rate of cell death in transfected cells. To determine which transduction path- channels by selected flavonoids and functional consequences of this regulation. ways are activated by G592R Kv3.3 we carried out an in situ kinase profiling Our studies with mitoplasts isolated from primary human dermal fibroblast screen to compare levels of activity of protein kinases in the cerebella of cells suggest that application of 10 mM naringenin leads to an increase in G592R-knock-in mice with those of wild type animals. The activity of TBK1 mitoBKCa channel activity. Statistical analysis of these results showed that was found to be significantly elevated in the cerebellum but not the cerebral opening probability of the channel decreased from 0.95 in the control condi- cortex of G592R Kv3.3 mice. This result was confirmed by western blotting. tions (100 mMCa2þ) to 0.06 at low calcium level (1 mMCa2þ) and increased We found that enhanced TBK1 activity accounts for the slowed inactivation to 0.81 after application of naringenin. Also, it has been observed that activity of the mutant channel. Pharmacological inhibition of TBK1 increased the of the mitoKATP channel increases after application of 10 mM naringenin.

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This study was supported by a grant MERIS PBS1/B8/1/2012 from the Na- and most diverse group of ion channels; yet the number of high resolution struc- tional Centre of Research and Development, Poland and partially by The tures of Kþ channels is limited, due to difficulties in their recombinant overex- Nencki Institute of Experimental Biology, Poland. pression and purification. Our group has developed a cost-effective method for stable expression of Kþ channels in Saccharomyces cerevisiae, as well as a 2000-Pos Board B320 technique for purification of correctly folded protein. The aim is to explore Mechanosensitivity of Mitochondrial Potassium Channels the functionality of a number of Kþ channels produced in yeast, including Piotr Koprowski, Agnieszka Kielbasa, Bogusz Kulawiak, Adam Szewczyk. hERG, Slick, Slack as well as the uncharacterized malaria potassium channels Department of Biochemistry, Nencki Institute of Experimental Biology, PfKch1 and PfKch2, by the planar patch clamp technique. Channels were Warsaw, Poland. tagged with GFP and histidine to follow their expression in Saccharomyces cer- Mitochondria are known for their unique membrane architecture with the inner evisiae and allow purification by Ni-affinity chromatography, after solubiliza- membrane folded into cristae. It is also known for long that mitochondria un- tion in FOS-12. Purified proteins were reconstituted into giant unilamellar dergo drastic changes in volume. These volume changes depend on potassium vesicles (diphyntanoylphosphatidilcholine þ 10% cholesterol) and proteolipo- fluxes. Influx via mitochondrial potassium channels (e.g. mitoBKCa or mito- somes were used for recording of single-channels on planar lipid bilayers with a KATP) results in swelling of mitochondria and unfolding of the cristae, which Port-a-Patch (Nanion Technologies). Continued recordings of hERG single- could be potentially translated into changes in the inner membrane tension. channel currents revealed voltage sensitivity and a slope conductance of However, little is known about mechanobiology of mitochondria including ex- 16 pS. Slick and Slack single-channel activity was enhanced by Naþ and istence of mechanosensitive channels. showed a conductance of 138 and 140 pS respectively. We measured for the Mitochondrial large conductance calcium activated potassium channel BKCa first time full length PfKch2 and a C-terminus truncated PfKch1, which showed þ (mitoBKCa) resides in the inner mitochondrial membrane (IMM) and plays Ca2 modulation and conductance of around 28 and 13 pS, respectively. In an important role in cytoprotection. KCNMA1 gene encodes for both mito- conclusion, our high-yield expression of Kþ channels expressed in and purified chondrial and plasmalemmal versions of BKCa channels, which share common 2þ from yeast showed similar current characteristics as single channels previously features including large conductance, synergistic gating by voltage and Ca recorded from mammalian cells, Xenopus oocytes or artificial bilayers. ions. However, it was suggested that one of plasmalemmal splice variants of BKCa containing STREX exon was uniquely mechanosensitive. Here we show that mitoBKCa formed by another splice variant BKCa-DEC is also sen- TRP Channels III sitive to stretch. We discuss potential physiological role of mechanosensitivity 2003-Pos Board B323 of mitoBKCa during mitochondria swelling. TRPC5 Channels are Inhibited by Wt Presenilin1 but not the Alzheimer’s 2001-Pos Board B321 Disease Mutant M146V Identification of Large-Conductance Calcium-Regulated K Channel in Sukhjinder Kaur, Zuleen Chia-Chang, Paris Hanson, Laura Yorke, Human Dermal Mitochndria Zafir Buraei. Adam Szewczyk1, Anna Kicinska2, Bartlomiej Augustynek1, Biology, Pace University, New York, NY, USA. Bogusz Kulawiak1, Wieslawa Jarmuszkiewicz2, Piotr Bednarczyk3. Alzheimer’s disease (AD) is a progressive neurodegenerative disease associated 1Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental with the abnormal accumulation of Amyloid-b (Ab) plaques and neurofibrillary Biology, Warsaw, Poland, 2Laboratory of Bioenergetics, Adam Mickiewicz tangles, and neuronal loss in various brain regions, especially the hippocampus. University, Poznan, Poland, 3Department of Biophysics, Warsaw University The amyloid plaques result from the accumulation of Ab, generated following of Life Sciences (SGGW), Warsaw, Poland. the proteolytic cleavage of Amyloid Precursor Protein (APP) by b- and g-secre- Potassium channels have been found in the inner mitochondrial membrane of tases. Presenilin-1 (PS1) is a membrane protein in the g-secretase complex, and various cells. These channels regulate the mitochondrial membrane potential, its mutations cause early onset AD. PS1 also resides on the Endoplasmic Retic- respiration and the production of reactive oxygen species. In this study, we ulum, where it regulates the efflux of Ca2þ from the ER, either directly, by form- þ identified the activity of a large-conductance Ca2 -regulated potassium chan- ing Ca2þ leak channels, or by controlling Inositol-Triposhpate-3 Receptor nel (mitoBKCa channel) in mitoplasts isolated from a primary human dermal (IP3R) signaling. Based on this, we hypothesized that PS1 could impact the fibroblast cell line. A potassium selective current was recorded with a mean function of TrpC5 channels, which contribute to normal hippocampal function, conductance of 280 5 2 pS in a symmetrical 150 mM KCl solution. The because they are activated by receptor-mediated Ca2þ efflux from the ER. Using 2þ mitoBKCa channel was activated by the Ca and by potassium channel opener Two-Electrode Voltage Clamp we show that PS1 significantly decreases TrpC5 NS1619. The channel activity was irreversibly inhibited by paxilline, a selec- channel activation. Surprisingly, the PS1 mutant associated with the severe and tive inhibitor of the BKCa channels. In isolated fibroblast mitochondria early onset familial AD, M146V, was ineffective in reducing TrpC5 currents. NS1619 depolarized the mitochondrial membrane potential, stimulated non- Thus, PS1 may use multiple signaling pathways to contribute to cell physiology phosphorylating respiration and decreased superoxide formation. Additionally, and AD pathophysiology. the a and b subunits (predominantly in the b3 form) of the BKCa channels were identified in fibroblast mitochondria. Our findings indicate, for the first time, 2004-Pos Board B324 the presence of a large-conductance Ca2þ-regulated potassium channel in the Atypical TRP Channel Pharmacology in Schistosomes inner mitochondrial membrane of human dermal fibroblasts. We show that a Swarna Bais, Corbett T. Berry, Xiaohong Liu, Bruce D. Freedman, large-conductance Ca2þ-regulated potassium channel functions in the inner Robert M. Greenberg. mitochondrial membrane of human dermal fibroblasts. The electrophysiolog- Pathobiology, University of Pennsylvania, Philadelphia, PA, USA. ical and biochemical properties of this channel is similar to previously Parasitic flatworms of the genus Schistosoma cause schistosomiasis, a ne- described mitochondrial potassium channels from other cell types. glected tropical disease affecting over 200 million people. Only one drug is This study was supported by a grant MERIS PBS1/B8/1/2012 from the Na- available for treatment and control, and there is an urgent need for new or re- tional Centre of Research and Development, Poland and partially by grant purposed antischistosomal agents. Ion channels are validated targets for current 2015/17/B/NZ1/02496 from Polish National Science Center. anthelmintics, but the roles and pharmacological sensitivities of transient re- ceptor potential (TRP) channels are essentially unexplored in schistosomes 2002-Pos Board B322 and other parasitic helminths. We are using TRP channel modulators, RNA D Single-Channel Recordings of K Channels Expressed in Saccharomyces interference, and heterologous expression to examine the function and pharma- Cerevisiae cology of TRP channels in schistosomes. Though the S. mansoni genome pre- Maria A. Tejada1, Karen Molbaek2, Halim Cazimi2, Peter Ellekvist3, dicts representatives of most TRP channel subfamilies, TRPV channels are Kirstine Calloe1, Claus H. Nielsen4, Per Amstrup Pedersen2, Dan A. Klaerke1. notably absent. Despite this lack of TRPV-like channels, both adult and larval 1IKVH-Physiology and Biochemistry, University of Copenhagen, schistosomes exhibit dramatic hyperactivity in response to selective activators Frederiksberg C, Denmark, 2Department of Biology, University of of mammalian TRPV1 such as capsaicin and resiniferatoxin. Capsaicin also Copenhagen, Copenhagen OE, Denmark, 3Herlev Hospital, Herlev, causes rapid separation of paired adult males and females, perhaps indicative Denmark, 4Department of Environmental Engineering, Technical University of a nociceptive response. The response to capsaicin is blocked by selective of Denmark, Kgs. Lyngby, Denmark. TRPV1 antagonists, further evidence for TRPV1-like pharmacology. Surpris- The study of ion channels has been traditionally based on expression in ingly, the capsaicin-induced hyperactivity in adult worms is virtually elimi- mammalian cell lines or Xenopus laevis oocytes and their functions have nated by knockdown of SmTRPA, a schistosome TRPA1-like channel. The been investigated by various electrophysiological techniques. Recently the TRPA1 activator AITC also elicits significant hyperactivity that is abolished structures of several ion channels have been determined and this has increased by knockdown of SmTRPA1, indicating that SmTRPA exhibits mixed the understanding of ion channel function. Potassium channels are the largest TRPV1/TRPA1-like pharmacology. Consistent with this hypothesis, exposure

BPJ 7855_7859 Tuesday, February 14, 2017 407a of SmTRPA-expressing mammalian cells to capsaicin or AITC elicits an influx Despite their importance to survival, cold-sensing TRPM8þ neurons represent of calcium, as measured by Fura-2 ratiometric imaging. These results suggest an understudied sensory neuron population, comprising only 8-10% of all tri- that some TRPV1-mediated sensory functions may be fulfilled by schistosome geminal (TG) and dorsal root ganglion (DRG) neurons. However, available TRP channels from other subfamilies, and that at least one schistosome TRP transgenic mouse lines that label TRPM8þ sensory neurons have important ca- channel has atypical pharmacological sensitivities that could be selectively tar- veats that hinder their use in functional studies. Thus, a mouse model that eases geted for development of new antischistosomal therapeutics. Other schisto- identification of these neurons would facilitate investigation of their unique some TRP channels might also be exploitable for targeting. physiological properties. To this end, we have found a novel use for the Vglu- t3iCre;Rosa26Ai14 mouse line in rapidly identifying menthol-sensitive sensory 2005-Pos Board B325 neurons in electrophysiological studies. In this mouse line, Vglut3 lineage neu- Pharmacology of Drosophila Melanogaster TRPA1 Isoforms rons express cytoplasmic tdTomato and are easily identifiable without immu- Brett Boonen. nolabeling. Menthol is a potent activator of TRPM8 channels and thus a KU Leuven, Leuven, Belgium. good proxy for identifying cold-sending neurons in vitro. Calcium micro- The TRPA1 cation channel functions as a broadly-tuned chemonociceptor in fluorimetry experiments on cultured Vglut3iCre;Rosa26Ai14 DRG neurons many species. Recent studies identified several splice variants of Drosophila from adult mice (<24 h in culture) revealed 100% of neurons responding to TRPA1 (from dTRPA1(A) to (D)). These were reported to display distinct menthol (100 mM) were small-diameter and of the Vglut3 lineage chemical and thermal sensitivities and distinct expression patterns. Some dif- (tdTomatoþ.) These neurons did not respond to capsaicin (1 mM), a TRPV1 ferences in the chemical sensitivities between insect and mammalian ortho- channel agonist, or chloroquine (1 mM), which activates TRPA1 signaling, logues have also been reported. Given the usefulness of Drosophila as consistent with the TRPM8þ sensory neuron phenotype. Furthermore, in animal model for studying TRPA1 chemosensation in vivo, we re-evaluated current-clamp recordings, menthol evoked a train of action potentials in the effects of three mammalian TRPA1 modulators (nicotine, citronellal and 100% of labeled neurons with a capacitance of < 15 pF (n = 5). Action poten- menthol) on dTRPA1 splice variants in HEK293T cells. Using intracellular 2þ tials were tetrodotoxin-sensitive, confirming these neurons were not nocicep- Ca fluorimetry and whole-cell patch-clamp, we confirmed that the electro- tors. Thus, by targeting small (<15 pF), tdTomatoþ DRG neurons in our philic agonist allyl isothiocyanate activates all splice variants, whereas nicotine electrophysiological recordings, we were able to increase the probability of activates all variants except dTRPA1(B). Citronellal robustly activated all identifying and recording from TRPM8þ neurons by 10-fold. In conclusion, splice variants. The activation by nicotine and citronellal could be inhibited the Vglut3iCre;Rosa26Ai14 is a useful model for rapidly identifying TRPM8þ by the TRPA1 antagonist HC030031. On the other hand, menthol did not acti- sensory neurons and could facilitate electrophysiological studies of cold- vate any dTRPA1 isoform. Our results demonstrate that the mammalian heter- responding sensory neurons. ologous expression system HEK293T can be used for functional studies on insect TRPA1 channels, and that the chemosensory properties of these channels 2008-Pos Board B328 may strongly differ from those of the mammalian homologues. Our findings Analog-To-Digital Conversion of Differential Metabotropic Inputs to serve as starting point for further structure-function studies for the determina- Lateral Septal Neurons by the Combined Action of TRPC4 and GIRK tion of the structural bases of chemical sensitivity in TRPA1 channels. Michael X. Zhu, Jin-bin Tian. 2006-Pos Board B326 Department of Integrative Biology and Pharmacology, University of Texas Novel Role of PUFAs as Essential Cofactors for TRPV4 Function Health Science Center at Houston, Houston, TX, USA. Rebeca C. Caires, Valeria Vasquez, Julio F. Cordero-Morales. In a neural network, neurons frequently receive multiple coincidental trans- Physiology, University of Tennessee, Memphis, TN, USA. mitter inputs representing varying activities of different brain regions. These Blood pressure regulation relies on the ability of membrane proteins to trans- inputs not only vary in the signal pathways they activate but also in the duce variations in physical and chemical stimuli into electrical signals. The strengths for each of the pathways. However, it remains unclear how neurons transient receptor potential vanilloid 4 (TRPV4) is a cation channel that has decipher co-incident inputs of varying strengths and encode discernible output been implicated in endothelium-dependent vasorelaxation and modulation of messages. The vast majority of neurotransmitters signal through G protein- systemic blood pressure. TRPV4 is widely expressed in the vasculature, being coupled receptors, which activate heterotrimeric G proteins to alter neuronal found in smooth muscles, vascular endothelial cells, and perivascular sensory function via a limited set of effectors, including G protein-activated and/or neurons. Activation of TRPV4 downstream of cell swelling via polyunsaturated receptor-operated channels that are capable of modulating neuronal excit- fatty acids-derived mediators promotes arterial vasodilation. The precise mech- ability. For receptors that couple to Gq/11 and Gi/o, whereas the activation of anism by which polyunsaturated fatty acids and eicosanoids regulate TRPV4 Gq/11-phospholipase C pathway, which hydrolyzes phosphatidylinositol 4,5-bi- sphosphate, suppresses Gi/o-mediated activation of G protein-activated function remains elusive. Here, we use C. elegans to precisely alter fatty þ acid content in vivo; worms allow for easy and accurate manipulation of lipid inwardly rectifying K (GIRK) channels, the phospholipase C signaling is content, have the ability to incorporate lipids back via diet supplementation, required for Gi/o stimulation of the nonselective cation channel, Transient Re- and a collection of lipid-deficient mutants. We show that chemical activation ceptor Potential Canonical 4 (TRPC4). Thus, coincident activation of Gq/11 and of a transgenic worm that expresses rat (r) TRPV4 in the sensory amphid neu- Gi/o pathways differentially affects GIRK and TRPC4 channel activities, rons (ASH) by GSK1016790A (TRPV4 selective agonist), but not wild type, shaping excitability in a manner that is very sensitive on the relative strengths elicits withdrawal responses; therefore, providing a chemical strategy to study of Gq/11 and Gi/o signaling. Using whole-cell slice recordings and agonist or the activation mechanism of TRPV4 using C. elegans. Using this assay, we electrical stimulation of lateral septal neurons, we demonstrate that varying determine that TRPV4 is not functional in the background of lipid-deficient stimulation intensities at Gq/11 and Gi/o-coupled receptors give rise to distinc- mutant’s worms (fat-3, lacking long polyunsaturated fatty acids; PUFAs). tive membrane potential waveforms that include burst firing, plateau depolari- These results suggest that PUFAs are obligatory cofactors of TRPV4 function. zation and hyperpolarization of varying durations resulting from combined Using confocal microscopy, we established that channel distribution and actions of TRPC4 and GIRK channels. These differential responses alter morphology of ASH neurons are indistinguishable between TRPV4::GFP neuronal firing in distinguishable patterns. We propose that the conversion of and TRPV4::GFP; fat-3 worms. Moreover, diet supplementation experiments concurrent Gq/11 and Gi/o signaling of differential strengths into discernible suggest that EET rescues 20 % less the withdrawal responses of TRPV4; fat- electrical responses by joint actions of TRPC4 and GIRK channels is vital 3 mutants when compared to arachidonic acid (AA), implicating AA as a for lateral septum to serve as the information processing center of cerebrum key fatty acid for TRPV4 function. By knocking down lysophospholipid acyl- for higher-order brain functions. transferase enzyme (MBO-6), we found that membranes phospholipids lacking AA negatively regulate TRPV4 function. Our results support a model whereby 2009-Pos Board B329 membrane composition and lipid metabolism modulates TRPV4 function. Crosstalk of Pain and Pleasure: TRPV1 is a Novel Oxytocin Receptor Yelena Nersesyan1, Lusine Demirkhanyan1, Tyler Dawson1, 2007-Pos Board B327 Swapna Asuthkar1, Deny Cabezas-Bratesco2, Sebastian Brauchi2, The Vglut3Icre;Rosa26Ai14 Mouse Model as a Tool for Studying TRPM8D Eleonora Zakharian1. Sensory Neurons 1Cancer Biology & Pharmacology, University of Illinois, Peoria, IL, USA, Theanne N. Griffith1,2, Javier E. Marquina-Solis2,3, Adrian C. Thompson2,4, 2Department of Medicine, Universidad Austral de Chile, Valdivia, Chile. Blair A. Jenkins1,2, Ellen A. Lumpkin1,2. The Transient Receptor Potential Vanilloid 1 (TRPV1) channel is a receptor for 1Physiology, Columbia University, New York, NY, USA, 2Neurobiology a wide range of noxious stimuli. In the peripheral nervous system, TRPV1 can Course, Marine Biological Laboratory, Woods Hole, MA, USA, 3David be sensitized by heat, capsaicin, and acidic pH. However, the physiological Rockefeller Graduate Program, The Rockefeller University, New York, NY, regulation of TRPV1 by endogenous agonists is not well understood. In the pre- USA, 4School of Medicine, University of Tasmania, Hobart, Australia. sent work, we discovered a new endogenous agonist of the TRPV1 channel,

BPJ 7855_7859 408a Tuesday, February 14, 2017 which appeared to be a neuropeptide hormone oxytocin. Oxytocin is produced B perforated patch-clamp electrophysiology on freshly-isolated UBSM cells in the hypothalamus and plays regulatory roles in various behaviors, including in a combination of isometric tension recordings of UBSM isolated strips social bonding and sexual reproduction in both genders. Oxytocin is also and the selective TRPM4 channel inhibitor, 9-phenanthrol. UBSM samples known to affect nociception. The parvocellular oxytocin neurons project to were obtained from experimental animals (rats and guinea pigs) and the brainstem and spinal cord, where the hormone is known to modulate inflam- patient-donors without preoperative overactive bladder symptoms. The results matory pain processing. We found that oxytocin elicited TRPV1 activity both demonstrated significant species-related differences in TRPM4 channel activ- in the native and the heterologous expression systems, evidenced in Ca2þ-im- ity in human UBSM compared to rat and guinea pig UBSM. Pharmacological aging and whole-cell patch clamp recordings. This activity was absent in the inhibition of TRPM4 channels with 9-phenanthrol caused a significantly cells with the knockdown of TRPV1, but not the classical oxytocin receptor, larger decrease in human UBSM excitability and contractility compared to indicating that this potentiation is TRPV1 specific. Furthermore, oxytocin- rats and guinea pigs. The whole-cell 9-phenanthrol sensitive TRPM4 current induced Ca2þ-uptake was abolished by the treatment with TRPV1 antagonist was greater in human UBSM cells compared to guinea pig UBSM cells. In capsazepine. Dissecting whether the agonistic effect of oxytocin on TRPV1 UBSM isolated strips, pharmacological inhibition of TRPM4 channels with is direct, we examined its action in the reconstituted system. Using planar lipid 9-phenanthrol had significantly higher inhibitory effect on UBSM sponta- bilayers, we justified that oxytocin directly activates TRPV1. The channel neous phasic contractions in man compared to rat and guinea pig. demonstrated a strong voltage-dependence and rectification, reflected both in However, no statistical differences were noted in UBSM contractility between single channel conductance and open probability. In conclusion, our results rats and guinea pigs when TRPM4 channels were inhibited with 9-phenan- indicated that oxytocin is a novel direct TRPV1 agonist, where the throl. In conclusion, the data showed important interspecies-related differ- neuropeptide-channel actions could be implicated in various oxytocin- ences and revealed that TRPM4 channels have a more prominent dependent regulatory mechanisms in the nervous system. physiological role in the regulation of UBSM function in man compared to rodents. The study was supported by NIH R01-DK106964 grant to Georgi 2010-Pos Board B330 V. Petkov. The Role of TRPC Channels in Ischemic Neuronal Cell Death Jaepyo Jeon, Guanghua Sun, Jinbin Tian, Sung-Ming Ting, 2012-Pos Board B332 Jaroslaw Aronowski, Michael X. Zhu. TRPV1 Activation Stimulates Brown Adipose Tissue Activation and The University of Texas Health Science Center at Houston, Houston, Enhances Mitochondrial Biogenesis to Counteract Diet-Induced Obesity TX, USA. Padmamalini Baskaran1, Zhaojie Zhang2, Baskaran Thyagarajan1. Brain ischemia causes damage to neurons due to reduced oxygen and glucose 1Pharmacy, University of Wyoming, Laramie, WY, USA, 2Zoology and supply, i.e. oxygen and glucose deprivation (OGD). Cell death after cerebral Physiology, University of Wyoming, Laramie, WY, USA. ischemia may result from elevation in intracellular calcium (Ca2þ), loss of cell Obesity is a huge burden to healthcare and one third of the word population is ion homeostasis and glutamate-induced excitotoxicity, which can all result obese or overweight. Currently no single pharmacotherapy is available to from activation of non-selective cation channels, such as those formed by counter obesity. Recent research from our laboratory and from others suggest the Transient Receptor Potential (TRP) proteins. During ischemia/reperfusion that activation of transience receptor potential vanilloid subfamily 1 is a reli- to the brain, Ca2þ influx to neurons may be induced by the elevation of extra- able strategy to prevent and treat metabolic diseases including obesity. Our pre- cellular glutamate concentration, which elicits several neurotoxic events vious research work unambiguously shows that capsaicin triggers the through activation of glutamate receptors. Among them, the metabotropic molecular conversion of white to BRITE (BRown in whITE) phenotype by glutamate receptors are known to couple to TRPC channel activation, leading activating sirtuin-1-dependent deacetylation and interaction of peroxisome to membrane depolarization and sustained Ca2þ influx. To determine the proliferator activated receptor gamma (PPARg) and PR domain containing contribution of TRPC channels in glutamate-induced intracellular Ca2þ over- protein 16 (PRDM-16) via TRPV1-dependent mechanism. Extension of this 2þ 2þ load, We measured intracellular Ca concentration ([Ca ]i) changes in work suggest that capsaicin feeding significantly enhanced the expression of response to glutamate stimulation in cultured mouse cortical neurons. Gluta- thermogenic proteins including bone morphogenetic protein 8b (BMP8b), 2þ mate induced a robust increase in [Ca ]i, which was inhibited by ~50% mitochondrial uncoupling protein-1 (UCP-1), PPARg coactivator 1a (PGC- with the TRPC4/C5 blocker, ML204. In neurons prepared from TRPC4 1a), sirtuin-1 (SIRT-1) and PRDM-16 in brown adipose tissue (BAT) while 2þ knockout mice, the glutamate-induced [Ca ]i increase was also significantly high fat diet (HFD)-feeding increased body weight gain by suppressing these less than in that from the wild type (WT) controls. These results indicate the thermogenic proteins. Capsaicin also significantly enhanced the expression of 2þ likely involvement of TRPC4 channels in glutamate-induced [Ca ]i rise, a thermogenic markers and increased basal and forskolin stimulated lipolysis condition commonly associated with OGD. To test if the TRPC channel activ- in BAT. Capsaicin decreased serum glucose, triglyceride and cholesterol levels ity is linked to ischemic neuronal cell death, we used both an in vivo stroke and restored glucose intolerance and insulin resistance observed in HFD-fed model and an in vitro OGD model. Transient middle cerebral artery mice. These data suggest an enhancement of plasma GLP-1 level, BAT activa- (MCA)/common carotid artery (CCA) occlusion resulted in large infarct areas tion and a concomitant regulation of glucose homeostasis by capsaicin. Further, in WT mice. The infarct areas were significantly smaller in the brains of the upregulation of PGC-1a, a gene that metabolically regulates mitochondrial TRPC4-/- mice. In cortical neuron cultures prepared from TRPC1-/-, biogenesis, by capsaicin is associated with an increased number of mitochon- TRPC4-/- and TRPC1/C4/C5/C6-/- mice, OGD-induced neuronal cell death dria in BAT. Also, capsaicin supplementation in diet increased the metabolic was significantly less than that from WT mice. In addition, the treatment activity, respiratory quotient and heat production in live mice. These data pro- with ML204 reduced OGD-induced death of WT neurons, showing protection vide evidence for the regulation of BAT activation and enhancement of mito- to ischemic damage. These data suggest that TRPC channels play a significant chondrial biogenesis by capsaicin to increase energy expenditure and heat role in neuronal death induced by OGD. Our study reveals that TRPC channels production to counter obesity. Further research work is in progress to under- significantly contribute to ischemic cell death through calcium overload and stand how TRPV1 activation couples to mitochondrial biogenesis by enhancing that inhibition of TRPCs may be neuroprotective against brain damage the expression and activation of PGC-1a. following ischemic stroke. 2013-Pos Board B333 2011-Pos Board B331 TRPV1 Expression in Adipose Tissues Determines Metabolic Activity and Species Differences in Urinary Bladder Smooth Muscle TRPM4 Channel Energy Expression to Counter Diet-Induced Obesity Activity: Comparison between Rat, Guinea Pig, and Man Baskaran Thyagarajan, Padmamalini Baskaran. Georgi V. Petkov. School of Pharmacy, University of Wyoming, Laramie, WY, USA. Drug Discovery and Biomedical Sciences, University of South Carolina, Obesity foreshadows metabolic syndrome comprising of type 2 diabetes, dys- Columbia, SC, USA. lipidemia, hypertension and cardiovascular diseases. Contemporary research The Ca2þ-activated transient receptor potential melastatin-4 (TRPM4) chan- suggests the involvement of transient receptor potential vanilloid subfamily nel is highly permeable for monovalent cations (Naþ and Kþ) but imperme- 1 (TRPV1) channel protein in diet-induced obesity. The effects of both the able to anions and divalent cations, including Ca2þ. Using a multidisciplinary expression/activation of TRPV1 and the loss of TRPV1 have been shown to experimental approach, my laboratory has recently identified and character- counter diet-induced obesity. However, these results were based on the type ized the TRPM4 channels as critical regulators of urinary bladder smooth of high fat diet that was used in the study. Hence, this study evaluated the muscle (UBSM) function. The present study provides new insights into the role of TRPV1 activation in diet-induced obesity and determined whether differential regulatory roles of TRPM4 channels in UBSM isolated from an- the lack of TRPV1 indeed altered the expression of adipogenic and thermo- imal species in comparison to man. The study employed the amphotericin- genic genes in white as well as brown adipocytes. We performed experiments

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-/- with normal chow diet or 60% high fat diet-fed wild type and TRPV1 mice showed the half maximal effective concentration (EC50)of24mM and the with the objective of analyzing the role of endogenous TRPV1 expression on maximum rate of 0.71 mM/s. The EC50 value is similar to that reported for metabolic activity, thermogenesis, expression of adipogenic and thermogenic recombinant TRPM7 overexpressed in HEK293 cells (Pflug Arch 466:2177- genes in the inguinal or brown fat pads. We measured the respiratory quotient, 2189, 2014). Thus, our pharmacological study suggests that the TRPM7 chan- heat production, and locomotion activity in these mice. Also, we isolated nel constitutes physiological Mg2þ influx pathways in cardiac myocytes. epididymal, subcutaneous (inguinal) and brown fat pads (EF, SCF and BF, respectively) from these mice and analyzed the expression of adipogenic 2016-Pos Board B336 and thermogenic genes. Endogenous lack of TRPV1 significantly decreased Dissecting Local and Systemic Effects of TRPV1 on Bladder Contractility the expression of several genes that are implicated in lipolysis and thermogen- in Diabetes 1,2 1 1 esis in mice that were fed normal chow diet. This is associated with decreased Bizhan R. Sharopov , Kseniya L. Gulak , Igor B. Philyppov , 1 1,2 locomotor activity in TRPV1-/- mice compared to wild type. Also, high fat Anna V. Sotkis , Yaroslav M. Shuba . 1Department of Nerve & Muscle Physiology, Bogomoletz Institute of diet caused a more significant down-regulation of thermogenic proteins in 2 the adipose tissues of TRPV1-/- compared to wild type mice. Analyses of loco- Physiology NASU, Kyiv, Ukraine, International Center for Molecular motion activity revealed that normal chow diet-fed TRPV1-/- mice exhibited Physiology NASU, Kyiv, Ukraine. less activity compared to wild type mice. Our data reveal that lack of Transient receptor potential vanilloid 1 (TRPV1) ion channel, which is mostly > TRPV1 strikingly increases metabolic impairment compared to wild type known as a sensor of noxious heat ( 42 ) and a receptor of active pungent mice. Our data suggest that the expression and activation of TRPV1 will ingredient of hot chilly paper, capsaicin (CAP), is nowadays attracting help in shedding new light into a strategy to counteract obesity. increasing attention as crucial regulator of physiological and pathological func- tions of the internal organs, particularly urinary bladder. In this study, we for 2014-Pos Board B334 the first time dissect contributions of two roles of TRPV1, as regulator of local Analysis of Role of TRPV1 in Exercise, Energy Intake and Obesity events and as one of the key determinants of reflex arc, in diabetic bladder Baskaran Thyagarajan, Padmamalini Baskaran, Vivek Krishnan. dysfunction (DBD), a bothersome disorder of micturition afflicting up to School of Pharmacy, University of Wyoming, Laramie, WY, USA. 80% of patients suffering from diabetes mellitus. Obesity is one of the biggest challenges encountered by the society today. Life- By using retrograde fluorescent labeling and whole-cell patch clamp, we style modifications like diet regulation and exercise are popular strategies to showed that TRPV1-mediated current (ITRPV1) in dorsal root ganglia (DRG) counter obesity but they are limited in their effectiveness. Many research neurons innervating the urinary bladder is increased by 42% in rat model of studies have shown that exercise can decrease appetite and energy intake streptozotocin(STZ)-induced diabetes compared to the mock-treated controls. through the regulation of expression, secretion and activity of various orexi- This observation is further supported by qPCR analysis which demonstrated genic and anorexigenic neuropeptides. Dietary supplementation of Capsaicin the 63% increase of Trpv1 transcription level in DRGs obtained from diabetic (CAP) has been shown to prevent high fat diet induced weight gain and vs. control animals, thus providing an explanation for ITRPV1 enhancement. improve insulin sensitivity via Transient Receptor Potential Vanilloid receptor On the contrary, tensiometric measurements of the contractions of isolated 1 (TRPV1)-dependent mechanisms without altering food intake. Here, we eval- multicellular bladder detrusor smooth muscle (DSM) strips revealed the unex- uated whether exercise and capsaicin can exhibit synergism to counter obesity. pected decrease of basal tone enhancement (DFC) in response to exogenous Therefore, we exercised NCD or HFD 5 CAP-fed wild type (WT) and CAP application in preparations from STZ-treated vs. control rats. This effect TRPV1-/- mice on a treadmill or a rotarod. Mouse body weights, food and water was observed at all concentrations of the agonist tested (105 to 107 M) and intake were recorded weekly. We found that CAP feeding reduced the weight constituted 3-to-10-fold difference between two groups. gain in both sedentary and exercised WT but not in TRPV1-/- mice. Food intake Therefore, our findings suggest the dual role of TRPV1 ion channel in patho- did not change between NCD and HFD (5 CAP)-fed mice in the sedentary genesis of DBD. On the one hand, the increased functional expression of conditions whereas in the exercised mice decreased food intake was observed TRPV1 in bladder-innervating DRG neurons suggests upregulation of the in the HFD (5 CAP)-fed condition compared to NCD. We also analyzed the micturition reflex arc initiated by TRPV1 activation in bladder afferents and neuromuscular functions of NCD or HFD (5 CAP)-fed WT and TRPV1-/- terminated by the release of key efferent DSM-contracting neurotransmitters, mice on a rotarod. CAP rescued the HFD-diet induced loss of muscular ACh and ATP, from bladder efferents, which would be consistent with urge in- coordination in WT but not in TRPV1-/- mice. CAP feeding also decreased continence. On the other hand, local TRPV1-mediated contractile signaling the number of falls in HFD-fed WT but not TRPV1-/- mice. These data suggest associated with ’’efferent function‘‘ of TRPV1-expressing afferents prompting that TRPV1 activation is also critical in the maintenance of neuromuscular them to release DSM-contracting tachykinins (e.g. substance P, SP) in response functions which were suppressed by HFD. From our data, we hypothesize to TRPV1 activation becomes compromised. Thus, our results may explain the that CAP feeding and exercise could synergistically decrease weight gain. disparity of DBD symptoms in diabetic patients by varying contribution of the However, the decreased HFD (5 CAP)-food intake in exercised mice did two effects. not contribute to their decreased weight gain. Further studies are in progress to analyze the mechanism underlying this effect. 2017-Pos Board B337 A Putative Nuclear/Nucleolar Signal Sequence Directs the Localization of 2015-Pos Board B335 TRPM7 Kinase Effects of an Activator of TRPM7, Naltriben, on Magnesium Influx of Rat Ceredwyn E. Hill, Adenike Ogunrinde, Christiane Whetstone, Ventricular Myocytes Evalina Williamson. Michiko Tashiro, Hana Inoue, Masato Konishi. Biology, Queen’s University, Kingston, ON, Canada. Dept. Physiology, Tokyo Med.Univ., Tokyo, Japan. The channel-kinase TRPM7 supports the survival, proliferation, and differenti- It has been suggested that TRPM7, a member of the melastatin family of tran- ation of many cell types. Both plasma membrane channel activity and kinase sient receptor potential channels, plays an important role in intracellular Mg2þ function have been implicated in these roles, although the specific functions homeostasis in mammalian cells. To evaluate physiological roles of the channel of either have not been clearly established. The kinase has been invoked in in native cardiac cells, we studied the effect of naltriben, a relatively specific the regulation of protein synthesis, chromatin status, actomyosin contractility, activator of TRPM7, on the Mg influx rate in rat ventricular myocytes. Acutely allergy and inflammation. Identified proteinaceous substrates are localized isolated single myocytes were loaded with a fluorescent Mg2þ indicator furap- throughout the cytoplasm and nucleus, lending credence to the observation 2þ 2þ tra to estimate intracellular Mg concentration ([Mg ]i)at25C. Extracel- that the soluble kinase domain can be cleaved from the channel (Krapivinsky 2þ 2þ lular application of 50 mM naltriben caused little change in [Mg ]i in Ca - et al. 2014). The same study reported that a kinase construct (1510-1863), het- free Tyrode’s solution that contained 140 mM Naþ. When Naþ- dependent erologously expressed in HEK293 cells, appears in the nucleus. We recently Mg2þ efflux was inhibited by substitution of extracellular Naþ with showed that hepatoma cells express a soluble C-terminal immunoreactive N-methyl-D-glucamine, however, naltriben (50 mM) significantly raised domain of TRPM7 in the nucleus and nucleolus, whereas non-dividing hepato- 2þ [Mg ]i from 0.8350.01 mM to 1.3350.09 mM in 2 hours (n=4). For quan- cytes express nuclear envelope but not nucleoplasmic, chanzyme. To further 2þ 2þ titative analyses of the rate of Mg influx, [Mg ]i was lowered by soaking our understanding of the cellular role(s) of the kinase domain and establish the cells with a high-Kþ and Mg2þ-Ca2þ-free solution, and the time course how the kinase is targeted to specific nuclear compartments, we used genetic 2þ 2þ of [Mg ]i recovery in the Ca -free Tyrode’s solution was fitted with a double and immunofluorescence approaches to identify the subcellular location(s) of exponential function. The first derivative of the recovery function at time zero TRPM7 kinase constructs containing putative subcellular localization se- was taken as the initial rate of Mg2þ influx (0.2250.03 mM/s, n=11). Admin- quences. HEK293T cells heterologously expressing the isolated kinase domain istration of 50 mM naltriben increased the initial rate of Mg2þ influx to (1510-1863) confirmed the nucleoplasmic localization. Contrary to the endog- 0.5250.10 mM/s (n=9). The concentration dependence of the drug’s effect enous labelling in hepatoma, this protein did not appear in the nucleolus.

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However, a larger construct containing a putative nucleolar signalling sequence Channelopathies result from ion channel dysfunction caused by mutation in (K1147-K1151) labelled the nucleoplasm and the nucleolus, where it colocal- either pore-forming subunits or regulatory proteins. The widespread use of ge- izes with fibrillarin. Western blotting of fractionated nuclei confirmed these re- netic and genomic testing has led to an explosive growth in the number of sults. Thus the TRPM7 kinase appears to take different forms, possibly cell newly discovered ion channel variants associated with human diseases and specific, allowing it to localize to specific nuclear locations. We surmise that in reference populations. For example, in congenital long QT syndrome TRPM7 and its cleavage products distribute between the plasma membrane (LQTS), hundreds of variants have been discovered in KCNQ1 and and nucleus in proliferating hepatoma cells. Nucleolar kinase activity may be KCNE1, which encode the pore-forming subunit and accessory protein involved in nucleolar functions such as ribosome synthesis. required to generate the slow delayed cardiac rectifier current (IKs). Functional annotation experiments (e.g., patch-clamp recording) have become the gold 2018-Pos Board B338 standard in assessing the likely pathogenicity of ion channel variants, but TRPV1 Contributes to Acrolein-Induced Toxicity the extreme time- and labor-intensity of this approach is insufficient to tackle 1 1 1 2 Yeranddy A. Alpizar , Brett Boonen , Maarten Gees , Pieter Uvin , hundreds, if not thousands, of known variants. To overcome this challenge, 1 2 2 1 Thomas Voets , Dirk De Ridder , Wouter Everaerts , Karel Talavera . we have implemented a work flow that combined high efficiency electropora- 1Department of Cellular and Molecular Medicine, KU Leuven, Leuven, 2 tion to achieve transient co-expression of ion channel subunits in cultured Belgium, Department of Development and Regeneration, KU Leuven, cells with automated planar patch clamp recording performed in a 384-well Leuven, Belgium. format. We demonstrated the success of this approach by determining the Acrolein is a toxic and highly reactive unsaturated aldehyde, often found in functional properties of 51 KCNQ1 variants co-expressed with KCNE1 in cigarette smoke and vehicle exhaust gases. Likewise, acrolein derived from CHO cells. Channel subunits were expressed from plasmid vectors encoding cyclophosphamide-treated patients constitutes the major culprit of bladder irri- either EGFP (KCNQ1) or DsRed (KCNE1) enabling quantification of trans- tation during chemotherapy in cancer patient. Although, initially, its toxicity fection efficiency by flow cytometry. Approximately 2/3 of cells were typi- and inflammatory properties have been related to the activation of the transient cally co-transfected with both subunits. Automated patch-clamp recording receptor potential A1 (TRPA1) in nociceptive neurons, recent evidences sug- was performed before and after application of the specific IKs blocker gests that other receptor may also play a role in acrolein-induced toxicity. HMR1556 (20 mM), enabling offline subtraction of non-specific currents. Here we show that, acrolein induces chemical irritation and nocifensive Typically, ~80-90% of wells exhibited cell capture, high seal resistance response in the absence of TRPA1. Ratiometric calcium measurements and (>0.5Gohms) and low series resistance. Semi-automated data handling rou- patch-clamp demonstrate that acrolein activates TRPV1 but, unlike TRPA1 tines allowed for rapid analysis of current density, voltage-dependence of that desensitizes immediately after activation, acrolein-induced activation of activation and gating kinetics. Our results indicate a successful implementa- TRPV1 is prolonged in time. Furthermore, we identify the N-terminal amino tion of a robust workflow that enables rapid functional annotation of human acid residue C157 as key for acrolein-induced TRPV1 activation. Taken ion channel variants. together, our results reveal a mechanism underlying the major role of TRPV1 as mediator for the acrolein-induced toxicity, unveiling TRPV1 as a 2021-Pos Board B341 potential therapeutic target in a wide spectrum of noxious conditions, from The Effects of Extracellular Protons on the hERG Potassium Channel exposure to smoke to cancer treatment. Stacey L. Wilson, Neil V. Marrion, Jules C. Hancox. Physiology and Pharmacology, University of Bristol, Bristol, United Ion Channels, Pharmacology, and Disease I Kingdom. The a-subunit of channels mediating the cardiac rapid delayed rectifier current 2019-Pos Board B339 (I ) is encoded by the human Ether-a`-go-go-Related Gene (hERG). Macro- State- and Use-Dependent Binding of KCNQ Channel Openers Kr scopic hERG current (I ) amplitude is reduced and deactivation kinetics Caroline K. Wang, Alice W. Wang, Harley Kurata. hERG are accelerated with extracellular acidosis. We have investigated the single University of Alberta, Edmonton, AB, Canada. channel basis for the effects of acidic external pH (pH ) on the isoforms of Retigabine is the first approved anticonvulsant that acts via activation of e I expressed in myocytes (hERG1a and 1b). Patch clamp recordings KCNQ2-5 voltage-gated potassium channels, caused by a hyperpolarizing shift hERG were made at room temperature with the extracellular superfusate (whole- in the voltage-dependence of activation. An important unexplored feature of re- cell) or pipette solution (cell attached) acidified to pH 6.3 compared with con- tigabine and its derivatives is their state- and use-dependent properties. Drugs trol (pH 7.4). A decrease in pH to 6.3 caused acceleration in deactivation and that exhibit use-dependence may have stronger effects with more frequent e a reduction in maximal whole-cell conductance of ~34% for I (n=8 channel stimulation, enabling selective targeting of hyperexcitable cells. We hERG1a cells) and of ~36% for I (n=5 cells). Single channel recordings were aimed to generate a detailed understanding of the mechanism of action of hERG1b made with isotonic potassium (140 mM) bathing the cells and in the electrode. various KCNQ channel openers by testing their state- and use-dependent ef- Channel amplitude and open state kinetics were measured at a series of repo- fects on KCNQ2 channels. We assessed drug binding to pre-open states by larisation voltages following a depolarising command to þ40mV. Slope applying drugs at different holding potentials, and also subjected cells to conductance values derived from amplitude current-voltage relationships be- frequent repetitive stimulation to assess use-dependent drug interactions. WT tween 120 and 40mV were 12.350.2pS for pH 7.4 (n=10 cells) and KCNQ2 channels exhibit marked state- and use- dependent binding to the re- 9.350.1pS for pH 6.3 (n=9 cells) (P<0.01, two-tailed t-test) for hERG1a. tigabine analog ICA069673. This drug exclusively binds to activated channel The corresponding values for hERG1b were 11.450.2pS for pH 7.4 (n=6 states, with more pronounced effects observed with more frequent channel cells) and 7.650.4pS for pH 6.3 (n=5 cells) (P<0.0001; two-tailed t-test). stimulation. We have also demonstrated that the KCNQ2[A181P] mutant Open-time kinetics at 120mV for hERG1a were reduced from significantly diminishes the gating shift and state-dependent effects of 8.4951.0ms in control (n=8 cells) to 4.250.4ms in pH 6.3 (n=7 cells) ICA069673. Retigabine exhibits less pronounced gating effects than e (P<0.05; two-tailed t-test). The hERG1b open state kinetics were ICA069673, along with less pronounced state-dependence highlighted by its 5.750.6ms in pH 7.4 (n=6 cells) and reduced to 3.150.7ms in pH 6.3 ability to bind to KCNQ2 at negative holding potentials. We used fast solution e e (n=5 cells) (P<0.05; two-tailed t-test). Thus, it can be concluded that a exchange to assess kinetics of drug interactions. At positive voltages, reduction in the single channel conductance and acceleration of open- ICA069673 binds similarly to both WT KCNQ2 and A181P mutant channels. times contribute to the attenuation of macroscopic I when exposed to Upon repolarization to negative voltages however, ICA069673 dramatically hERG acidic pH . decelerates WT KCNQ2 closure, but has much weaker effects on KCNQ2 e [A181P]. These data suggest that the active conformation of KCNQ2 2022-Pos Board B342 [A181P] channels is less effectively stabilized by ICA069673. These findings The Roles of Selected Pore Residues in Pharmacological Inhibition of the highlight the dramatic state-dependence of certain KCNQ channel openers hERG Potassium Channel by a Minimally Structured Blocker and begin to reveal the underlying molecular determinants of these effects. Matthew V. Helliwell1, Jules C. Hancox2, Yi H. Zhang2, 2020-Pos Board B340 Christopher E. Dempsey1. 1 Functional Annotation of KCNQ1 Variants of Unknown Significance Department of Biochemistry, University of Bristol, Bristol, United 2 using Automated Electrophysiology Kingdom, Department of Physiology and Pharmacology, University of 1 1 1 2 Bristol, Bristol, United Kingdom. Carlos G. Vanoye , Reshma R. Desai , Katarina L. Fabre , Jens Meiler , þ Charles R. Sanders3, Alfred L. George1. hERG K channels are sensitive to pharmacological inhibition by structurally 1Pharmacology, Northwestern University Feinberg School of Medicine, diverse drugs. This can lead to acquired long QT syndrome. We have charac- Chicago, IL, USA, 2Chemistry, Vanderbilt University, Nashville, TN, USA, terised the interactions with hERG of a recently designed ‘‘minimally struc- 3Biochemistry, Vanderbilt University, Nashville, TN, USA. tured’’ high affinity hERG channel inhibitor, ‘‘Cavalli-2’’. Experiments

BPJ 7855_7859 Tuesday, February 14, 2017 411a were performed on HEK-293 cells expressing wild-type or mutant hERG trafficking to approximately 10% of wild type level, while the combination channels. Whole-cell patch clamp measurements of IhERG were made at of the small molecule VX809 with either the second site mutation or low tem- 37C and data are presented as mean 5 SEM, nR5. Commercially synthe- perature increased trafficking to nearly 40% of wild type level. The result indi- sized Cavalli-2 suppressed hERG tail currents (IhERG) with a half-maximal cates that the small molecule VX809 works synergistically with either the low inhibitory concentration (IC50) of 35.6 5 1.0 nM. Aromatic residues on temperature or the second site mutation. No statistical difference is seen be- the S6 helix are known to be structural determinants for high affinity hERG tween the cargo load in each trafficking vesicle of wild type and I539T/ channel block. Mutation of Y652 residues to alanine markedly reduced VX809 corrected F508 CFTR. Overall, the results suggest both low tempera- the affinity of Cavalli-2 for hERG (100 nM Cavalli-2 caused 61.5 5 5.0% tures and second site mutations lead to the stabilization of a pool of mutant block of WT IhERG but only 23.0 5 4.1% of Y652A IhERG). The pore CFTR in the ER. VX809 acts on this stabilized pool increasing trafficking to helical residues T623 and S624 have been reported to make important the plasma membrane. interactions with polar aromatic para-substituents of drugs. Despite the absence of polar para-substituents in the minimal hERG blocker, T623A IhERG 2025-Pos Board B345 þ (which required measuring inward tail currents in high [K ]at120 mV), Characterization of CFTR Activators and Inhibitors by the use of a Planar was blocked 47.5 5 4.2% by 100 nM Cavalli-2 compared to 73.8 5 7.1% Patch Clamp System 1 2 3 1 of inward WT IhERG and S624A IhERG was blocked by 29.1 5 3.8% Andrea Bruggemann€ , Soren Friis , Tim Strassmeier , Markus Rapedius , 1 1 1 1 (compared to 61.5 5 5.0% WT IhERG). Combined with computational model- Tom Goetze , Ilka Rinke , Claudia Haarmann , Nina Brinkwirth , ling of interactions of Cavalli-2 with the hERG pore, these results support Atsushi Ohtsuki4, Takayuki Oka4, Michael George1, Niels Fertig1. binding modes in which S624 stabilises the protonated amino group of 1Nanion Technologies, Munich, Germany, 2Nanion Technologies, Cavalli-2 near the internal K binding site and T623 may promote favourable Copenhagen, Denmark, 3Nanion Technologies, NJ, USA, 4Nanion pore geometries for drug binding, possibly by stabilising optimal conforma- Technologies, Tokio, Japan. tions of Y652. Cystic fibrosis is caused by malfunction of the chloride channel Cystic Fibrosis Transmembrane Regulator (CFTR). CFTR is expressed in the apical membrane 2023-Pos Board B343 of epithelial cells where it is involved in the regulation of fluid transport across Lamprey CFTR, an Evolutionary Ancestor of Human CFTR, Exhibits the epithelium. A large number of mutations in the protein are known to cause Numerous Biophysical Dissimilarities from Human CFTR CFTR to become dysfunctional and only a few pharmaceutical compounds Guiying Cui1, Brandon B. Stauffer1, Barry R. Imhoff1, Amit Gaggar2, have been developed to treat the disease by restoring the chloride conductance Nael A. McCarty1. of the channel (1). CFTR is activated by cAMP dependent phosphorylation and 1Pediatrics, Emory University, Atlanta, GA, USA, 2Division of Pulmonary, is gated by ATP. Activation is typically achieved using forskolin that activates Allergy & Critical Care Medicine, Department of Medicine, University of adenylate cyclase which then leads to phosphorylation of the channel via pro- Alabama at Birmingham, Brimingham, AL, USA. tein kinase A (PKA). Screening for CFTR activators is usually done in the Lampreys, an extant representative of the jawless vertebrates, diverged from absence of forskolin, but in the presence of low concentrations of cAMP. jawed vertebrates approximately 550 million years ago and possess a unique Studies in animal models support the development of CFTR inhibitors for anti- CFTR ortholog. Lamprey CFTR (Lp-CFTR) shares 46% sequence identity secretory therapy of enterotoxin-mediated diarrheas and polycystic kidney dis- and 65% sequence similarity with human CFTR (hCFTR). The biophysical ease. For this purpose an alternative approach can be used. In the presence of consequences of this dissimilarity remain unknown so we investigated the internal fluoride CFTR is activated as to the same degree as after forskolin acti- channel behavior and pharmacology of Lp-CFTR expressed in Xenopus vation. We here present data from Nanion’s SyncroPatch 384PE where we acti- oocytes. Like hCFTR, Lp-CFTR channel activity was PKA-dependent with vate the channel with either forskolin under fluoride free conditions or by whole cell currents stimulated by forskolin and IBMX, and excised macro- internal solution exchange where the fluoride is washed into the intracellular patch currents activated by intracellular exposure to MgATP and PKA. solution. Our results show that the activation of CFTR by either forskolin or Two blockers of hCFTR, NPPB and glibenclamide, blocked Lp-CFTR activ- intracellular F- is sensitive to glibenclamide and CFTRInh-172 in a dose - ity in a manner comparable to hCFTR. Surprisingly, GlyH-101 failed to block and/or voltage dependent manner. Taken together, these experiments show a Lp-CFTR at Vm = 60 mV and CFTRinh172 exhibited modest inhibition of stable and cost optimized approach to study the pharmacology of CFTR at Lp-CFTR in both the whole cell (TEVC) and inside-out macropatch configu- high throughput that might empowers new ways in the drug discovery on ration. The sole clinical hCFTR potentiator VX-770 (KalydecoTM) mildly CFTR. potentiated hCFTR in the presence of MgATP þ PKA, recorded with the inside-out macropatch technique. However, Lp-CFTR was significantly in- 2026-Pos Board B346 hibited by VX-770 under the same experimental conditions. Furthermore, Discovery of NAV1.7 Modulators from Marketed Drugs using High Lp-CFTR exhibited significant inward rectification, the most among all throuthput Automated Electrophysiological System Ionworks Barracuda the CFTR species tested thus far, when it was recorded in symmetrical Xueqin Chen, Sui Fang, Qiang Ding, Zhaobing Gao. 150 mM Cl- in inside-out macropatches. The single channel behavior of Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Lp-CFTR was also unique. Whereas open hCFTR channels occupy the full Shanghai, China. open state (f) over 98% of the time, Lp-CFTR randomly occupies the f, the The voltage-gated sodium channel Nav1.7 is a major contributor to pain subconductance 1 (s1), or subconductance 2 (s2) states. In summary, Lp- signaling in humans and a subject of extensive research programs as an impor- CFTR has unique pharmacological and biophysical characteristics compared tant target for developing anti-pain drugs. Finding good drug leads de novo to hCFTR. The differences may provide a tool for identifying the binding sites from large chemical libraries, real or virtual, is not an easy task. Especially and working mechanism of VX-770 and may shed light on understanding the for an ion channel drug target, using indirect high-throughput screening evolution of CFTR. (NIH R01-DK 056481). such as fluorescence-based assay is often plagued by a significant level of false positives, low hit rates and many leads that are toxic or exhibit poor 2024-Pos Board B344 bioavailability. Exploiting the secondary activity of marketed drugs, on the Small Molecule Induced Changes in the Trafficking of the Cystic Fibrosis other hand, may help in generating drug leads that can be optimized for Conductance Regulator the observed side-effect target, while maintaining acceptable bioavailability Zhihui Zhang, David Heidary, Chris Richards. and toxicity profiles. Here, we described an efficient high throughput electro- Chemistry, University of Kentucky, Lexington, KY, USA. physiology assay to discover hits to the Nav1.7 target from safe marketed Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane drugs. We screened a marketed drugs library(US Drug Collection 1280 drugs) conductance regulator (CFTR). Deletion of Phe508 (F508), the most prevalent using IonWorks Barracuda(IWB) platform(population patch clamp mode, mutation in CFTR, results in the failure of CFTR trafficking to plasma mem- PPC), the second generation of IonWorks instruments. Two different voltage brane. My work focused on understanding changes in the trafficking of F508 protocols were used to compare compounds inhibition between resting and CFTR to the plasma membrane due to the correction of the small molecule inactivation state of the channel. We tested throughput, reproducibility and VX809, low temperature (30C ) and second site mutations (I539T). Real uniformity of the Nav1.7/IWB assay and demonstrated that the assay had time total internal reflection fluorescence (TIRF) images were taken to identify met standard industry acceptance criteria as Z0 factor, signal window and whether correctors increased surface expression through increased trafficking inter-plate reproducibility of IC50 values for positive controls. Results showed or membrane turnover. The pH sensitive fluorescence protein, super ecliptic that 41 drugs has an inhibition percentage of more than 80% on Nav1.7 phluorin, was tagged to CFTR to quantify plasma membrane expression. The channel, some of them are anesthetic such as Tetracaine, Benoxinate and second site mutation I539T and low temperature alone increase F508 CFTR Diperodon.

BPJ 7855_7859 412a Tuesday, February 14, 2017

2027-Pos Board B347 TMYX is a traditional Chinese Medicine drug used to control cardiac rate in Structure-Based Discovery of Potential Two-Pore-Domain Potassium arrhythmic patients. The efficacy and safety of this substance is proved by Channel Task-3 (K2P9.1) Modulators its clinical use, but the molecular mechanism of action is unknown. TMYX David Ramı´rez1, Julio Caballero1, Leandro Zu´n˜iga2,Ba´rbara Are´valo1, (2 mg/ml) reduces spontaneous rate (21.950.8%, n=11) of pacemaker car- Aytug Kiper3, Susanne Rinne3, Niels Decher3, Wendy Gonzalez1. diomyocytes due to a selective reduction of the diastolic depolarization slope 1 Centro de Bioinforma´tica y Simulacio´n Molecular, Universidad de Talca, (49.353.6%, n=11) caused by a leftward shift of the activation curve of If Talca, Chile, 2Centro de Investigaciones Me´dicas (CIM), Escuela de (7.150.6, n=3) and associated current inhibition. Since a similar shift is 3 Medicina, Universidad de Talca, Talca, Chile, Institute for Physiology and typical of the muscarinic modulation of the If current, we tested for a possible Pathophysiology, Vegetative Physiology, Philipps-University of Marburg, functional interaction between ACh and TMYX. When tested alone both drugs Marburg, Germany. induced hyperpolarizing shifts of the If activation curve (ACh 1 mM: The two-pore domain potassium (K2P) channel TASK-3 is a highly expressed 9.551.4 mV; TMYX, 6 mg/ml: 6.850.6 mV). However, when TMYX in hippocampus, cerebellum and cortical areas of the brain. Besides regulating was delivered in the presence of ACh, no additional shift was observed neurotransmitter functions, TASK-3 has an oncogenic potential and it is over- (n=3). This suggests that the modulatory pathways activated by TMYX and expressed in different types of cancer cells, for this reason the development of ACh converge; however, the action of TMYX is not abolished by atropine new selective TASK-3 modulators could influence the pharmacological treat- (5.150.3, TMYX 6 mg/ml; 5.450.7, TMYX 6 mg/ml þ atropine 1mM), ment of cancer and several neurological disorder. In the present work we indicating that it must occur downstream the muscarinic receptor. Interest- have searched for potential TASK-3 modulators using a virtual screening ingly, the effect of TMYX is abolished when intracellular cAMP is buffered (VS) protocol that includes pharmacophore modeling, molecular docking, to a saturating concentration (100 mM, n=3), further suggesting that the and free energy calculations (MM/GBSA). At the end, 18 hits were identified, cAMP level is the final effector of TMYX action. To recapitulate the bradycar- these hits were screened against TASK-3 using Fluorometric Imaging Plate dic effect of TMYX observed in vitro, we tested the effect of TMYX on the Reader - Membrane potential assay (FLIPR-MPA), and two lead ligands intrinsic heart rate of pharmacologically denervated freely-moving mice. showing IC50s of about 40 mM were obtained as potential modulators. Under these conditions TMYX induced a long-lasting bradycardic effect (50.2% at the peak of effect, n=4). We conclude that TMYX causes intrinsic 2028-Pos Board B348 heart rate bradycardia by acting on the If current, and preliminary experiments Pharmacological Characterization of the NMDA-B-C in Parallel by Auto- in inside-out configuration suggest that TMYX could act by directly binding to mated Patch Clamp the internal part of f-channels. Ilka Rinke1, Søren Friis2, Claudia Haarmann1, Alison Obergrussberger1, Michael George1, Andrea Bruggemann€ 1, Niels Fertig1. 2030-Pos Board B350 1Nanion Technologies, Munich, Germany, 2Nanion Technologies, Computational Investigations of KATP Channel Pore Blockers Copenhagen, Denmark. Xingyu Chen, Eva-Maria Zangerl-Plessl, Anna Stary-Weinzinger. N-Methyl-D-Aspartate receptors (NMDAR) belong to the family of ionotropic Pharmacology and Toxicology, University of Vienna, Vienna, Austria. glutamate receptors and are one of the key players in basic and complex excit- Cantu´ syndrome is a rare disease affecting a small number of people world- atory neurotransmission in the mammalian CNS. Seven subunits of NMDAR wide. Characteristic disease features are distinctive facial features, hypertri- have been identified: NR1, NR2A-D and NR3A-B1. Assembled as a tetramer1, chosis, cardiac abnormalities and lymphedema. This genetic disorder is they consist of two NR1 subunits and either two NR2 subunits or a combination caused by dominant gain-of-function mutations in the ATP-dependent potas- of NR2 and NR3 subunits2. sium inward rectifier channel, KATP, that prevent channel closure. These Unique properties of NMDAR are the activation of the channel by simultaneous channels consist of a sulfonylurea transporter subunit (encoded by the binding of glutamate and glycine, a voltage-dependent block by magnesium ABCC9 gene), and the pore-forming KIR6.1 subunit (encoded by the ions, a relatively slow current kinetic and a predominantly calcium carried KCNJ8 gene). The aim of this study is to find selective KATP channel blocker conductance. The exceptional calcium entry through open NMDARs triggers using a combination of in silico methods including molecular dynamics sim- on the one hand a number of regulatory and modulatory processes important ulations, docking and pharmacophore modeling. Since no crystal structure of in development and synaptic plasticity, which also involves weakening and this channel is available, we build homology models based on KIR3.2 crystal strengthen neuronal connections during learning and memory3. On the other structures in different gating states. As a first drug molecules, we study PNU- hand NMDARs are proposed to play a pathophysiological role in a number of 37883A, a highly selective inhibitor of vascular smooth muscle KATP chan- neurological disorders such as epilepsy and Alzheimer’s3. Indeed, memantine nels and rosiglitazone, an anti-diabetic drug from the glitazone class. Our ms is an NMDA antagonist which has been approved for the treatment of moderate long MD simulations reveal putative binding sites for both compounds, which to severe Alzheimer’s1. Given the importance in the treatment of such neurolog- are also in agreement with ligand based pharmacophore models. From these ical diseases and also of neuropathic pain, major depression and Parkinson’s dis- studies we expect a better understanding of the function of the KATP channel ease1, it is of great interest developing clinically relevant NMDAR antagonists and improved possibilities for drug development, facilitated by in depth that would block excitotoxic NMDAR activation, without interfering with knowledge of the binding site. Further, this research will serve as basis for NMDAR function needed for normal synaptic transmission and plasticity. compound optimization. This study focuses on the basic biophysical properties of different NMDAR subtypes and the pharmacological relevance of modulation and antagonism 2031-Pos Board B351 of NMDARs. Among others, we show the activation kinetics of the NR2A, Rational Design of KCa2 Channel Activators NR2B, NR2C and NR2D subunit containing NMDARs. Moreover, we present Heesung Shim1, Brandon Brown2, Hai M. Nguyen2, data from the target screen of the positive allosteric modulator CIQ and subtype Vladimir Yarov-Yarovoy3, Heike Wulff2. 1UC Davis, Davis, CA, USA, 2Pharmacology, UC Davis, Davis, CA, USA, selective blockers such as Ifenprodil which were recorded with high 3 throughput. All experiments were performed on the SyncroPatch384PE, the Physiology and Membrane Biology, UC Davis, Davis, CA, USA. Small-conductance (KCa2) and intermediate-conductance (KCa3.1) calcium- only APC device, which is able to stack the solutions inside a pipette and þ rapidly apply it to the cell, allowing a brief and accurate solution exchange activated K channels are voltage-independent and share a common calcium/ (<10ms) and exposure times (<200ms). With that procedure, we could reliable calmodulin mediated gating mechanism. Existing positive gating modulators activate slowly acting NMDARs, repetitively. We found a clear difference in like EBIO, NS309 or SKA-31 activate both KCa2 and KCa3.1 channels with the activation kinetics and blocking effects among the different receptor sub- similar potency. We previously performed a structure activity relationship types. The experimental layout of the 384 well plates allow the target and (SAR) study with the aim of optimizing the benzothiazole pharmacophore of counter screen to be done in parallel. SKA-31 towards KCa3.1 selectivity and have identified SKA-121 (5-methyl- naphtho[2,1-d]oxazol-2-amine), which displays 41-fold selectivity for KCa3.1 2029-Pos Board B349 (EC50 109 nM 5 14 nM) over KCa2.3 (EC50 4.4 5 1.6 mM). In order to under- A Traditional Chinese Medicine Drug (TMYX) Controls Heart Rate by stand why 2-amino benzothiazole/oxazol system could achieve such a signifi- Modulation of the Pacemaker (F) Channels cant gain in selectivity for KCa3.1 over KCa2.3 we are currently using the 1 1 1 1 Manuel Paina , Chiara Piantoni , Luca Carnevali , Anthony Frosio , Rosetta Ligand method and the structure of the KCa2.2 CaM-BD/CaM in com- 1 1 2 3 3 Annalisa Bucchi , Andrea Barbuti , Liu Sheng , Wang Yanyan , Yi Wang , plex with EBIO to generate models of SKA-121 binding to both the KCa3.1 and 1 1 Dario DiFrancesco , Mirko Baruscotti . KCa2.3 C-terminal CaM-BD/CaM and are mutating channel residues suggested 1Biosciences, University of Milan, Milano, Italy, 2Department of Physiology to be within 4A˚ of SKA-121. We made mutations to confirm the model and we 3 and Pathophysiology, Tianjin Medical University, Tianjin, China, Tianjin found that the NH2 group of SKA-121 forms hydrogen bonds with M51 and University of Traditional Medicine, Tianjin, China. E54 on calmodulin. E54 is part of a hydrogen bond network including R362,

BPJ 7855_7859 Tuesday, February 14, 2017 413a

E295 and N300 of the KCa3.1 channel. We hypothesize that it is this hydrogen 2034-Pos Board B354 bond network that is responsible for the selectivity of SKA-121 for KCa3.1 over Expression and Characterization of Wildtype and Mutant Forms of Xenopus KCa2. Based on this study, we are starting to design new KCa2 selective acti- Human Connexin31 using Oocytes vators and are attempting to predict whether the compounds would show selec- Rasheed Bailey, Husain Bneed, Dawn Fernandez, Shelby Rarick, tivity for KCa2 over KCa3.1 channels using Rosettaligand docking. We are Shahd Kadhim, Martha Skerrett. proposing to disrupt the hydrogen bond between the -NH2 group of the Biology, Buffalo State College, Buffalo, NY, USA. benzothiazole ring, and the CaM M51 and E54 residues, which are present in Connexin31 is a gap junction protein encoded by the GJB3 gene in humans. both the KCa3.1 and KCa2.3 models of SKA-121, and to add various substit- Mutations in the gene lead to a rare skin disorder known as erythrokeratoder- uents on new lead compound. Supported by U54NS079202 and U01 HL122202 mia variabilis (EKV) a disease characterized by figurate red patches and gen- from NIH. eral hyperkeratosis. While a few mutations have also been linked to deafness and neuropathy, over twenty mutations, mostly point mutations within the 2032-Pos Board B352 coding region, have been associated with EKV. Oocytes from Xenopus laevis Investigation of the Ion Channels TMEM16A and TRPC5 and their were used for expression and analysis of Cx31 and mutations. As far as we Modulation by Intracellular Calcium know this is the first time Cx31 has been expressed and characterized in Nina Brinkwirth1, Søren Friis1, Tom Goetze1, Markus Rapedius1, oocytes. Wildtype Cx31 displayed gating properties similar to those reported James Costantin2, Andrea Bruggemann€ 1, Michael George1, Niels Fertig1. after expression in mammalian cells but with some interesting differences. 1Nanion Technologies, Munich, Germany, 2Nanion Technologies, Several skin disease mutations induced leaky membranes associated with Livingston, NJ, USA. cell death, an effect that was both time- and concentration-dependent. It TMEM16A/Anoctamin1 and TRPC5 are ion channels activated by elevated was hypothesized that aberrant hemichannel behavior was responsible for cytosolic calcium concentrations and as both fulfill central physiological func- leaky membranes, which was supported by the observation that addition of tions their electrophysiological characterization is of great interest in the phar- calcium or cobalt to the extracellular media enhanced viability. Other muta- maceutical industry. tions induced behavior ranging from failure to form functional channels to As a calcium-activated chloride channel, TMEM16A has a broad functional expression and gating similar to wtCx31. As well as providing insight into spectrum in processes like trans-epithelial ion transport, olfaction, photo- the mechanisms underlying skin disease, deafness and neuropathy, we will transduction, smooth muscle contraction, nociception, cell proliferation and discuss the role of Cx31 mutants as tools to assess connexin interactions in control of neuronal excitability. TRPC5 on the other hand is a calcium- skin. permeable cation channel predominantly expressed in neuronal cells, but also 2035-Pos Board B355 in the kidney and the cardiovascular system. It plays an important role in cal- Modulation of Gaba(A) Receptors by General Anesthetics: A Combina- cium flux and its biological function ranges from neurotransmission and control toric Model Predicts Twenty Years of Concentration-Response Data of axon guidance to vascular smooth muscle cell migration and contractility. (With a Few Interesting Exceptions) Changing the intracellular calcium concentration to initiate channel activity Grace Brannigan. during the course of a patch clamp experiment is usually challenging and there- Physics and Center for Computational and Integrative Biology, Rutgers fore often done in inside-out Patches. The current amplitude can limit the use of University - Camden, Camden, NJ, USA. this method as well as run down that often is more pronounced in excised The gamma-aminobutyric acid receptor (GABAAr) is one of the most well- patches. studied targets for general anesthetics. General anesthetics such as propofol Here we present data of whole cell measurements where the intracellular solu- or sevoflurane potentiate the GABA(A)r at clinical concentrations, but tion is exchanged in a very robust manner. At the same time the extracellular concentration-response curves are frequently complex and even non- solution is still accessible for compound application. The 384-well format of monotonic. These are often fit with Hill functions with non-unity Hill coeffi- the patch clamp platform allows measurements in a highly parallel manner. cients, which have no well-defined physical meaning. Likewise, multi-site This allows the measurement of your target cell lines and controls to be per- mechanisms have complicated efforts to detect binding sites, although formed in one run. recent photoaffinity labeling studies have consistently identified a few areas The data shown will summarize our results on Calcium sensitivity and pharma- of the GABA(A)r as potential binding regions, particularly intersubunit sites cological modulation. at the subunit interfaces. Rigorous physics-based binding affinity calculations carried out by my research group repeatedly indicated that the dissociation 2033-Pos Board B353 constant for the second highest affinity site was closest to the experimental Structural Dynamics of the MSCL C-Terminal Domain EC50, for several distinct general anesthetics, including propofol and 1 2 3 4 Adam D. Martinac , Navid Bavi , Marien D. Cortes , Omid Bavi , sevoflurane. 5 2 6 Takeshi Nomura , Boris Martinac , Eduardo Perozo . Here I present a discrete, combinatoric model inspired by that observation, 1University of Queensland, Brisbane, Australia, 2Victor Chang Cardiac 3 which uses computed binding affinities and requires that any two intersubunit Research Institute, Sydney, Australia, Department of Cell Physiology, sites must be bound for potentiation to occur. I will compare the resulting pre- Texas Tech University Health Sciences Center, Lubbock, TX, USA, dictions for propofol and sevoflurane concentration-response at various 4Institute for Nanoscience and Nanotechnology, Sharif University of 5 GABA concentrations, including additive effects of both propofol and sevo- Technology, Tehran, Iran, Islamic Republic of, Department of Molecular flurane, to experimental data from numerous research groups using a range Cell Physiology and Bio-Ionomics, Prefectural University of Medicine, 6 of GABAAr subunits and cell lines. Most predicted concentration-response Kyoto, Japan, Department of Pediatrics, Institute of Molecular Pediatric curves are in excellent agreement with experimental potentiation data, without Sciences, University of Chicago, Brisbane, IL, USA. requiring any empirical fit parameters. Interesting exceptions and possible The large conductance mechanosensitive channel (MscL), acts as an osmopro- origins of the discrepancies will also be presented, as well as potential impli- tective emergency valve in bacteria that gates in response to membrane ten- ˚ cations for future interpretation of GABA and modulator concentration- sion to open a large, water-filled pore of ~ 30 A in diameter. In its closed response data. configuration, the last 36 residues at the C-terminus form a bundle of five a-helices co-linear with the five-fold axis of symmetry. However, the physi- 2036-Pos Board B356 ological relevance of this bundle has been put in doubt given the unorthodox Subtype Selectivity of Tets on GABA-A Receptors crystallographic conditions (pH 3.6-3.8). To resolve the controversy, we Brandon Pressly, Hai M. Nguyen, Heike Wulff. examined in this study the structural dynamics of the C-terminus of EcMscL Pharmacology, UC Davis, Davis, CA, USA. using side-directed spin labelling electron paramagnetic resonance (SDSL Tetramethylenedisulfotetramine (TETS), is a potent convulsant that is listed EPR) spectroscopy. To complement the experimental results, we have also as a possible threat agent by the Department of Homeland Security. TETS employed molecular dynamics (MD) simulations and finite element (FE) is an ‘‘ideal’’ poison due to its relatively high solubility in water, difficulty modelling. Our data show that under physiological conditions, the C-terminus of detection and low LD50 of 0.1 mg/kg. The mechanism of action for is indeed an a-helical bundle, located near the five-fold symmetry axis of the TETS is thought to be inhibition of GABA-A receptors. However, in previ- molecule. Both experiments and computational modelling demonstrate that ously performed electrophysiological experiments TETS has been shown to only the top part of the C-terminal domain (from the residue A110 to block GABA-A receptors at surprisingly high concentrations of roughly E118) dissociates during channel gating, while the rest of the C-terminus stays 20 mM and has not been found to be very potent in displacing standard intact. This suggests the C-terminus role as a molecular sieve and stabilizer of GABA-A receptor ligands in binding assays. Yet, [14C]-TETS displaces the oligomeric MscL structure. cold TETS at 80 nM in rat brain synaptosomes, suggesting that there is a

BPJ 7855_7859 414a Tuesday, February 14, 2017 higher affinity binding site. In order to identify whether TETS exhibits sub- 2039-Pos Board B359 type selectivity for a particular GABA-A receptor combination we are using Biophysical Insight into the Substrate Permeation through the Major Acinetobacter baumannii whole-cell patch-clamp to determine the IC50 of TETS for the major synaptic Outer Membrane Channels of and extrasynaptic GABA-A receptors associated with convulsant activity. The Satya Prathyusha Bhamidimarri1, Michael Zahn2, active component of picrotoxin, picrotoxinin, is serving as a standard control. Jigneshkumar Dahyabhai Prajapati1, Ulrich Kleinekathoefer3, While picrotoxinin in our hands shows some preference for b2 containing Bert van den Berg2, Mathias Winterhalter1. 1 GABA-A receptors (IC50 4.5 mM for a1b2g2L; IC50 > 30 mM for Department of Life Sciences and Chemistry, Jacobs University Bremen, 2 a1b1g2L), TETS so far exhibited the highest activity on a2b3g2L (IC50 Bremen, Germany, Institute for Cell and Molecular Biosciences, Newcastle 3 480 nM). TETS showed lower activity on a6b3g2L (IC50 900 nM), a4b3d University, Newcastle, United Kingdom, Department of Physics, Jacobs (IC50 4.4 mM), and a1b2g2L (IC50 13 mM), while a1b1g2L was insensitive University Bremen, Bremen, Germany. (> 30 mM). Overall, TETS therefore seems to preferentially affect a2 and Among other mechanisms the cause for Multidrug Resistant (MDR) bacteria is b3 containing GABA-A receptor combinations. Further testing of additional their reduced permeability for antibiotics in particular in Gram-negative bacte- GABA-A subtypes will provide a clearer understanding of the molecular ria such as Acinetobacter baumanii. Owing to the low permeability (100 fold mechanism of TETS activity and should aid with the identification of a spe- less compared to that of E. coli) and genetic plasticity to adopt to the external cific countermeasure against TETS intoxication. Supported by U54NS079202 environment accompanied with robust efflux pumps aids these bugs in limiting and T32GM099608 from NIH. the intracellular active concentration of the antibiotic to minimum. Here we characterize transport of small water soluble molecules across channels in 2037-Pos Board B357 the outer membrane (OM) of A. baumanii. We use Single Channel Electrophys- Analysis of Partial and Complete Block of Amantadine and Rimantadine iology as a main tool for the biophysical characterization of the majorly ex- in Influenza a M2 S31N by Electrophysiology and Curve Fitting Methods pressed channels from A. baumanii. Combining our study with high Kelly L. McGuire, David D. Busath. resolution X- ray crystallography and molecular dynamics simulations, we pro- PDBio, BYU, Provo, UT, USA. vide insight into the OM of A. baumanii. The mutation from serine to asparagine at residue 31 of M2, which is found in [1] Zahn M, Bhamidimarri SP, Basle A, Winterhalter M, van den Berg B. 2016. the majority of influenza strains circulating in humans, inhibits amantadine þ Structure 24:221-31 (ADA-NH3 ) block. But, from simulations it is unclear whether the inhibition results from weak binding or incomplete block. We addressed the question of 2040-Pos Board B360 whether there is aminoadamantane ‘‘binding without block’’ of proton conduc- Three-Step Gating Triggered by Conformational Changes of the Motor tance experimentally. Two-electrode voltage clamp (TEVC) was used to mea- Channel Common to Viral DNA Packaging Motor of T3, T4, Spp1, and sure aminoadamantane block of M2 WT or S31N proton conductance at Phi29 unusually high drug concentrations. Curve fitting methods were used to obtain Zhouxiang Ji, Shaoying Wang, Farzin Haque, Peixuan Guo. rate constants for both the onset and relief of block. Results showed that the College of Pharmacy and College of Medicine, The Ohio State University, S31N M2 channel is completely blocked at 10 mM, proving that there is no Columbus, OH, USA. ’’binding without block‘‘ for amantadine in the influenza M2 channel. Both It was reported recently that bacterial virus Phi29 utilizes the ‘‘one-way the on and off rate constants for amantadine are significantly increased in the revolving’’ mechanism for genomic DNA packaging through a portal channel. mutant M2 as compared to the WT M2. In contrast rimantadine (ADA- If the channel acts as a one-way inward valve, the question as to how dsDNA þ CH(NH3 )-CH3), which blocks the wild-type M2 S31 channel better than ejects during infection, however, is still a puzzle. Nanopore technology opens amantadine, achieves a modest maximal level of block in the S31N M2 channel a new door to the study of the conformational change of DNA and protein. at 1 mM, with no further increase in block at 10 mM drug. The on and off rate Herein, we reported an electrophysiological approach of studying protein constants for rimantadine are significantly increased in the mutant M2 as conformational changes by inserting the portal protein channels into the lipid compared to the WT M2. We conclude that rimantadine, but not amantadine, membrane. The conductance measurement showed that portal proteins binds without block in the S31N M2 channel. These findings can further including bacterial viruses T3, T4, SPP1, and Phi29 displayed a three-step constrain simulations designed to evaluate the mechanism of reduced M2 channel gating induced by high voltage. Each step of robust channel gating S31N block. caused current reductions of 32%, 64% and 96% after the clean signal of three discrete steps. Our results suggested that the channel of the viral DNA packaging motor transits from open during DNA packaging to closed Other Channels I after DNA packaging by gating and prepares for a new conformation for the dsDNA to exit during host infection. 2038-Pos Board B358 Reference: Voltage- and Current Clamp on Induced Pluripotent Cardiomyocytes with 1.Wang, S., Ji, Z., Yan, E., Haque, F., & Guo, P. (2016). Three-step channel Automated Patch Clamp conformational changes common to DNA packaging motors of bacterial vi- Mads P.G. Korsgaard. ruses T3, T4, SPP1, and Phi29. Sophion Bioscience, Ballerup, Denmark. 2.Wendell, D., Jing, P., Geng, J., Subramaniam, V., Lee, T. J., Montemagno, Induced stem cell-derived cardiomyocytes (iPS CM) have many of the pheno- C., & Guo, P. (2009). Translocation of double-stranded DNA through typic properties of authentic cardiomyocytes and great interest has been shown membrane-adapted phi29 motor protein nanopores. Nature nanotechnology, in their possibilities for modeling human diseases. For drug discovery iPS CM 4: 765. provide an exciting new tool for e.g. cardiac target discovery and cardiac safety 3.Guo, P., Noji, H., Yengo, C. M., Zhao, Z., & Grainge, I. (2016). Biological studies. IPS Cardiomyocytes were biophysically and pharmacologically char- Nanomotors with a Revolution, Linear, or Rotation Motion Mechanism. Micro- acterized with planar automated patch clamp. Here we present the results ob- biology and Molecular Biology Reviews, 80:61. tained during assay optimization of the cell culture, assay set-up, as well as biophysical and pharmacological validation of the cardiac currents. We present 2041-Pos Board B361 how iPS Cardiomyocytes performs in voltage as well as current clamp assays. Probing the Ion Permeability of the Bacterial Translocon with a Locked We demonstrate how action potentials can be induced by using mixed voltage/ Translocation Intermediate at the Single Molecule Level current protocols and how the modulation of these with pharmacology can be Denis G. Knyazev, Roland Kuttner, Mirjam Zimmermann, Christine Siligan, detected and analyzed using Sophion analysis software. Implementing optimal Peter Pohl. cell culture routines, the cardiomyocytes demonstrated induced rhythmical cur- Institute of Biophysics, Johannes Kepler University, Linz, Austria. rents indicating functional properties of adult cardiomyocytes. Three different The bacterial translocon SecYEG is responsible for membrane insertion or the types of currents were studied including sodium, calcium and potassium. By translocation of most of membrane and secretory proteins. The idle translocon using specific buffer solutions and changing the voltage protocols it was is sealed to ions by a hydrophobic ring of six isoleucines in its middle and a possible to characterize calcium, and sodium currents from the same cell. short helix, the so-called plug (1). Insertion of a polypeptide chain dislocates Our study showed that iPS Cardiomyocytes successfully can be applied to the plug. By engulfing the chain in a gasket-like manner, the ring is thought APC systems. The recorded currents are similar to adult human cardiomyo- to keep the barrier to ions (2). When testing this hypothesis, we found that cytes and the response to known pharmacology is as expected. Using auto- the complex of SecYEG and a stalled translocation intermediate possesses mated patch clamp we believe that iPS Cardiomyocytes have great potential ion channel activity (3). Physiological values of the membrane potential Dc for safety screening and other cardiovascular investigations early in the drug reduced the conductivity, suggesting that the translocation intermediate either discovery process. moved out of the lateral gate into the lipid phase or slid back into the donating

BPJ 7855_7859 Tuesday, February 14, 2017 415a compartment, so that the plug and pore ring could again seal the channel. Here VSD in different conformations. In the accompanying abstract (Kennedy, we prevented the intermediate from either backsliding or exiting through the Zhao, et al.), we describe isolation of C5 and C6 from ~1 million member lateral gate by (i) stalling a highly charged helix, the voltage sensor domain phage display library constructed on an inhibitor cysteine knot (ICK) scaffold, S4 of the potassium channel KvAP in the SecYEG pore and (ii) locking it be- a backbone found in many gating modifier neurotoxins. As expected, the S3-S4 tween the ribosome on one side of the membrane and calmodulin on the other loop of hHv1 was found to be the major binding epitope for C6 toxin. Whereas, side. Dc was unable to gate this complex. We observed several conductivity ci-Hv1 channels from sea squirt were insensitive to 1 mM C6, transplanting the levels, suggesting that the S4 helix was free to sample between the aqueous hHv1 S3b-S4a motif to ci-Hv1 conferred the chimera with sensitivity (maximal environment of the pore and the lipid interior. This observation (i) confirms blockade 70%; half maximal inhibition at 35 nM); the hHv1 motif was also the general view of how the translocon distinguishes membrane proteins able to confer C6 blockade to otherwise insensitive Kv2.1. Scanning the motif from secretory proteins and (ii) disproves the hypothesis that the pore ring in hHv1 by Cys scanning across 12 residues revealed four sites that decreased acts to maintain the membrane barrier in an active translocon. The work was affinity of C6 over 3-fold, one site that increased inhibition 75-fold (and per- supported by a grant from the Austrian science fund to Peter Pohl (P28213). mits 100% inhibition), and one site that allows C6 binding to activate the chan- 1. Saparov SM, et al. (2007) Determining the conductance of the SecY protein nel by 30% (half maximal activation at 250 nM). Consistent with a shared VSD translocation channel for small molecules. Mol Cell 26(4):501-509. 2. Park E & trapping mechanism, C6 inhibits whereas C5 activates, yet both modify the ki- Rapoport TA (2011) Preserving the membrane barrier for small molecules dur- netics of activation and deactivation as well as the voltage-dependence of ing bacterial protein translocation. Nature 473(7346):239-242. 3. Knyazev DG, gating, and while they are 39 and 41 residues, respectively, the C-terminal Winter L, Bauer BW, Siligan C, & Pohl P (2014) Ion conductivity of the bac- 31 residues are identical. A tethered-toxin strategy has been employed to terial translocation channel SecYEG engaged in translocation. J Biol Chem map the C6 interaction surface with hHv1 as a prelude to designing more 289(35):24611-24616. potent inhibitors and activators; ten important residues that are charged or polar have been identified. 2042-Pos Board B362 A Designer Peptide Toxin Isolated by Phage Display that Inhibits the 2044-Pos Board B364 Human Voltage-Gated Proton Channel, hHv1 HCN3 Channel Expression in Human Leukocytes Kelleigh Kennedy1, Ruiming Zhao1, Qufei Li2, Eduardo Perozo2, Chiara Piantoni1, Angelica Gualdoni1, Claudia Bazzini1, Gerardo A. De Blas3, Martin A. Pavarotti3, Rodolfo Arias3, Luis S. Mayorga3, Raffaella Milanesi1, Miryam Adelfio1, Annalisa Bucchi1, Andrea Barbuti1, Steve A.N. Goldstein1. Matteo Pecchiari2, Mirko Baruscotti1, Dario DiFrancesco1. 1Brandeis University, Waltham, MA, USA, 2University of Chicago, Chicago, 1Department of Biosciences, Universita` degli Studi di Milano, Milan, Italy, IL, USA, 3National University of Cuyo, Mendoza, Argentina. 2Department of Pathophysiology and Transplantation, Universita` degli Studi hHv1 is critical to immune defense, maintaining pH homeostasis in white di Milano, Milan, Italy. blood cells by extruding protons during respiratory burst to compensate for Ivabradine is a heart rate-reducing agent, which specifically and selectively reactive oxygen species (ROS) production, and during sperm capacitation blocks pacemaker HCN channels. Strong evidence shows that Ivabradine causing cytosolic alkalization. Hindering basic and clinical research, hHv1 is also reduces inflammatory processes and the suggested mechanism of an orphan receptor without a potent and specific blocker. hHv1 is homodi- action is the modification of the vascular shear stress caused by the pri- meric; each subunit has a conduction pathway and is homologous to the four mary bradycardic effect of the drug (Li et al., 2008 Plos one 11(2): membrane-spanning segments that form the voltage sensing domains (VSD) e0149694.doi:10.1371). Leukocytes, the key players of the immune in KV and NaV channels. To produce a toxin ligand for hHv1, we employed response, express several ion channels (Kv1.3,KCa3.1,TRPM4,ORAI1and a phage display strategy whereby ~1 million novel peptides were fabricatedon others) which control the functional state of the cell and are therefore an inhibitor cysteine knot (ICK) scaffold, a backbone stabilized by three disul- involved in cell activation and inflammatory processes. Since HCN channels fide bonds and found in nature to be rich in VSD-directed toxins. Designed by are important determinants of membrane potential in both excitable and combinatorial permutation of 110 venom toxins, phage sorting was performed non-excitable cell types, we investigated the presence of these channels in on purified recombinant hHv1 and specificity of binding validated by ELISA. leukocytes. Although qualitative PCR experiments revealed a positive Five novel peptides were identified (C2-C6), synthesized, and studied by amplification of all HCN mRNA isoforms (HCN1-2-3-4), preliminary quan- external application to hHv1 channels expressed in HEK293T cells. Proton titative PCR carried out in total human leukocytes showed a reliable signal currents measured by whole cell patch clamp were inhibited by C6. Consistent only for HCN3. We therefore proceeded with the investigation of HCN3 with VSD trapping, C6 slows activation, accelerates deactivation, and shifts channel expression in human leukocytes. Immunolabelling with commercial activation to more depolarized voltages. Inhibition by C6 is partial, showing anti-HCN3 antibody confirmed the presence of this isoform in human total at most 50% block at þ40 mV (1 mM) with a Ki of 150 nM. Partial block leukocytes. Flow cytometry analysis revealed that the major sources of was not due to partial occupancy of the dimer: two C6 were seen per dimer HCN3 signal appear to be granulocytes and lymphocytes. We finally tested by single particle photobleaching using C6 labeled with 5,6-TAMRA and the effect of Ivabradine (1 mM) on the activation process of human total leu- hHv1 tagged with TFP. Moreover, a monomeric hHv1 channel was also kocytes. The results show a 29% (n=5) reduction in the activation readout blocked by only 50% with a Ki of 110 nM and bound one fluorescent C6 pep- parameters in the presence of Ivabradine. These results raise the possibility tide. Of note, a point mutation in the channel epitope where C6 binds increased that the mechanism by which Ivabradine reduces inflammation is a direct toxin affinity and produced complete blockade (accompanying abstract by binding to HCN3 channels in human leukocytes and we therefore suggest Zhao, Kennedy et al). C6 was isolated on purified hHv1 protein but shown that HCN3 channels may play a functional role in specific human leukocytes to inhibit two cellular responses proposed to depend on native hHv1 function. subpopulations. Future electrophysiological experiments will further investi- Human whole blood cells stimulated by PMA release ROS and this was sup- gate this possibility. pressed in a dose dependent manner by C6 (IC50 100 nM). C6 at 20 mM also specifically suppressed the cytosolic calcium increase and acrosome reaction 2045-Pos Board B365 triggered by progesterone in human sperm. Both ROS generation and capaci- Silica Nanoparticles Induce Calcium-Permeable Pores in Plasma Mem- þ branes tation were insensitive to 10 or 20 mM of scorpion toxin blockers of Kv1.3 K channels. Alicia Sanchez, Kateryna Demydenko, Peter Hoet, Karel Talavera. KU Leuven, Leuven, Belgium. 2043-Pos Board B363 Amorphous silica nanoparticles (SiNPs) are extensively used for their benefi- Identification of Activating and Inhibiting Gating Modifier Toxins that cial properties in cosmetics and food industry as an additive and fining agent. Target the S3-S4 Loop of the Human Proton Channel, hHv1 However, the high levels of exposure have raised concerns about health haz- Ruiming Zhao1, Kelleigh Kennedy1, Qufei Li2, Eduardo Perozo2, ards, and it has been shown that SiNPs can penetrate tissues and cells resulting Steve A.N. Goldstein1. in health problems. In this study we first evaluated the effects of SiNPs on intra- 1 2 2þ 2þ Brandeis University, Waltham, MA, USA, University of Chicago, Chicago, cellular Ca concentration ([Ca ]i) in different cell types (HEK293, CHO IL, USA. and mouse airway epithelial cells). We found that extracellular application of 2þ Human Hv1 channels (hHv1) are implicated in a wide variety of cellular func- 9 nm SiNPs (LudoxÒ)at25 C produced a pronounced increase in [Ca ]i tions including reactive oxygen species production, sperm capacitation and cell when the cells were co-stimulated with arachidonic acid or LPS. These effects proliferation in cancer. Each channel has two hHv1 subunits that are homolo- were not observed when Ca2þ was omitted in the extracellular solution, indi- gous to the S1-S4 voltage sensing domains (VSD) in voltage-gated Kþ and cating that SiNPs induce a Ca2þ entry pathway through the plasma membrane. Naþ channels. The S3-S4 loop of VSD is a hot spot for interaction of known Similar results were obtained when cells were exposed to SiNPs and heating gating modifier toxins, which either activate or inhibit by stabilizing the from 25 to 35 C, suggesting that SiNPs are sufficient to induce the Ca2þ entry

BPJ 7855_7859 416a Tuesday, February 14, 2017 pathway at physiological temperatures. Whole-cell patch-clamp experiments 2. Zhou, et al & Guo, P. (2016). Oriented single directional insertion of nano- revealed that SiNPs trigger large currents that could be blocked by the channel of bacteriophage SPP1 DNA packaging motor into lipid bilayer via po- cation channel blocker ruthenium red in a voltage-dependent manner. Analysis lar hydrophobicity. Biomaterials, 105: 222. of the selectivity properties of this current showed that they are mainly 3. Wendell, et al & Guo, P. (2009). Translocation of double-stranded DNA carried by cations. Taken together, our results demonstrate that SiNPs through membrane-adapted phi29 motor protein nanopores. Nature nanotech- induce Ca2þ-permeable pores in the plasma membrane, and that this phenom- nology, 4:765. enon is enhanced by factors that increase membrane fluidity. We propose that 4. Haque, et al & Guo, P. (2012). Real-time sensing and discrimination of sin- this Ca2þ entry pathway may be relevant for the toxicological properties of gle chemicals using the channel of phi29 DNA packaging nanomotor. ACS SiNPs. nano, 6:3251.

2046-Pos Board B366 2049-Pos Board B369 How to Get Large Drugs through Small Pores? Exploiting the Porins Perturbation of Bilayer Surface Tension Differentially Modulates Mecha- Pathway in Pseudomonas Aeruginosa nosensitive Ion Channels Susruta Samanta1, Tommaso D’Agostino1, Ishan Ghai2, Monisha Pathania3, Navid Bavi1,2, Charles D. Cox1,2, Omid Bavi3, Boris Martinac1,2. Silvia Acosta Gutierrez1, Mariano Andrea Scorciapino1, Igor Bodrenko1, 1Victor Chang Cardiac Research Institute, Sydney, Australia, 2St. Vincent’s Richard Wagner2, Bert van den Berg3, Mathias Winterhalter2, Clinical School, University of New South Wales, Sydney, Australia, Matteo Ceccarelli1. 3Institute for Nanoscience and Nanotechnology, Sharif University of 1Physics, University of Cagliari, Monserrato, Italy, 2Jacobs University, Technology, Tehran, Iran, Islamic Republic of. Bremen, Germany, 3University of Newcastle, Newcastle upon Tyne, United The lipid composition of biological membranes modulates the activity of inte- Kingdom. gral membrane proteins (1). This is particularly important for mechanosensi- The main focus of our study is to identify the structural features responsible tive (MS) channels, regardless of their gating paradigm. Here we use the for the transport of molecules through substrate-specific channels of Gram surface active agent 2,2,2-trifluoroethanol (TFE) as a pharmacological tool negative bacterium Pseudomonas aeruginosa. We present a precise molecular to study the effect of surface tension perturbations on an array of bacterial analysis of the structure and dynamics of OccK8 to explain the translocation and mammalian MS channels including MscL, Piezo1 and TREK-1. We chose of natural amino acid residues and antibiotics of two different chemical TFE (a general anaesthetic) due to the fact that its effect on the bacterial chan- families through it. We used molecular dynamics simulations to obtain infor- nel MscS has already been studied. In particular TFE facilitates MscS activa- mation on the molecule/channel interactions and proposed quantitative tion from the periplasmic side, while it abolishes MscS current from the structure-function relationships based on them. We found some physical cytoplasmic side (2, 3). Here, we demonstrate that 2 % v/v TFE can also facil- chemical properties of molecules playing an important role in modulating itate the activation of: (i) MscL if TFE is added to either bilayer leaflet, (ii) the translocation through OccK8. Molecules with zwitterionic structures Piezo1 only if added to the cytoplasmic side and (iii) TREK-1 only from have an affinity towards the polar eyelet region and presence of negative the extracellular side. Our molecular dynamics simulations revealed TFE in- charge help interaction with the basic ladder facilitating translocation. A creases the surface tension and the first moment of the transbilayer pressure favorable chemical structure helps to widen the otherwise small constriction profile markedly and hence facilitates activation of MscL. Using our molecular region helping translocation of relatively large antibiotics. Whenever possible, dynamics, energetic analysis and collective experimental data, we postulate liposome swelling assays and electrophysiology at single-molecule level were there is a close relationship between MS channel shape and its activation used to confirm our data. mechanism by surface tension perturbations. The activation curve of MscL, which is a cylindrical protein, was shifted to the left (activated easier) upon 2047-Pos Board B367 addition of TFE from either side. MscS and TREK on the other hand, which Molecular Transport through Large Diameter DNA Origami Channels are conical, were only facilitated from the extracellular side. The activation Swati Krishnan, Friedrich Simmel. curve of Piezo1 was also shifted to left only when TFE was added to the cyto- Physics, TU munich, Munich, Germany. plasmic side. Given surface active drugs are adsorbed onto cell membranes, We introduce a large DNA membrane channel with a z 4 nm diameter pore, these findings provide a mechanistic understanding of their non-specific which spontaneously inserts into flat lipid bilayer membranes using hydropho- impact on the function of different membrane proteins, particularly MS ion bic functionalizations or streptavidin linkages between biotinylated channels channels. and lipids. Using a dye influx assay, we demonstrate formation of membrane 1. Cantor RS (1998) The lateral pressure profile in membranes: a physical pores in giant unilamellar vesicles. The versatility of DNA nanotechnology mechanism of general anesthesia. Toxicol Lett 100-101:451-458. makes these channels promising molecular devices for synthetic biology and 2. Akitake B, Spelbrink RE, Anishkin A, Killian JA, de Kruijff B, & Sukharev biosensing. S (2007) 2,2,2-Trifluoroethanol changes the transition kinetics and subunit in- teractions in the small bacterial mechanosensitive channel MscS. Biophys J 2048-Pos Board B368 92(8):2771-2784. Fingerprinting and Differentiation of Small Proteins with a Large Channel 3. Nomura T, Cox CD, Bavi N, Sokabe M, & Martinac B (2015) Unidirectional of Bacteriophage PHI29 DNA Packaging Motor incorporation of a bacterial mechanosensitive channel into liposomal mem- Zhouxiang Ji, Shaoying Wang, Zhengyi Zhao, Zhi Zhou, Farzin Haque, branes. FASEB J 29(10):4334-4345. Peixuan Guo. The Ohio State University, Columbus, OH, USA. 2050-Pos Board B370 Insertion of biological nanopores to the lipid membranes has inspired single Novel Properties of LRRC8-Mediated VRAC Currents molecule sensing of peptides and proteins. Most biological membrane nano- Antonella Gradogna1,He´ctor Gaita´n-Pen˜as2, Lara Laparra-Cuervo3, pores are around 1.2 nm, too small for the folded protein to pass through the Carles Solsona4,Vı´ctor Ferna´ndez-Duen˜as5, Alejandro Barrallo-Gimeno2, nanopore. A well-studied bacteriophage phi29 DNA packaging motor with a Francisco Ciruela6, Melike Lakadamyali3, Rau´l Este´vez2, Michael Pusch1. large channel has been inserted into the lipid membrane, served as the biosensor 1Istituto di Biofisica, Genoa, Italy, 2Unitat de Fisiologı´a, Departament de of nucleic acid and chemical. Herein, we reported that the large channel of Cie`ncies Fisiolo`giques II, IDIBELL-Universitat de Barcelona, Barcelona, bacteriophage phi29 packaging motor can be used for fingerprinting of peptides Spain, 3The Barcelona Institute of Science and Technology, ICFO-Institut de or small proteins. Different peptides can be distinguished well with the Cie`ncies Foto`niques, Barcelona, Spain, 4Laboratory of Neurobiology, blockage, dwell time or ionic signature, and the oligomeric states of peptides IDIBELL-Universitat de Barcelona, Barcelona, Spain, 5Unitat de can be investigated in real time. The translocation was confirmed by single Farmacologia, Departament Patologia i Terape`utica Experimenta, IDIBELL- molecule florescence imaging. Further, Ni-NTA nanogold binding assay Universitat de Barcelona, Barcelona, Spain, 6Unitat de Farmacologia, showed that peptides were translocated from the N-terminal to the C-terminal Departament Patologia i Terape`utica Experimental, IDIBELL-Universitat de of the channel. Single direction insertion of the nanopore into the channel was Barcelona, Barcelona, Spain. controlled by polar hydrophobicity of the N or C terminal. Our results demon- Heteromers of the LRRC8A protein with other LRRC8 members (B, C, strate the potential of this nanopore system for the detection of peptide bio- D, E) form the volume regulated anion channel (VRAC). We used the markers related to certain diseases. Xenopus oocyte system to study functional and structural properties of these Reference: channels. 8A/8C, 8A/8D, and 8A/8E heteromers gave rise to small volume 1. Ji, Z. et al & Guo, P. (2016). Fingerprinting of Peptides with a Large Channel stimulated currents. We discovered that adding fluorescent proteins to the of Bacteriophage Phi29 DNA Packaging Motor. Small, 12: 4572. C-terminus resulted in constitutive anion channel activity that was further

BPJ 7855_7859 Tuesday, February 14, 2017 417a stimulated by hypotonicity reaching currents larger than 10 microA. Using where OmpF/OmpC orthologs constitute the main influx routes for polar an- these constructs we investigated ion selectivity, osmosensitivity, single tibiotics, but also the intriguing Major Outer Membrane Porin (MOMP) of channel conductance, and carbenoxelone (CBX) sensitivity. A8/8D hetero- Campylobacter jejeuni for which its role was not clear, and some specific mers had significantly larger permeability for taurine and glycine compared channels from the Occ family from Pseudomonas Aeruginosa. The electro- to A/C and A/E heteromers. CBX was found to act from the extracellular static characterization of such porins suggests that the molecular dipole side, binding to probably more than one site. We further found that moment of the diffusing molecules modulates its permeablity. The electro- LRRC8 heteromers mediate glutamate and ATP flux from oocytes. Single static characterization of channels may have important implications for molecule photobleaching experiments suggested that the stoichiometry of modeling permeability inside gram-negative pathogen, allowing the explora- LRRC8 heteromers is variable, with a number of subunits R6andthat tion of the chemical space to find drugs with improved permeation properties the heteromer composition depends on the relative expression of different in rational drug designing. subunits. The system described here enables easy structure-function analysis of LRRC8 proteins. 2053-Pos Board B373 Hydrophobic Gating and Functional Annotation of Ion Channel Struc- 2051-Pos Board B371 tures by Molecular Dynamics Simulations Transport of Small Amino Acids by the Outer Membrane Protein OPRG 1,2 2,3 1,2 Pseudomonas Aeruginosa Gianni Klesse , Jemma Trick , Sivapalan Chelvaniththilan , of 1,2 4 1 2 1,2 1,2 Prafulla Aryal , Jayne Wallace , Stephen Tucker , Mark S.P. Sansom . Patrick Seelheim , Raghavendar Reddy Sanganna Gari , 1Clarendon Laboratory, Department of Physics, University of Oxford, Iga Kucharska1,2, Lukas K. Tamm1,2. 2 1 Oxford, United Kingdom, Department of Biochemistry, University of Molecular Physiology and Biological Physics, University of Virginia, 3 2 Oxford, Oxford, United Kingdom, Department of Physics, Kings College Charlottesville, VA, USA, Center for Membrane and Cell Physiology, London, London, United Kingdom, 4Oxford Nanopore Technologies Ltd, University of Virginia, Charlottesville, VA, USA. Oxford, United Kingdom. Pseudomonas aeruginosa is an opportunistic human pathogen that is respon- Ion channels play key roles in cell membranes, with recent advances in e.g. sible for a growing number of nosocomial infections and the main cause of cryo-electron microscopy yielding an increasing number of structures. How- death in patients with cystic fibrosis. Its very stable outer membrane lacks ever, the functional relevance of these structures is often unclear and better unspecific porins and poses an efficient permeation barrier to both hydrophilic computational tools are required for their functional annotation. Many ap- and lipophilic compounds rendering P. aeruginosa resistant to many common proaches to functional annotation rely upon a simple description of the phys- antibiotics. ical dimensions of the predicted pore. However, in many sub-nanometer pores The x-ray crystal structure of the P. aeruginosa outer membrane protein OprG (such as those found in most ion channels), hydrophobic gating has been b revealed a tall 8-stranded -barrel extending far into the extracellular space and shown to promote dewetting to produce a functionally closed (i.e. non- featuring a proline-rich region near the membrane interface. It has been spec- conductive) state. Using the serotonin (5-HT3R) and glycine receptor struc- ulated that this proline-region may function as a gate for the transport of small tures as examples, we demonstrate the use of molecular dynamics to aid the hydrophobic compounds across the outer membrane. functional annotation of channel structures via simulation of the behaviour However, we found that OprG facilitates the transport of small, uncharged of water within the pore. Our approach correctly predicts the 5-HT3R crystal amino acids both in vitro and in vivo as shown by liposome swelling assays structure to represent a functionally closed (i.e. non-conductive) state. We also and P. aeruginosa growth curves. When determining the NMR solution struc- illustrate application of water equilibrium density simulations to predict the tures of wild-type OprG and the transport incompetent P92A mutant OprG, we functional status of different conformational states of a glycine receptor b found shorter -barrels with long disordered extracellular loops in both pro- thereby demonstrating the applicability of our approach to structures of rela- teins. However, the P92A mutation led to an asymmetric elongation of the bar- tively modest structural resolution. Overall we will describe how this method- rel and changes in the loop dynamics. ology can be developed to provide a more accurate description of ion channel As the lumen of OprG is too small to accommodate even the smallest amino pores and how it may be integrated into existing platforms for functional anno- acid glycine, we hypothesized that the active entity is a multimer facilitating tation of structures. transport along a yet to be defined interface between individual OprG proto- mers. We set out to elucidate the transport mechanism by structure-driven mutagenesis, chemical crosslinking, and quantitative atomic force microscopy 2054-Pos Board B374 imaging. Our data so far show a significant population of dimers, trimers Evolutionary Analysis Discriminates between Alternative ASIC Structures Marina Kasimova1, Timothy Lynagh2, Daniele Granata1, Stephan Pless2, and higher-order multimers in preparations of transport-competent OprG 1 liposomes. Vincenzo Carnevale . 1Temple University, Philadelphia, PA, USA, 2University of Copenhagen, 2052-Pos Board B372 Copenhagen, Denmark. Filtering with the Electric Field: A Story on Protein Channels Electro- Acid-sensing ion channels (ASICs) are neuronal ion channels that facilitate statics sodium currents across the membrane in response to elevated extracellular Silvia Acosta-Gutierrez1, Giuliano Malloci1, Igor Bodrenko1, proton concentrations. Several crystal structures of ASICs have revealed the Mariano Andrea Scorciapino2, Matteo Ceccarelli1. overall architecture of these channels: they are composed of three subunits, 1Physics, Cagliari University, Cagliari, Italy, 2Biomedical Sciences- each having two transmembrane helices (TM1 and TM2) and a large extracel- Biochemistry, University of Cagliari/Istituto Officina dei Materiali del lular domain. Some of these structures are characterized by the presence of a Consiglio Nazionale delle Ricerche (IOM-CNR), Cagliari, Italy. domain swap at the level of the transmembrane domain: TM2 unfolds in the There is an urgent need of new drugs for combating multidrug-resistant bacte- middle, forming a kink and establishing contacts with the TM1 helix of the ria, especially for Gram-negative bacteria where the presence of the outer mem- adjacent subunit. At present it is not known whether the activated channel brane (OM) hinders the access of molecules to internal targets. To date neither a populates both conformations (with and without the domain swap) or only robust screening method for permeation nor defined physical/chemical rules one of them. With this question in mind, we analyzed the available structural governing permeation through the OM are available, leading to the design of information in light of a phylogenetically informed extensive sequence anal- molecules with rather low permeability that require high doses usually causing ysis. Using advanced statistical approaches, such as critical variable selection toxicity issues. Recent works in literature pointed the electrostatics of the chan- and direct coupling analysis, we inferred the likely orientation of TM2 and the nel to be responsible for its filtering mechanism and some theoretical investi- residue-residue interactions. We then tested experimentally these predictions gations are already reported. using conventional mutagenesis, unnatural amino acid incorporation and We combined all-atom simulations with enhanced sampling techniques to electrophysiology. Together, these findings allow to discriminate between characterize the electrostatics properties of different porins. We focused distinct channel conformations and tentatively support a native domain- initially in the calculation of the internal electrostatics of Enterobacteriaceae, swapped conformation.

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Ligand-gated Channels I 2057-Pos Board B377 Multiple Subconductance States of Tarped AMPA Receptors Revealed by 2055-Pos Board B375 Slow Dissociation of Antagonist Identification and Characterization of the Binding Pocket for Negative Ian Coombs1, Andrij Tarasiuk2, Lauren Marconi2, Chris Shelley2, Allosteric Modulators in AMPA Receptors Mark Farrant1, Stuart G. Cull-Candy1. 1Neuroscience, Physiology, and Pharmacology, University College London, Charlotte Stenum-Berg, Sergei C. Abiega, Christine L. Thisted, 2 Anders S. Kristensen. London, United Kingdom, Biology, Franklin and Marshall College, Drug Design and Pharmacology, University of Copenhagen, Copenhagen O, Lancaster, PA, USA. Denmark. Fast excitatory synaptic transmission in the central nervous system is mediated AMPA receptors (AMPARs) are present throughout the CNS and mediate mainly by AMPA-type glutamate receptor-channels (AMPARs) associated with the majority of fast excitatory neurotransmission. AMPARs are implicated auxiliary transmembrane subunits such as TARPs and cornichons. AMPARs in CNS diseases such as Alzheimer’s, Parkinson’s, and epilepsy. Recently, assemble as tetrameric ion channels, and each subunit contains a single agonist the first AMPAR-selective drug was approved in the form of perampanel binding site. They are unusual as individual channels open to multiple stable for treatment of epilepsy. Perampanel is a negative allosteric modulators conductance and subconductance states, and despite decades of work there is still (NAMs), inhibiting AMPAR activity via a poorly understood mechanism. no consensus on the number of subconductance states AMPARs can adopt, with Little is known about the location and structure of binding sites for peram- three or four distinct levels most commonly reported. Additionally, while TARPs panel or other prototypical AMPAR NAMs such as GYKI-53,655 and CP- increase the conductance of AMPARs, it’s still unclear whether this is achieved by 465,022. Identifying NAM binding sites and binding modes is prerequisite increasing the proportion of openings to larger conducting states, or whether to understanding the molecular mechanism that underlies negative allosteric TARPs increase the conductance levels themselves. To identify the activity of in- modulation of AMPAR function and is important for future development of dividual ion channels, outside-out patches obtained from cells transfected with a other NAMs. GluA1 - TARP g2 tandem construct were bathed in the slowly-dissociating This project aims to characterize the binding sites and binding modes of peram- competitive antagonist ZK200775 to block all currents and the desensitization panel and other NAMs. AMPARs are tetrameric assemblies of subunits with a blocker, cyclothiazide. Fast switching to extracellular solution containing satu- highly modular structure comprising two large extracellular domains - the rating glutamate, cyclothiazide, but no antagonist resulted in step-wise increases amino-terminal domain and the ligand-binding domain - connected to the in subconductance states, up to a maximum single channel conductance of approx- transmembrane domain via three flexible linkers (denoted S1-M1, S2-M3, imately 40 pS. We were able to characterize at least four distinct conductance and S2-M4). Previous work has identified positions within these linkers to con- levels, the frequency of transitions between conductance states, and the dwell- trol NAM potency (Balannik et al., 2005, Neuron). To identify additional res- times of the different conductance states. Despite the presence of cyclothiazide idues that participate in NAM interaction, mutational scanning was performed and saturating levels of glutamate during the agonist jump, clear subconductance of the S1-M1 and S2-M4 linker regions. 48 point mutants were functionally states were often observed subsequent to the step-wise increase in single channel characterized using an intracellular calcium imaging assay. For the 37 mutants current, indicating that subconductance states are not due to dissociation of gluta- mate from individual binding sites. Our findings also demonstrate that in the pres- with intact receptor function, IC50 was determined for four prototypical NAMs, including perampanel, to identify mutant effects on NAM potency. Several mu- ence of TARPs, AMPARs can open toenhanced conductance states and do not just show an increase in the proportion of larger openings. tants were found having >5-fold change in IC50 of one or more NAMs and further characterized by two-electrode voltage clamp electrophysiology. The 2058-Pos Board B378 mutational dataset will be used to guide molecular modeling studies of NAM AMPA Receptor Modulation by Stargazin binding modes to the receptor. Sana A. Shaikh1, Drew M. Dolino1, Garam Lee1, Sudeshna Chatterjee2, David M. MacLean1, Charlotte Flatebo2, Christy F. Landes2, 2056-Pos Board B376 Vasanthi Jayaraman1. Phosphorylation in AMPA Receptor Carboxy-Terminus: Structure, 1Biochemistry and Molecular Biology, The University of Texas Health Function, and Lipid Regulation Science Center, Houston, TX, USA, 2Chemistry, Rice University, Houston, 1 2 1 1 Caitlin E. Nurik , Suma Devi , Sana Shaikh , Drew Dolino , TX, USA. Darren Boehning1, Vasanthi Jayaraman1, James Howe2. The alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) 1Biochemistry and Molecular Biology, University of Texas Health Science 2 receptors are ion channels activated by the neurotransmitter, glutamate. They Center, Houston, TX, USA, Pharmacology, Yale Univeristy, New Haven, mediate fast excitatory synaptic transmission in neurons and play a role in syn- CT, USA. aptic plasticity that forms the basis of learning and memory. AMPA receptors The a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor are a target for ischemic stroke therapy, where their excessive stimulation by is one of the two non-NMDA-type ionotropic glutamate receptors. It is the accumulated glutamate triggers neuronal injury and death. In their native state, primary contributor to fast excitatory transmission in neurons, which is key AMPA receptors are associated with auxiliary proteins such as stargazin, which to learning and building memory. Its dysfunction is indicated in a wide array modulate receptor trafficking and gating. The mechanism by which stargazin of pathophysiologies including Parkinson’s, stroke, Alzheimer’s, and schizo- enhances AMPA receptor function is however poorly understood. To determine phrenia. The AMPA receptor can be divided into four domains. Extracellularly, the placement of stargazin on the AMPA receptor, we used luminescence reso- there is the amino terminal domain, and the more membrane-proximal ligand- nance energy transfer (LRET) measurements to map distances between the full- binding domain, to which activators bind and induce channel opening. The length AMPA receptor and stargazin. By nanopositioning using these LRET transmembrane domain serves as the actual ion-channel pore. Of these four do- distances, we were able to place stargazin below the AMPA receptor ligand- mains, the structure of the outermost three has been shown in detailed crystal binding domain. Additionally, we determined the ensemble structural changes structures. However, very little is known about the structure of the cytoplasmic in the receptor brought about by stargazin. We also used single molecule fluo- domain. Although it is widely considered disordered, it is unknown whether rescence resonance energy transfer to study the structural and conformational local order (levels of secondary and/or tertiary structure) may exist in the distribution of the AMPA receptor, and the changes in distribution caused by carboxy-terminus, or whether structure changes may occur as conformational stargazin. Our data show that stargazin acts as a scaffold below the AMPA shifts due to functional modifications. Previous studies have established protein receptor ligand-binding domain and promotes long-range conformational kinase C phosphorylation sites at residues in the GluA1 subtype receptor, and changes causing the activation of the receptor by destabilizing its desensitized confirmed their effect to enhance the function of the receptor. Our initial studies state. examined a membrane-proximal section of the GluA1 carboxy-terminus to consider local structural changes brought about by the phosphorylation events. 2059-Pos Board B379 The peptides were examined using Fourier transform infrared spectroscopy, Design of a Glutamate-Activated Potassium Channel upon Fusion of the which showed a conversion to lesser helix content, more extension, in the phos- Ligand-Binding Domain of the Mammalian AMPA Receptor Glua1 to phomimetic sample. Studies were conducted in a lipid context and showed that the Channel Pore of the Viral ATCV-1 KCV KD Channel the change in secondary structure is enhanced in the context of close proximity Michael Schoenrock1, Alina Poci1, Gerhard Thiel2, Bodo Laube1. to a lipid membrane. Single-channel electrophysiological studies on expressed 1Neurophysiology and Neurosensory Systems, TU Darmstadt, Darmstadt, wild-type and phosphomimetic channels confirmed that the functional effect of Germany, 2Plant Membrane Biophysics, TU Darmstadt, Darmstadt, phosphorylation depends on interaction with the charged lipid membrane. Germany. Ongoing imaging studies are examining the mechanism of this interaction in Tetrameric ionotropic glutamate receptors (iGluRs) are cation channels that are þ þ whole cells. permeant to Na and K with an extracellular, clamshell-like ligand-binding

BPJ 7860_7863 Tuesday, February 14, 2017 419a domain (LBD), which opens the transmembrane ion pore upon glutamate- ranging from the hippocampus, striatum, and spinal cord, increase the promise induced closure. The core pore region of the iGluRs shares a high sequence of KAR specific therapies for circuit disorders such as epilepsy. Consequently, homology with tetrameric voltage-gated Kþ channels with a remarkably structure-function studies of KARs are needed to inform drug design. From similar secondary structure, indicating an evolutionary link between the two these studies we have learned that each subunit of these tetrameric proteins channel structures. However, the channel of these two families of membrane consists of an N-terminal domain (NTD), ligand binding domain (LBD), proteins differs strikingly concerning i) its orientation within the cell membrane transmembrane domain (TMD) and a C-terminal intracellular tail. Moreover, (inverted), ii) the induction of channel opening (voltage- vs. ligand-gated) and the LBD-LBD/NTD-NTD contacts are influential sites in shaping the KAR iii) its cation permeability. So far, despite the similar channel architecture, no conformational landscape and excellent allosteric targets. However, native experimental work has been performed to substantiate a putative functional KARs were recently found to be frequently accompanied by gain-of-function compatibility of the pore structures of Kþ and iGluR channels. First to analyze auxiliary proteins known as NETOs. Since there is no structural data for whether potassium channels are also functional upon an inverted insertion into NETO proteins the mechanism underlying their structural modulation is the cell membrane, we provided a small 82 amino acid long viral Kcv ATCV-1 unknown. In this study we investigated the structural basis of Neto action by potassium channel, which displays all the major features of Kþ channels, with examining the inter-subunit distances of the KAR (GluK2) LBD and NTD an N-terminal signal peptide sequence of HLA class1 gene resulting in an with and without Neto2 using luminescence energy resonance transfer opposite orientation within the cell membrane. Functional analysis of this (LRET). The LRET data shows that the inter-subunit distances in both the construct upon heterologous expression revealed that inversion does not influ- LBD and NTD are decreased in the presence the auxiliary protein, constraining ence the conductivity or selectivity of the channel. In a second step, we engi- the KAR into conformations more conducive to activation. Thus the auxiliary neered chimeric constructs which possessed the LBD of the ionotropic protein acts by supporting the receptors extracellular domains, cradling them glutamate receptor (GluA1) combined with the membrane-spanning domain into closer inter-domain proximity while promoting structural organization. of the Kcv ATCV-1. The chimeras functioned as a Kþ-selective, glutamate- gated channels displaying both, the characteristics of ligand recognition of 2062-Pos Board B382 the iGluR and the selectivity of the Kcv ATCV-1. Thus, by linking a LBD Functional Coupling of Bacterial Binding Protein with the Channel Pore of of a mammalian iGluR to the channel pore of a viral Kþ channel, our results an Ionotropic Glutamate Receptor are consistent with a conserved activation mechanism of the pore region within Max Bernhard, Bodo Laube. € the two families and supports a phylogenetic link. Neurophysiology and Neurosensory Systems, Technische Universitat This study was funded by the LOEWE project iNAPO of the Hessen State Darmstadt, Darmstadt, Germany. Ministry of Higher Education, Research and the Arts. Prokaryotic solute binding proteins (SBP) facilitate chemotaxis and substrate uptake of a large variety of small molecules and ions by binding their ligands 2060-Pos Board B380 with high specificity and affinity. Despite a low sequence identity, SBP are Cryo-Electron Microscopy Reveals Structural Basis of Kainate Subtype structural homologous to the ligand binding domain (LBD) of ionotropic gluta- Glutamate Receptor Desensitization mate receptors (iGluR) and share a common ligand binding mechanism. SBP Sagar Chittori1, Joel R. Meyerson1, Alan Merk1, Prashant Rao1, and LBD consist of two lobed domains connected by a hinge forming a clam- Tae H. Han2, Mihaela Serpe2, Mark L. Mayer2, Sriram Subramaniam1. shell like structure. Ligand binding takes place at the interface between the two 1NCI, National Institutes of Health, Bethesda, MD, USA, 2NICHD, National domains, inducing a domain closure. This conformational change function as a Institutes of Health, Bethesda, MD, USA. key element in the transition of ligand recognition and ion channel gating in Ionotropic glutamate receptors (iGluRs) are ligand-gated tetrameric channels iGluR. Due to the wide range of recognised ligands and the conserved binding that mediate synaptic transmission in the central nervous system and are instru- mechanism, SBP may function as a component for analyte recognition in the mental in learning and memory formation in vertebrates. iGluRs can be broadly design of a new class of biosensors. Here we describe an approach to design classified into AMPA, kainate, NMDA and d (delta) receptor families, formed novel ligand activated ion channels by substituting the LBD of iGluRs with by homo- and hetero-oligomeric assembly of four subunits consisting of an SBP. Substitution of the LBD of the prokaryotic iGluR GluR0 with the ectoine amino-terminal domain (ATD), an agonist-recognizing ligand-binding domain binding protein EhuB resulted in ectoine-activated channels with nanomolar af- (LBD), an ion-channel forming transmembrane domain (TMD), and an intra- finity. Thus, by functional linking of a bacterial binding protein to the channel cellular carboxy-terminal domain (CTD). These receptors function by opening pore of an iGluR, our results substantiate the compatibility of SBPs and the a cation-selective ion pore in response to ligand binding and subsequent to pore structures of iGluR channels for the design of new biosensors for specific channel activation, the receptor adopts a desensitized state in which the ion analyte recognition. This study was funded by the LOEWE project iNAPO of channel is closed but the ligand remains bound to the receptor. Although the Hessen State Ministry of Higher Education, Research and the Arts. near-atomic resolution structures have been reported for the AMPA receptor subtype GluA2 in apo, antagonist-bound and pre-activated states, a high- 2063-Pos Board B383 resolution structure of the desensitized state of any glutamate receptor subtype Probing the Channel Gating of a Glutamate Receptor with Photoactive has remained elusive. Thus, the central question of how a closed ion channel Unnatural Amino Acids 1,2 1 3 can be accommodated in both resting and desensitized states in the context Mette Poulsen , Anahita Poshtiban , Viktoria Klippenstein , 1 1,4 of markedly different quaternary structure of the LBD layer has remained un- Valentina Ghisi , Andrew Plested . 1FMP-Berlin, Berlin, Germany, 2University of Copenhagen, Copenhagen, answered. Here, by determining the structure of the glutamate receptor subtype 3 4 GluK2 in its desensitized state by single particle cryo-electron microscopy Denmark, IBENS, Paris, France, NeuroCure Charite´, Berlin, Germany. (cryo-EM) at 3.8 A˚ resolution, we show that desensitization is characterized Ionotropic glutamate receptors (iGluRs) are responsible for fast synaptic trans- by the establishment of a ring-like structure in the LBD layer of the receptor. mission throughout the nervous system. Despite considerable study, the confor- Formation of this ‘desensitization ring’ is mediated by staggered helix contacts mational change of the transmembrane domain (TMD) underlying ion channel between adjacent subunits, which leads to a pseudo 4-fold symmetric arrange- activation remains unclear. Here, we aim to explore the function and dynamics ment of the LBD layer, illustrating subtle changes in symmetry that are impor- of the transmembrane region of AMPA-type glutamate receptors using unnat- tant for the gating mechanism. Disruption of the desensitization ring is ural amino acid (UAA) photo-cross-linkers, p-benzoyl-L-phenylalanine (BzF) probably the key switch that enables restoration of the receptor to its resting and p-azido-L-phenylalanine (AzF). Using mammalian cells as expression sys- state, thereby completing the gating cycle. tem, AzF and BzF were individually introduced throughout the TMD of the AMPA receptor GluA2 by genetically-encoded UAA mutagenesis. Outside- 2061-Pos Board B381 out patch clamp recording of receptors activated by a fast-perfusion system Reduced Structural Dynamics in Kainate Receptors through Auxiliary was combined with synchronized exposures to UV light via epi-illumination, Protein Modulation to characterize the functionality of the AzF and BzF containing iGluR con- Douglas B. Litwin, David M. MacLean, Vasanthi Jayaraman. structs, as well as their individual photo-controllable profiles. AzF and BzF Biochemistry and Molecular Biology, University of Texas Health Science were individually inserted in 23 sites throughout the TMD. Glutamate induced Center, Houston, TX, USA. currents could be measured from 18 constructs containing AzF at different Ionotropic glutamate receptors (iGluRs) are the primary neurotransmitter sites, while only 10 constructs containing BzF resulted in a glutamate- receptors involved in excitatory synaptic transmission. Amongst the iGluR activated current. The glutamate activated currents of mutant receptors had family, kainate receptors (KARs) are the most diverse in function but also similar characteristics to wild type channels. Exposing channels harboring the least understood. KARs contribute to postsynaptic responses, to regulation AzF to UV light had a range of effects, from inhibition to potentiation, depen- of cellular excitability as well as control over the presynaptic release of both dent on the insertion site. Exposure of channels incorporating BzF in the TMD excitatory and inhibitory neurotransmitters. These multiple roles, in tissues to UV light did not have any effect on currents. In contrast, photoactivation of

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BzF in the Pre-M1 linker was found to increase peak current by up to 50%. Our potency of a series of ibuprofen analogues at ASIC1a, ASIC2a and ASIC3 results demonstrate that UAAs can be incorporated site specifically in the TMD and (ii) identify the molecular determinants of ibuprofen sensitivity in and can trap key moving parts of the ion channel. ASIC1a. We found that ibuprofen and most analogues inhibited each sub- type with low potency (high micromolar IC50 values for inhibition), how- 2064-Pos Board B384 ever, certain moieties of the ibuprofen molecule were more important for Investigating Coupling between Proton Sensors and Channel Motions in inhibition of certain subtypes. For example, p-tolyl propionic acid, which Acid-Sensing Ion Channels lacks the isobutyl ‘‘tail’’ of ibuprofen, inhibited ASIC2a but not ASIC1a. Maria Musgaard1, David M. MacLean2, Douglas B. Litwin2, In contrast, the carboxylic acid appears crucial for inhibition of all subtypes, Vasanthi Jayaraman2, Philip C. Biggin1. as its replacement with a nitro group in the novel compound 1-isobutyl-4- 1 Biochemistry, University of Oxford, Oxford, United Kingdom, (1-nitro-ethyl)benzene abolished inhibition at all subtypes tested. The role 2 Biochemistry and Molecular Biology, University of Texas, Houston, TX, of the carboxylic acid prompted us to substitute all 22 surface-accessible USA. basic side chains in ASIC1a via site-directed mutagenesis to identify molec- Acid-sensing ion channels (ASICs) are proton-gated sodium channels found ular determinants of sensitivity. We observed that mutation of three vicinal throughout the central and peripheral nervous systems. The human genome residues, each of which orient into a cavity just above the level of the mem- contains four ASIC genes, and the channels are among other things involved brane, abolished inhibition by 1 mM ibuprofen. Together, these data suggest in pain signaling. ASICs operate on a millisecond-to-second timescale and that ibuprofen inhibits ASIC1a through an interaction between the drug open upon acidification of the synaptic cleft. At least three different states carboxylate and a site at the interface of extracellular and channel domains are involved in the ASIC functional cycle; a deprotonated closed state; a pro- in ASIC1a. tonated open state; and a protonated but closed desensitized state. Several crys- tal structures for the two protonated states, open and desensitized, have been 2067-Pos Board B387 solved, whereas the structure of the deprotonated closed state is still unknown. Stoichiometry of Toxin Binding to Acid-Sensing Ion Channels Therefore, the overall coupling mechanism between protonation and channel Christian B. Borg, Timothy Lynagh, Linda M. Haugaard-Kedstro¨m, opening is currently not well understood structurally. At least two proton- Kristian Strømgaard, Stephan A. Pless. sensing regions in the large extracellular domain (ECD) have been described, Department of Drug Design and Pharmacology, University of Copenhagen, at distances of around 20 A˚ and 60 A˚ from the channel domain, respectively. Copenhagen, Denmark. To investigate possible large-scale slow motions coupling the ECD, and thus Acid-sensing ion channels (ASICs) are homo- or heterotrimeric proton-gated potentially the proton sensing regions, to the ion channel, we have performed cation channels, which have been identified as one of the crucial players in an analysis of the elastic network modes calculated for different open and de- the initiation of pain. A number of peptide toxins have been shown capable sensitized crystal structures and compared the structural changes observed in of modulating ASIC function and therefore have great therapeutic potential these modes to inter-residue distances measured with luminescence resonance for the management of pain. Psalmotoxin-1 (PcTx1), extracted from spider energy transfer (LRET). Modes, which at the same time fulfil experimental venom, is the most potent and specific inhibitor of homomeric ASIC1a distance measurements from LRET and illustrate potential channel motion, channels. It has been shown to inhibit the channel in heterologous expression have been identified. Furthermore, we have used atomistic molecular dynamics systems and rodent nociceptive neurons, however, the precise mechanism un- simulations to study the effect of protonation and deprotonation of the proton- derlying the inhibitory effect of PcTx1 is still elusive. In this study we have sensing regions at higher resolution, allowing us to study the changes in the im- determined the stoichiometry of the PcTx1-ASIC1a complex and provided mediate environment around the proton-sensors upon binding and unbinding of mechanistic insight into toxin binding to ASIC channels. This was achieved protons with the potential for capturing longer-distance communication to the by using a series of concatenated ASIC1a constructs, containing a mutation ion channel. known to abolish PcTx1 inhibition in one, two, or all three subunits. These constructs were assessed for their toxin sensitivity by two-electrode voltage 2065-Pos Board B385 clamp electrophysiology, as well as for their structural integrity using Acid Sensing Ion Channels have Agonist-Dependent Deactivation biochemistry. Our results show that binding of the first two PcTx1 molecules David M. MacLean, Vasanthi Jayaraman. to ASIC1a channels result in an approximately equal and additive inhibitory Biochemistry, UT- Houston, Houston, TX, USA. effect, whereas binding of a third toxin molecule shows no further effect on Acid sensing ion channels (ASICs) are trimeric cation-selective ion channels the inhibition. These data strongly suggest that two PcTx1 molecules are suf- activated by protons in the physiological range. Recent reports have revealed ficient to elicit the full inhibitory effect. In conclusion, we have, for the first that postsynaptically-localized ASICs contribute to the EPSC by responding time, provided direct insight into the stoichiometry of the PcTx1-ASIC1a to the transient acidification of the synaptic cleft which accompanies neuro- complex. transmission. In response to such brief acidic transients, both recombinant and native ASICs show extremely rapid deactivation in outside-out patches 2068-Pos Board B388 when jumping from a pH 5 stimulus to a single resting pH of 8. However, Channel Open Probability Controls Allosteric Modulation of Potency and the resting pH of the synaptic cleft is highly dynamic and depends on recent Efficacy synaptic activity. By rapidly jumping outside-out patches from pH 8 through Riley E. Perszyk1, Kevin K. Ogden1, Katie L. Strong1,2, Dennis C. Liotta2, pH 5 for approximately 1 ms to a third pH ranging from pH 8 to 6.6 we can Stephen F. Traynelis1. 1 2 approximate this range of cleft pHs and explore the deactivation kinetics of Pharmacology, Emory University, Atlanta, GA, USA, Chemistry, Emory ASICs under these conditions. Strikingly, the deactivation of ASICs span University, Atlanta, GA, USA. nearly three orders of magnitude from extremely fast (~ 0.4 ms) at pH 8 Kinetic models of NMDA receptors (NMDARs) that separate binding and to very slow (~ 400 ms) at pH 7. To our knowledge this is the first example gating into two distinct steps predict that the enhancement of open proba- of a ligand-gated ion channel whose deactivation is so strongly dependent on bility will shift the equilibrium to activated states and therefore enhance agonist concentration. Kinetic simulations and further mutagenesis provide agonist EC50. The tetrahydroisoquinoline CIQ is a positive allosteric modulator evidence that ASICs show such steeply agonist-dependent deactivation (PAM) of GluN2C- and GluN2D-containing NMDARs, increasing the current because of strong cooperativity in proton binding. This capacity to signal response to maximal effective concentrations of agonist by 3-fold. Surpris- across such a large synaptically-relevant bandwidth provides ASICs with ingly, CIQ did not detectably alter glutamate potency at GluN2D (Mullasseril the ability to shape synaptic activity in response to recent history of the et al., 2010). We recently identified a CIQ analogue, (-)1180-55, that also synapse. potentiates GluN2B-containing NMDARs. This compound enhances the response of GluN2D-containing NMDARs by 3-fold but again has no detect- 2066-Pos Board B386 able effect on glutamate EC50 for GluN2D-containing NMDARs, which was Molecular Basis for Inhibition of Acid-Sensing Ion Channels by Ibuprofen 0.550.07 mM (n=19) in control and 0.450.01 mM (n=13) in 20 mM (-) Timothy Lynagh, Jose L. Romero-Rojo, Stephan A. Pless. 1180-55. By contrast, (-)1180-55 increased the response of GluN2B- University of Copenhagen, Copenhagen, Denmark. containing NMDARs to maximally concentrations of agonist by 2-fold and Activation of acid-sensing ion channels (ASICs) is known to elicit pain, increased glutamate potency by 2-fold from 2.050.16 mM (n=30) in control prompting the search for small molecule inhibitors as potential analge- to 1.250.07 mM (n=22) in 20 mM (-)1180-55. To explore why this PAM sics. Curiously, non-steroidal anti-inflammatory drugs (NSAIDs) such as showed variable actions on agonist potency in GluN2B and GluN2D ibuprofen have been shown to inhibit ASICs, but a systematic assessment NMDARs, we modelled the potentiation of efficacy for a de Castillo and of subtype selectivity and potency of different NSAIDs, as well as any bind- Katz model of channel function that separated binding and gating steps. Eval- ing site information, are lacking. We therefore aimed to (i) characterize the uation of the shift in EC50 as a function of open probability revealed that the

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PAM-induced changes in agonist potency following an increase in efficacy are tem. In the latter, P2X4 and P2X7 receptors are involved in regulation of only detectable when the open probability is greater than 0.1. For exceptionally inflammation and nociception. While functional heteromerization is estab- low open probability receptors such as GluN2D, doubling open probability lished for P2X2 and P2X3 receptors expressed in sensory neurons, controver- from 0.01 to 0.02 does not detectably alter the EC50. The relationship between sial reports have been published with respect to a functional interaction open probability and allosteric modulation is thus critical to understanding between P2X4 and P2X7 receptor subtypes. whether PAMs will alter agonist potency by shifting the gating/binding To address this issue, we coexpressed in Xenopus leavis oocytes P2X4 and equilibria, or whether their binding pose directly impacts the ligand binding P2X7 receptor constructs and investigated physical and functional interaction domain. by means of blue native polyacrylamide gel electrophoresis (BN-PAGE), Fo¨rster resonance energy transfer (FRET) and by the two-microelectrode 2069-Pos Board B389 voltage clamp technique. BN-PAGE analysis provided evidence for the forma- Effects of Ligand Conformations and Hetero-Liganded States on P2X2 tion of heterotrimeric P2X4/7 receptors. Coexpression of P2X4 and P2X7 with Receptors GFP and TagRFP located in corresponding extracellular receptor domains led Federica Gasparri1, Jesper Wengel2, Thomas Grutter3, Stephan Pless1. to significant FRET signals. On the other hand, ATP concentration-dependent 1 Drug Design and Pharmacology, University Of Copenhagen, Copenhagen, effects as well as the effects of P2X4R- or P2X7R-specific agonists or antago- 2 3 Denmark, University of Southern Denmark, Odense, Denmark, University nists on oocytes coexpressing P2X4 and P2X7 could be explained by non- Of Strasbourg, Strasbourg, France. interacting hP2X4 and hP2X7. Coexpression of P2X4 with P2X7 constructs P2X receptors (P2XRs) belong to the family of ligand-gated ion channels and which display only very small ion currents after activation by ATP largely are activated by ATP. They are expressed throughout the human body and decreased the P2X4-dependent currents which may be explained by an excess mediate crucial physiological roles, such as modulation of synaptic transmis- of the P2X4 expression capacity of the oocytes due to the coexpressed P2X7 sion, inflammation and neuropathic pain. Despite recent breakthroughs in our construct or by formation of P2X4/P2X7 heteromers which do not conduct understanding of the P2XR structure through the crystallization of P2XRs, ATP-induced currents. many molecular aspects of ATP binding remain unclear. Here we combined In conclusion, although coexpression of P2X4 and P2X7 subunits seems to whole cell and single channel electrophysiological recordings using a range result in heteromeric receptor assemblies, no new electrophysiological P2X of ATP analogs with an extensive mutagenesis screen to elucidate 1) whether receptor phenotype is produced. the chemical nature or conformational flexibility of agonist affects the channel open state and 2) if the three agonist binding sites are functionally coupled. Our 2072-Pos Board B392 data show that other nucleotide triphosphates, such as CTP, GTP and UTP, act A Novel, Spectroscopic Window into Nucleotide Activation of KATP as partial agonists that lead to an open state identical to ATP with regards to Channels single channel conductance and flickering behavior. Subtle ATP analogues Michael C. Puljung, Natascia Vedovato, Frances M. Ashcroft. with either increased or decreased conformational flexibility of the ribose Department of Physiology, Anatomy & Genetics, University of Oxford, ring also show identical open channel properties, suggesting that chemical Oxford, United Kingdom. nature and binding pose of the agonist have little effect on the conformation The KATP channel comprises four pore-forming Kir subunits and four sulfonyl- of the open pore. Further, we show that ATP-evoked responses are potentiated urea receptor (SUR) subunits. Each component of the complex binds adenine by co-application of partial agonists at sub-saturating concentrations. This nucleotides. Binding of ATP/ADP to Kir is inhibitory, whereas binding to phenomenon is present also in the absence of divalent cations and when muta- the two nucleotide binding sites (NBSs) on SUR stimulates channel opening, þ tions are introduced within the binding site or at the subunit interface. These but only in the presence of Mg2 . Attempts to understand nucleotide handling data shed new light on how P2XRs can be modulated in a presence of multiple by KATP have been confounded by the fact that most techniques (e.g. electro- potential agonists, e.g. in a synaptic environment. physiology, ATPase assays) measure the summed activity of all nucleotide binding sites working together. We have devised a novel method to isolate 2070-Pos Board B390 the distinct contributions of each nucleotide binding site to channel activity. Point Mutants of ATP-Gated P2X Receptors Cast Doubt on the This combines expression of channels tagged at a single type of nucleotide Inclusiveness of the Reservoir Theory of Apparent Pore Dilation binding site (via site-specific incorporation of the non-native, fluorescent amino Laura Janks. acid ANAP) with FRET between ANAP and a fluorescent nucleotide (TNP- Saint Louis University, Affton, MO, USA. ATP or TNP-ADP) in ‘‘unroofed’’ plasma membrane sheets. We found that P2X receptors (P2XRs) are ATP-gated ion channels that modulate cell excit- TNP-ATP and TNP-ADP are capable of binding at either NBS of SUR1 co- ability in healthy and diseased tissues. Short applications of ATP evoke a rapid expressed with Kir6.2 in the membranes of HEK293 cells. Binding occurs in þ þ þ depolarizing current as small cations like Na ,K , and Ca2 flow down their both the absence and presence of Mg2þ. However, the dissociation rate for electrochemical gradients. Longer applications (a few seconds) evoke a second nucleotides is much slower in the presence of Mg2þ. This suggests that when phase of current, traditionally thought to result from a gradual dilation of a both Mg2þ and nucleotides are present, the nucleotide binding domains un- narrow part of the pore. Recent work suggests an alternative explanation: the dergo a conformational change that prevents ATP/ADP dissociation and pro- second phase of current reflects the redistribution of ions across the cell surface motes channel opening. This approach will allow direct insight into the membrane. In the work presented here, we use mutant P2XRs and a stable normal regulation of KATP by metabolic flux within the cell and the effects P2XR cell line to investigate the hypothesis that apparent pore dilation is an of mutations in KATP that result in diseases of insulin secretion. Furthermore artifact of a common method used to measure ionic permeability. Our findings this technique is readily adaptable to study binding in any protein with an support the contention that significant ion redistribution occurs using bi-ionic ATP-binding site (e.g. purinergic receptors, ABC transporters), or indeed any þ þ NMDG o/Na i solutions. However, we also find that mutant P2X2Rs with protein for which there is a suitable fluorescent ligand. large inward Naþ currents show no apparent pore dilation under bi-ionic con- ditions, suggesting that the change in the ionic composition of the cytoplasm 2073-Pos Board B393 does not fully explain the positive shift in the reversal potential of the ATP- Independent Activation of Ion Conduction Pores in the Double-Barreled 2D - gated current seen in the wild-type P2X2R. Our experiments leave open the Ca -Activated Cl Channel TMEM16A possibility that a slow, time-dependent change in the conformation of the Andy K.M. Lam, Novandy K. Lim, Raimund Dutzler. P2XR pore does indeed occur during long applications of ATP. Department of Biochemistry, University of Zurich,€ Zurich,€ Switzerland. TMEM16 proteins constitute a family of membrane proteins that include Ca2þ- 2071-Pos Board B391 activated lipid scramblases and Cl- channels. Due to high sequence conserva- Interaction of Purinergic P2X4 and P2X7 Receptor Subunits tion, members from both these branches probably share a common architecture, Fritz Markwardt1, Markus Schneider1, Kirsten Prudic1, Anja Pippel1, which is defined by our recent structure of the lipid scramblase nhTMEM16. þ Manuela Klapperstuck€ 1, Christa Muller€ 2, Michaela Stolz3, The structure reveals that each subunit in the homodimer contains a Ca2 - Michaela Schumacher3,Gunther€ Schmalzing3. binding site and also a potential site of catalysis. Here, we ask whether similar 1JB Institute for Physiology, Martin-Luther University, Halle (Saale), organization may also be present in the Ca2þ-activated Cl- channel TMEM16A. Germany, 2Pharmaceutical Institute, University of Bonn, Bonn, Germany, We generated concatemeric constructs containing subunits with distinct ac- 3Molecular Pharmacology, RWTH University, Aachen, Germany. tivation and conduction properties and characterised these constructs with Like other members of the P2X family, homotrimeric P2X4 and P2X7 patch-clamp electrophysiology. We show that TMEM16A exhibits biphasic 2þ receptors operate as non-selective cation channels gated by extracellular activation in response to Ca with EC50’s in the sub-mM and sub-mM range. ATP. P2X4 and P2X7 subtypes are co-expressed in several cell types including This biphasic behaviour is a consequence of a further increase in open secretory epithelia, bone cells and cells of the immune and inflammatory sys- probability at high Ca2þ concentrations with an unaltered unitary conductance

BPJ 7860_7863 422a Tuesday, February 14, 2017 as revealed by non-stationary noise analysis. In both activations, the current for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, reverses close to the Nernst potential of Cl-, suggesting that TMEM16A re- Basel, Switzerland. mains highly anion-selective during both activations. We found that the func- Eukaryotic cyclic nucleotide-modulated ion channels perform various physio- tional behaviour of constructs containing either two wild-type (WT) subunits or logical roles by channel opening in response to cyclic nucleotide binding to a one WT subunit paired with a second subunit with compromised activation specialized cyclic nucleotide-binding domain (CNBD). Despite increasing closely resembles TMEM16A. Concatemeric constructs that combine a WT knowledge regarding function and structure, a full picture of the gating pro- subunit with a subunit with decreased potency exhibit activation properties cess is still missing. Here, we report the direct monitoring of ligand-induced that can be described by a linear combination of the activation profiles of conformational changes in single cyclic nucleotide-modulated channels from their constituents. Consistent with this observation, mutation of a putative Mesorhizobium loti (MloK1), in real-time and native-like conditions. Using pore-lining residue that changes the conduction properties of the channel can high-speed atomic force microscopy (HS-AFM) imaging we show that in the selectively suppress the activation of the subunit containing this mutation. presence of cAMP most channels are in a stable conformation characterized Our results strongly suggest that TMEM16A contains two ion conduction pores by a windmill-like arrangement of the CNBDs within the channel tetramer. þ that are independently activated by Ca2 binding to sites that are embedded in A few molecules, however, are observed to dynamically switch back and forth the transmembrane part of each subunit. (blink) between states corresponding to at least two CNBD conformations that differ in their height, or distance from the membrane. Upon cAMP depletion 2074-Pos Board B394 more channels start blinking, and the blinking height of an individual channel 2D Mutagenesis Study of the Ca Sensitivity of SK2 Channels increases over time suggestive of slow, progressive loss of ligands from each Young Woo Nam, Benjamin J. Whitmore, Razan S. Orfali, Miao Zhang. CNBD within the tetramer. Thus, as a fully liganded MloK1 tetramer is Chapman University School of Pharmacy, Irvine, CA, USA. exposed to low cAMP, it slowly loses ligands one-by-one from each of the 2þ Small conductance Ca -activated potassium (SK) channels play a significant CNBDs, which start blinking independently from each other, yielding a highly role in modulating the membrane excitability of neurons. In animal model mobile state. We propose that during gating, the channel transitions from a mo- studies, SK channels have been identified as a potential drug target for the bile set of conformations in the absence of ligand to a stable conformation in the therapy of movement disorders such as ataxia. Previous studies suggest that presence of ligand and that these conformations are important for determining the neuronal activity of the Purkinje cells are abnormally elevated in ataxia the open and closed states of the conducting pore. mouse models. Positive allosteric modulators (PAMs) of SK channels have been shown to increase the Ca2þ sensitivity of these channels, thus reducing 2077-Pos Board B397 the excitability of the cells and exerting neuroprotective effects. The SK chan- Identification of an Odorant-Binding Site Residue in an Olfactory nels’ Ca2þ sensitivity is conferred by interaction with calmodulin (CaM), a Receptor of the Malaria Vector Mosquito Ca2þ binding protein. Our previous research has identified the binding pocket Suhaila Rahman, Charles W. Luetje. of the SK channels’ PAMs to be located between the CaM binding domain Molecular and Cellular Pharmacology, University of Miami Miller School of (CaMBD) and CaM. These findings have led us to introduce mutations into Medicine, Miami, FL, USA. the PAM binding pocket, resulting in changes in the Ca2þ sensitivity of SK Many insect behaviors are driven by olfaction, making odorant receptors channels. (ORs) appealing targets for control of disease vector mosquitoes. Insect ORs are odorant-gated ion channels composed of two subunit types; one of many 2075-Pos Board B395 odorant binding subunits and a conserved co-receptor subunit (Orco). We are Role of Individual Camp Binding Sites on Relieving the Autoinhibition in exploring the structural basis for odorant binding by ORs of the human malaria HCN Channels vector mosquito, Anopheles gambiae. Two structurally and functionally Mallikarjuna Rao Sunkara, Jana Kusch, Klaus Benndorf. similar odorant binding subunits Agam\Or13 and Agam\Or15 (82% amino Institute of Physiology II, University Hospital Jena, Jena, Germany. acid identity) were expressed in Xenopus oocytes, each in combination with HCN channels are tetrameric cation channels, activated by hyperpolarizing Agam\Orco, and assayed by two-electrode voltage clamp electrophysiology. voltages and modulated by binding of cyclic nucleotides (CN). Each subunit Both ORs were activated by acetophenone, but displayed distinct sensitivities carries an intracellular binding site (CNBD), connected to the transmembrane to antagonism by (-)-fenchone. To identify residue(s) responsible for this dif- portion via the C-linker (CL). Several studies proposed that the tetrameric ference in antagonist sensitivity, we assayed a series of mutant Agam\Or15 CNBD-CL structure exerts an autoinhibitory effect on the channel, which is subunits in which each residue that differed between Agam\Or13 and relieved by CN binding. The molecular mechanism behind this gating mecha- Agam\Or15 in the transmembrane and extracellular regions was changed nism is still elusive. We want to contribute to a better understanding by study- from the Agam\Or15 residue to the AgamrOr13 residue. Position 195 (Ala ing the contribution of each individual binding step. We monitored HCN2 in Agam\Or15, Ile in Agam\Or13), located at the interface between the pre- channel activation following hyperpolarizing voltage jumps in inside-out dicted second extracellular loop and fourth transmembrane domain, was found macro patches with and without saturating [cAMP]. We used concatenated to alter (-)-fenchone sensitivity. The reverse mutation in Agam\Or13 could also channel constructs with either one, two, three or four disabled binding sites alter (-)-fenchone sensitivity. To determine whether residue 195 is located at (compare Ulens & Siegelbaum, Neuron, 2003, 40:959-70). We found that occu- the odorant binding site of these receptors, we employed thermodynamic pation of two of the available four CNBDs is sufficient to stabilize the open mutant cycle analysis. Concentration-inhibition analyses for (-)-fenchone and state. This finding is supported by three types of data: Two ligands were suffi- six structurally related compounds were performed for Agam\Or15 subunits cient (1) to reach the maximum current amplitude increase, (2) to slow down with Ala (wt), Val, Leu and Ile at position 195. High interaction coefficient depolarization-induced deactivation, and (3) to cause a persistent current dur- values obtained for several cycles indicate a close physical proximity between ing cAMP wash-out. However, to reach maximum open probability, energy residue 195 and ligand. provided by voltage had to be higher than at full occupation. In contrast, to evoke the full cAMP-induced shift of the steady-state activation curve to less 2078-Pos Board B398 hyperpolarizing voltages, occupation of all four CNBDs was required. Modulating O2 Permeability of the Central Pore of RH50 by in Silico Site Thereby, each binding site contributed to a similar extent to the shift of the Directed Mutagenesis voltage of half-maximum activation (V1/2): one: DV1/2=4.2 mV; two, cis: Eric Shinn, Emad Tajkhorshid. DV1/2=9.9 mV, two, trans: DV1/2=9.5 mV; three: DV1/2=14.3 mV; four: University of Illinois at Urbana-Champaign, Urbana, IL, USA. DV1/2=20.9 mV). Remarkably, both options for half-occupation, cis and trans, The Rhesus (Rh) family of integral membrane proteins exhibit ubiquitous led to a similar shift. Together, these data led us conclude that for a complete expression among animals and their structural homology to ammonium trans- relief of autoinhibition full occupation of the tetrameric CNBD is required. In port (Amt) proteins suggests their function as ammonia/ammonium trans- partially liganded channels, the relative position of the occupied CNBDs is not porters. They have also been implicated in facilitating permeation of gaseous important for their effect. species, such as dioxygen and carbon dioxide, across the cell membrane. Rh50 proteins are expressed in erythrocytes, as well as in epithelial tissues 2076-Pos Board B396 of organs such as the kidneys where critical nitrogen processing occurs. Using Monitoring the Conformational Changes of Individual Cyclic Nucleotide- the solved Rh50 structure from bacterial homologue NeRh50 found in Nitroso- Gated Ion Channels by High-Speed Atomic Force Microscopy monas europaea, we employed a combination of molecular dynamics and free Martina Rangl1, Atsushi Miyagi1, Julia Kowal2, Henning Stahlberg2, energy approaches including implicit ligand sampling, explicit ligand sam- Crina M. Nimigean1, Simon Scheuring1. pling, umbrella sampling, and in silico mutagenesis to characterize O2 perme- 1Departments of Anesthesiology, Physiology and Biophysics, and ation pathway, mechanism, and energetics. Free-energy calculations reveal Biochemistry, Weill Cornell Medical College, New York, NY, USA, 2Center high-energy barriers both in the monomeric pore as well as the pore formed

BPJ 7860_7863 Tuesday, February 14, 2017 423a in the trimerization domain similar to those previously reported for CO2 and by gas molecules. The free energy calculations indicate no barriers for the tran- NH3. Based on the the free-energy calculation results, we identified key amino sition of gases from the bulk to the side pores. acids potentially responsible for the high barrier. Bioinformatic analysis of these sites shows that some of the residues lining the high-energy region of 2081-Pos Board B401 the pore exhibit a high degree of conservation only for members of the Rh50 Modeling Ion Selectivity in Transmembrane Domain of the NMDA family, with the exception of NeRh50. Residues Thr199 in Arg202, which Receptor Samaneh Sayede Mesbahi1, Jon W. Johnson2, Maria Kurnikova1. are localized near the pore opening, exist as hydrophobic amino acids in the 1 2 protein’s eukaryotic counterparts and Leu206 in NeRh50 presents itself primar- Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA, University ily as an isoleucine. Mutation of these residues to the conserved amino acids of Pittsburgh, Pittsburgh, PA, USA. results in a reduction in the energy barrier. This suggests a permeation pathway N-methyl-D-aspartate receptors (NMDARs) are members of the ionotropic for gaseous species not observable in the native NeRh50 structure which can be glutamate receptor family that mediate excitatory synaptic transmission in verified by functional characterization of the mutants. the central nervous system. The channels of NMDARs are permeable to Ca2þ but blocked by Mg2þ - a distinctive property that underlies essential 2079-Pos Board B399 processes such as induction of synaptic plasticity. However, due to limited Symmetry Match in Design of Multivalent Inhibitors of Anthrax Toxin structural information on the NMDAR transmembrane ion channel forming Sanaz Momben Abolfath1, Halle Welch1, Vladimir A. Karginov2, domain (TMD), the mechanism of divalent cation permeation and block is Ekaterina Nestorovich1. not yet well understood. We have developed a fully atomistic closed- 1Biology, The Catholic University of America, Washington, DC, USA, channel model of the NMDAR TMD composed of GluN1 and GluN2A sub- 2Innovative Biologics, Inc., Manassas, VA, USA. units (GluN1/2A receptors). The model was generated using (a) a homology Multivalency is a remarkable modern tool to transform relatively weak model based on the NaK channel and a partially resolved structure of an single interactions into a strong binding by creating multiple fine-tuned AMPA receptor, and (b) a partially resolved structure of the GluN1/2B points of contact between multivalent ligands and their receptor inter- NMDAR. Refinement and extensive Molecular Dynamics (MD) simulations faces. The centrosymmetric multisubunit organization is inherent to a num- of the NMDAR TMD model were performed in explicit lipid bilayer mem- ber of channel-forming bacterial exotoxins that oligomerize on a cell surface brane and water. The presented model is stable in all simulations and is constituting the ideal receptor target for rational design of multivalent anti- within the resolution of published NMDAR structures obtained by x-ray toxins. The existing efforts to develop the channel-blocking multivalent in- crystallography. Using the ANTON supercomputer, we performed extensive hibitors concentrate mainly on targeting the anthrax toxin, which is made of multi-microsecond simulations of the NMDAR TMD model structure with a þ þ protective antigen (PA), lethal factor (LF), and edema factor (EF). PA, after Mg2 or Ca2 ion bound to the putative divalent cation binding site in the being cleaved to PA63 and endocytosed, forms a heptameric/octameric chan- channel. The dynamics of the protein and the ions in the binding site are nel that facilitates translocation of LF and EF into the cytosol leading to host inferred from these simulations. NMDAR protein conformational flexibility death. PA channel is selective for cations and thus many positively-charged was similar with no ion bound to the divalent cation binding site and with þ þ ligands were shown to block the channel. This effect was greatly enhanced Ca2 bound, whereas Mg2 binding reduced protein fluctuations in the þ þ with tailor-made sevenfold symmetrical aminocyclodextrins (CDs). The pore region. Furthermore, free energy profiles of a Ca2 or Mg2 ion ap- original idea in design of these compounds was to build the sevenfold sym- proaching the divalent cation binding site were modeled using the Umbrella þ metrical blocker molecules to complement the heptameric structure of the Sampling method with a novel reaction coordinate designed for the Mg2 þ þ PA channel. However later it was shown, that the symmetry requirement ion. Our results reveal that Mg2 binds more strongly than Ca2 to the 63 þ was not strict because both 6þ aCD and 8þ gCD were able to inhibit chan- divalent cation binding site, and that Mg2 experiences a larger free energy nel conductance, with aCD bonding being noticeably weaker and gCD bind- barrier for escaping the binding site. We hypothesize that these differences 2þ 2þ ing being comparable to 7þ bCD. Moreover, PA63 was shown to form establish the mechanistic basis for Ca permeation but Mg block of octamers. To investigate the role of the blocker/target symmetry match, NMDAR channels. we quantify kinetic parameters of the PA63 blockage using aCD, bCD, and gCD. We show that the residence time of the aCD inside PA63 is 6 times Cardiac Muscle Regulation II lower compared to bCD, whereas for bCD and gCD, tres is comparable. In 2082-Pos Board B402 all three systems, tres is an exponential function of the applied voltage. The on-rate shows only weak voltage dependence and changes proportion- Effect of Stimulation Frequency Modification on Kinetic Parameters of ally to the CD’s diameter. Explanted Human Myocardium Jae-Hoon Chung1, Nima Milani-Nejad1, Tallib Karaze1, Ahmet Kilic2, 2080-Pos Board B400 Peter Mohler1, Paul Janssen1. Identification and Energetic Characterization of Gas Permeation 1Department of Physiology and Cell Biology, The Ohio State University, Pathways through a Plant Water Channel Columbus, OH, USA, 2Department of Cardiac Surgery, The Ohio State Ahmad Raeisi Najafi, Paween Mahinthichaichan, Emad Tajkhorshid. University, Columbus, OH, USA. Department of Biochemistry, University of Illinois at Urbana-Champaign, The rate of cross-bridge cycling represents the speed at which actin and myosin Urbana, IL, USA. attach and detach in cardiac myofilaments. This rate is thought to be decreased Aquaporins are homotetrameric membrane channels consisting of four in dilated cardiomyopathy, but little is known about how it changes at different monomeric pores, which facilitate the transfer of water into and out of cellular stimulation frequencies. This may be important in heart failure because the pa- compartments, and a central pore. They also have multiple functions other than tients typically exhibit symptoms due to physical exertion as the heart cannot maintaining cellular osmotic balance. Some aquaporin species mediate the appropriately respond to the increased heart rate to increase contractility. We permeation of gases such as O2 and CO2. Other interesting structural features hypothesized that failing hearts will have slower cross-bridge cycling rates of aquaporins are the side pores, located between each pair of monomers close compared to non-failing hearts as stimulation frequency is increased. We iso- to the protein-lipid interface, with the probability of permeating gases. The lated intact human left ventricular trabeculae from failing hearts and measured current study investigates the permeability of the spinach plasma membrane the rate of tension redevelopment (ktr), an often-used index of rate of cross- aquaporin SoPIP2;1 to O2 and CO2, which are important elements in plant bridge cycling. Here, we present our preliminary data that suggest that ktr in physiology. To provide a complete map of gas migration pathways inside failing left ventricular trabeculae (n=5, 9.3 s1) collected at maximal calcium SoPIP2;1, we employ multi-scale molecular dynamics simulations, namely activation is significantly slower at high stimulation frequency compared to ktr explicit ligand sampling simulations, implicit ligand sampling, umbrella sam- in non-failing left ventricular trabeculae (n=7, 14.0 s1). However, the baseline pling, and in silico mutagenesis. The simulations show that the central pore ktr at 0.5 Hz stimulation frequency were not significantly different between has a very low permeability to gases, and exhibits a drastically different profile failing and non-failing trabeculae (n=5, 11.0 s1 and n=7, 13.8 s1 respec- from permeability for mammalian aquaporins (e.g., AQP1, AQP4, and AQP5). tively). We also found that the maximal force that can be attained via potassium The free energy calculations identify two bottlenecks in the central pore asso- contracture is not significantly different failing and non-failing LV trabeculae ciated with residues Trp79 and Leu75. Trp79 is unique in SoPIP2;1 and is re- across stimulation frequencies we tested (0.5 Hz, 1 Hz, 2 Hz, and 3 Hz, n=4 placed by aliphatic amino acids in eukaryotic aquaporins. We also carried out for failing, and n=7 for non-failing). Failing LV trabeculae had impaired in silico mutagenesis of Trp79 to several mutants and compared the energetics contraction kinetics at 3 Hz, showing higher time-to-peak (TTP) compared to and gas permeability of the mutants to the wild-type protein. The water pores non-failing trabeculae (n=6, 143.5 s and n=5, 127.6 s, respectively). This pre- have low permeability to gases due to the presence of a high energy barrier at liminary data on contractile dysfunction may be explained by the slowed ktr at the selectivity filter. The side pores, on the other hand, can be readily permeated high stimulation frequency.

BPJ 7860_7863 424a Tuesday, February 14, 2017

2083-Pos Board B403 potential propagation with a red-shifted voltage sensitive dye (di-4- The Effect of Ribonucleotide Reductase Overexpression on Cardio- ANBDQPQ) in whole mouse hearts. Control of the electrical activity was myocyte Metabolism achieved by employing transgenic mouse hearts expressing Channel Jason D. Murray1, Farid Moussavi-Harami2, Michael Regnier3. Rhodopsin-2 (ChR2). In order to draw arbitrarily-chosen ChR2 stimulation 1Physiology and Biophysics, University of Washington, Seattle, WA, USA, patterns with sub-millisecond temporal resolution, the macroscope was im- 2Cardiology, University of Washington, Seattle, WA, USA, 3Bioengineering, plemented with a random-access scanning head based on acousto-optic de- University of Washington, Seattle, WA, USA. flectors (AODs). AODs rapidly scan the laser beam across the whole field Our group has repeatedly reported that of 2-deoxy-ATP (dATP) can be used by of view exciting different volume with a commutation time of few ms. At myosin and significantly increases contraction of cardiac muscle at all levels of the end of one cycle the AODs return to the initial position and repeat the calcium activation. We have also demonstrated that overexpressing the enzyme stimulation cycle. Alternatively, a simpler optical solution based on digital ribonucleotide reductase (RNR) in cardiomyocytes using viral vectors in- micromirror device (DMD) in combination with a high power LED was creases the rate and magnitude of contraction and increases left ventricular used to manipulate light positioning in a real simultaneous manner. We em- contraction in normal and infarcted hearts. This increase in work likely results ployed the macroscope to study the mechanistic features of ventricular in a concomitant increase in energy utilization. Thus an important question in tachycardia and we designed mechanistically-based cardioversion/defibrilla- developing this approach as a therapy is to determine whether and how elevated tion patterns exploiting the transient refractoriness of myocardium produced dATP affects cardiac metabolism, which is the goal of this current study. by the ChR2 stimulation. Multiple regions of conduction block revealed to Preliminary data suggest that upregulating RNR results in an increase in the efficiently defibrillate arrhythmic hearts but with lower energy requirements relative contribution of succinate dehydrogenase to mitochondrial respiration as compared to whole ventricle interventions. To confirm that the cardiover- in the heart, possibly indicating an enhanced utilization of fatty acid oxidation sion efficiency is rigorously dependent on the mechanistic-based design, we despite no change in overall respiratory capacity. While mitochondria have a positioned multiple regions of conduction block regardless of the re-entry low affinity for dADP, creatine kinase phosphorylates dADP at a similar rate arrhythmic wavefront obtaining a dramatically reduced cardioversion rate. as ADP, allowing for rapid regeneration of dATP. The overexpression of In conclusion, this work demonstrates that defibrillation energies can be sub- RNR in cardiomyocytes normally results in a tenfold increase in cytosolic stantially reduced by applying discrete stimulation patterns and promotes the concentration of 2-deoxy-ATP (dATP), such that it becomes ~1% of the total investigation of new anti-arrhythmic strategies. ATP pool. We now have a viral vector for expressing a constitutively active form of the Rrm1 subunit (D57N mutation) that may increase cytosolic 2086-Pos Board B406 [dATP] as high as 10% of the ATP pool in cardiomyocytes and in vitro exper- Optogenetic Modulation of Cardiomyocyte Excitability iments indicate this level does not reduce mitochondrial respiratory capacity. Ramona Kopton1, Eva Rog-Zielinska2, Urszula Siedlecka2, Jonas Wietek3, Ongoing and planned experiments will compare the effects of overexpression Peter Hegemann3, Peter Kohl1, Franziska Schneider1. 1Institute for Experimental Cardiovascular Medicine, University Heart of RNR vs. D57N containing RNR on contractile and metabolic properties of 2 cardiomyocytes. Centre Freiburg - Bad Krozingen, Freiburg, Germany, National Heart and Lung Institute, Imperial College London, London, United Kingdom, 2084-Pos Board B404 3Experimental Biophysics, Institute for Biology, Humboldt-University Cardiac Fatty Acid Binding Protein (FABP3) Depletes SR Calcium Load Berlin, Berlin, Germany. in Ventricular Myocytes Optogenetics is a fast-developing technology, first applied to neuroscience Wenjie Li, Shaoran Zhang, Siwei Zhou, Lingling Jiang, Wei Wang. research. A number of studies have begun to transfer the optogenetic Biochemistry and Molecular Biology, Hebei Medical University, approach to cardiovascular research, with a focus on electrical regulation Shijiazhuang, China. of specific cell types in the heart. There is demand to develop tools to selec- Rationale: Cardiac fatty acid-binding protein (FABP3) is a cardiac-specific tively modulate electrical activity in cardiomyocytes (CM) and to interfere member of lipid-binding protein family and its expression level is often reduced with membrane currents in non-myocytes (NM) to advance our understand- in patients with diabetic heart diseases. Recent evidence suggests that FABP3 ing of heterocellular-electrotonic coupling in vitro and in vivo.Wehere suppresses calcium transient and shortening of isolated rat cardiomyocytes. report on cell-type specific activation of co-cultured CM and NM using However, the underlying mechanisms are largely elusive. Objective: To deter- the light-gated cation channel ChR2. Furthermore, we tested three anion- mine the role of FABP3 in regulating SR calcium release. Results: In STZ- selective channelrhodopsins (ACR) for their potential to inhibit action poten- induced type I diabetes mouse model (DM), protein expression level of tial initiation and propagation. FABP3 was elevated, cardiac function was reduced and negatively correlated Neonatal hearts of the lines WT1-VSFPþ;þ, aMHC-VSFPþ;þ and WT1-ChR2- with FABP3 expression level. Amplitudes of cell shortening and calcium tran- H134Rþ;þ were isolated and digested. Cultured cells were transfected with sient are both impaired in DM cardiomyocytes, which can be mimicked by cDNA of ACRs coupled to fluorescent marker proteins. applying FABP3 at pathological concentration to control cardiomyocytes. In order to analyse heterocellular coupling we performed whole-cell patch- FABP3 reduces calcium transient amplitude of cardiomyocytes in a dose clamp recordings on CM cocultured with NM from the WT1-ChR2- dependent manner (EC50 = 0.052 nmol/L). SR calcium content is reduced in H134Rþ;þ line. In a first set of experiments, pulsed ChR2 activation in NM DM cardiomyocytes and FABP3 depletes SR calcium content in cardiomyo- evoked action potentials in patched CM, indicating direct electrical coupling. cytes. FABP3 colocalizes with SERCA, inhibits SERCA activity with a greater Next, we tested three different ACR for their potential to hyperpolarise cardiac EC50 (0.49 nmol/L). Co-immunoprecipitation study suggests elevated FABP3 cells (GtACR1-eGFP, iCþþ mCherry, Phobos-mCherry). Transfected cells promotes binding between SERCA and phospholamban. FABP3 also colocal- were patch-clamped in the current-clamp mode to follow light-induced changes izes with RyR2, binds to RyR2 and promotes RyR2-mediated SR calcium leak. in membrane voltage. Notably, ACR activation lead to hyperpolarisation in iso- Conclusion: Increased FABP3 expression level in DM mice compromises car- lated cardiac NM and to depolarisation in CM. diac function by reducing SR calcium load via two independent approaches: We would like to inhibit cardiac electrical activity by optogenetic hyperpolar- (A) to reduce SERCA activity by promoting PLB-SERCA interaction and isation of NM. As we have shown ACR photocurrents depolarise CM and (B) to enhance RyR2-mediated SR calcium leak. evoke action potentials, different to the inhibitory effect reported in neurons. We expect that NM, which are connected to CM, have more negative poten- 2085-Pos Board B405 tials, resulting in depolarising ACR currents also in NM. Therefore, we are Optogenetics Design of Mechanistically-Based Stimulation Patterns for looking for a stronger tool to inhibit cardiac activity and plan to generate a Cardiac Defibrillation light-activated Kþ-channel. Claudia Crocini1, Cecilia Ferrantini2, Raffaele Coppini2, Marina Scardigli1, Ping Yan3, Leslie M. Loew3, Godfrey L. Smith4, Elisabetta Cerbai2, 2087-Pos Board B407 Corrado Poggesi2, Francesco S. Pavone1, Leonardo Sacconi1. Antihypertrophic Effects of Diazoxide Involves Changes in MIR-132 1LENS, Sesto Fiorentino, Italy, 2University of Florence, Florence, Italy, Expression in Adult Rat Cardiomycytes 3University of Connecticut Health Center, Farmington, CT, USA, 4University Gayathri Narasimhan, Elba Carrillo, Ascencion Herna´ndez, of Glasgow, Glasgow, United Kingdom. Maria C. Garcı´a, Jorge A. Sanchez. Current rescue therapies for life-threatening arrhythmias disregard the path- Pharmacology, Cinvestav, Mexico, D.F., Mexico. ological electro-anatomical substrate and base their efficacy on a generalized Diazoxide (DZX), a mitochondrial KATP channel opener has anti-hypertrophic electrical discharge. Here, we developed an all-optical platform to examine effects in cardiac hypertrophy models but the mechanism involved is unclear. less invasive defibrillation strategies. An ultrafast wide-field macroscope Here we report that DZX prevents cardiac hypertrophy induced by long-term operating at 2 KHz (100 x 100 pixel) was developed to optically map action beta-adrenergic stimulation by regulating miR-132 expression.

BPJ 7860_7863 Tuesday, February 14, 2017 425a

Methods: Hypertrophy was induced by injection of isoproterenol (ISO) ghrelin. Recently some reports indicate that des-acyl ghrelin has biolog- (5mg/kg/day). DZX (100 mM) actions were also assessed in adult rat cardio- ical activity, and inhibits DOX-induced cardiotoxicity. However, molecular myocytes, paced externally at 1 Hz. Rate of ROS (reactive oxygen species) pro- mechanisms of des-acyl ghrelin for the preventive effects of DOX-induced duction was measured by a fluorescent probe. mir-132 expression was assessed cardiotoxicity remains unclear because of lack of the molecular identification by the qRT-PCR technique as 2-DDCT. p-CREB and p-CaMKII protein levels of its specific receptor. In the present study, we sought to compare and deter- were analyzed by Western blot in total fractions of isolated cardiomyocytes. mine the effects of ghrelin and des-acyl ghrelin on DOX-induced cardio- Results: The heart/body weight ratio (mg/g) was 3.450.1 (5) in control exper- toxicity in in vitro system. H9C2 cardiomyocyte cells were treated with iments, 4.950.1 (6) in ISO treated rats and 4.150.1 (3) in ISO þ DZX treated Dox (0-1 mM) for 72h with and without ghrelin or des-acyl ghrelin (1 mM rats. ISO increased miR-132 expression from 1 to 1.550.1 (8) while expression each). Ghrelin and des-acyl ghrelin significantly reduced DOX-induced was 1.0 50.1 (8) in ISOþDZX experiments. The relative rate of ROS produc- cell damage and cell death, with greater effects at des-acyl ghrelin. In addi- tion increased to 3.050.6 (18) by ISO while in ISO and DZX experiments the tion, apoptosis assay showed that DOX induces cell damage and cell death ratio was 1.250.3 (10). H2O2 (100 mM) increased miR-132 expression to on H9C2 cells through apoptosis. Ghrelin and des-acyl ghrelin significantly 1.2450.05 (4) suggesting that miR-132 expression is regulated by ROS and inhibited DOX-induced apoptosis as well as cell damage and cell death. that the protective action of DZX against hypertrophy involves a reduction in Further investigation is ongoing to determine the mechanisms of cardiotox- ROS levels. icity prevented by ghrelin and des-acyl ghrelin and to demonstrate therapeu- ISO increased pCREB expression to 1.6 50.2 (4) which was prevented by tic effect of ghrelin and des-acyl ghrelin on the DOX-induced cardiotoxicity DZX (1.050.2 (3)). H2O2 also increased the expression pCREB suggesting using in vivo mouse model. that up-regulation of miR-132 expression by ROS is mediated by this tran- 2090-Pos Board B410 scription factor. Finally, ROS production induced by ISO was blocked by the 2D CaMKII inhibitor KN-93 (1.150.2 (8)), suggesting that CaMKII is involved Human AC8 Overexpression Increases the Ca Mediated Ensemble and In Vivo Ex Vivo at early stages. Imparts Improved Rhythmicity to the Sinoatrial Node and 1,2 3 1 1 Conclusion: Diazoxide reduces ISO- related hypertrophy by reducing miR-132 Jack M. Moen , Michael G. Matt , Syevda G. Sirenko , Kirill V. Tarasov , 1 1 1,4 1 expression in a ROS production dependent manner in which p-CaMKII and Ismayil Ahmet , Chris Ramirez , Oliver Monfredi , Kenta Tsutsui , 1 5 1 p-CREB play relevant roles. Bruce Ziman , Yael Yaniv , Edward G. Lakatta . 1NIA-LCS, NIH, Baltimore, MD, USA, 2Systems Biology Institute, Yale Supported by CONACyT grants 16946 to JAS and 250937 to MCG and fellow- 3 ship 595030 to GN. University, New Haven, CT, USA, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA, 4Institute of Cardiovascular Sciences, 2088-Pos Board B408 University of Manchester, Manchester, United Kingdom, 5Biomedical Designing Calcium Binding Proteins as Therapeutics against Muscle Engineering, Technion – Israel Institute of Technology, Technion, Israel. Diseases Current thought on mechanisms that underlie cardiac pacemaker cell function Svetlana Tikunova1, Sandor Gyorke1, Frank Brozovich2, includes the idea that cAMP-PKA signaling intrinsic to pacemaker cells þ Brandon Biesiadecki1, Paul Janssen1, Mark Ziolo1, Jonathan Davis1. generates spontaneous, diastolic local Ca2 releases to prompt the generation 1Physiology and Cell Biology, The Ohio State University, Columbus, OH, of rhythmic action potentials. To directly demonstrate the contribution of USA, 2Mayo Clinic, Rochester, MN, USA. Ca2þ- activated adenylyl cyclases (ACs) to intrinsic cellular coupled-clock The calcium ion is a universal second messenger that plays a critical role mechanisms we measured functional changes in isolated, single sinoatrial in numerous cellular processes. Within cells, the calcium signal is trans- nodal pacemaker cells, in SAN tissues, and changes in heart rate (HR) lated into a response by a plethora of calcium binding proteins. The most in vivo in mice overexpressing the human form of Ca2þ activated AC8. common calcium binding motif found in proteins is the helix-loop-helix RNA-Seq analysis confirmed a high expression level of the human AC8 trans- motif termed an EF-hand. We are designing EF-hand calcium binding gene in the TGAC8 mouse SAN (around 330 fragments per kilobase of tran- proteins to palliate, or potentially even cure various skeletal and cardiovas- script per million mapped reads (FPKM)). The level of RyR2 and HCN4 were cular diseases that arise from defects in contraction (via engineering of almost 2 fold higher than Ryr2 expression level and more than 10 fold higher troponin C), relaxation (via engineering of parvalbumin) or electrical then HCN4 expression level in TGAC8 SAN vs WT. To investigate the spe- signaling (via engineering of calmodulin). Since skeletal and cardiac muscle cific role of AC8 in calcium clock function in the absence of AP occurrence diseases have varying etiologies, genetic backgrounds and co-morbidities, and at a fixed free Ca2þ level, we analyzed spontaneous local Ca2þ releases development of personalized therapies is needed. As gene therapy or other (LCRs) in mouse permeabilized sinoatrial nodal cells. The average LCR size protein delivery techniques become better developed, designer proteins was increased 3-fold, and the LCR Ca2þ signal increased 2-fold in AC8 TG could open doors for novel personalized, and potentially, even generalized cells vs WT cells. The spontaneous beating rate of isolated SAN tissue was treatments. 396 5 74 BPM in WT and 503 5 75 BPM in TGAC8 (p-value < 0.05). Telemetry sensors were used to record 24 hour ECGs from wild type 2089-Pos Board B409 littermates (WT) and transgenic mice (TGAC8) overexpressing AC8 under Effects of Ghrelin and Des-Acyl Ghrelin on Doxorubicin-Induced Cardiac a normal light-dark cycle prior to and during i.p. drug administration. Toxicity Comprehensive analysis revealed TGAC8 mice basal heart rates were 1 2 2 Miki Nonaka , Nagomi Kurebayashi , Takashi Murayama , 448 5 31 BPM vs 648 5 88 BPM in WT mice (p-value < 0.0001), approx- 3 1 1 4 Masami Sugihara , Seiji Shiraishi , Kanako Miyano , Hiroshi Hosoda , imately 45% higher than that of WT mice. Moreover, the TGAC8 intrinsic 5 6 2 1,7 Shosei Kishida , Kenji Kangawa , Takashi Sakurai , Yasuhito Uezono . heart rate (i.e. the presence of autonomic blockade via combined atropine 1Division of Cancer Pathophysiology, National Cancer Center Research 2 and propranolol) was approximately 35% higher than in WT mice. Thus, Institute, Tokyo, Japan, Department of Cellular and Molecular our findings in the TGAC8 SAN cells substantiate the pivotal role of Ca2þ- Pharmacology, Juntendo University Graduate School of Medicine, Tokyo, activated cAMP signaling to drive the coupled-clock pacemaker cell system, Japan, 3Department of Clinical Laboratory Medicine, Juntendo University 4 and demonstrate that within intact mice a role of cAMP-dependent regulation School of Medicine, Tokyo, Japan, Department of Regenerative Medicine of intrinsic function of pacemaker cells in the generation of the heart rate and Tissue Engineering, National Cerebral and Cardiovascular Center in vivo. Research Institute, Osaka, Japan, 5Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 2091-Pos Board B411 Kagoshima, Japan, 6National Cerebral and Cardiovascular Center Research Assessment of Thyroid Hormone Induced Hemodynamic Alterations Institute, Osaka, Japan, 7Division of Supportive Care Research, National and Cardiac Irregularities following Antithyroid Drug Treatment and Cancer Center, Exploratory Oncology Research & Clinical Trial Center, Thyroxin Usage Recovery Tokyo, Japan. Nancy S. Saad, Kyle Floyd, Steven Repas, Paul Janssen, Doxorubicin (DOX) is a type of anthracycline antitumor antibiotic and exten- Mohammad Elnakish. sively used for cancer chemotherapy. However, it is known to induce severe Physiology and Cell Biology, Ohio State University, Columbus, OH, USA. adverse effects to non-tumor tissues, especially in the heart. DOX-induced Hyperthyroidism consorts with documented cardiovascular alterations assumed cardiotoxicity occurs in a dose-dependent manner and results in cardiomyop- to be inverted by anti-thyroid treatment. Nevertheless, cumulative data propose athy, and subsequent congestive heart failure often limits DOX-based noteworthy continuing cardiac mortality. Here, we studied the effect of a chemotherapy. Des-acyl ghrelin is the non-octanoylated form of the orexi- well-known anti-thyroid drug, propylthiouracil (20 mg/kg/day/I.P) as well genic hormone ghrelin and considered initially to be the inactive form of as 2 weeks of T4 treatment discontinuation on hemodynamic alterations

BPJ 7860_7863 426a Tuesday, February 14, 2017 and cardiac irregularitiesfollowing 2 weeks of thyroxin (500 mg/kg/day/I.P) reperfusion injury. TREK1 can be a potential target for new cardioprotective administration. Our data showed that thyroxin significantly increased mean agents. arterial pressure (125 5 3 mmHg; p < 0.001) compared to basal group (104 5 2 mmHg). Besides, it led to cardiac hypertrophy and destabilized the Kinesins, Dyneins, and Other Microtubule-based contractile myocardium responses both in-vivo and ex-vivo as indicated by significantly reduced left ventricular ejection fraction (53.29 5 1.70 %; Motors II p < 0.001) and maximal right ventricular isoproterenol response (1.21 5 0.13 fold; p < 0.05) compared to basal group (65.58 5 1.34 % and 2.02 5 2094-Pos Board B414 0.11 fold), respectively. PTU and T4 treatment discontinuation have partial Cooperativity of Kinesin Motor Proteins under External Loads Qian Wang1, Margaret Cheung2, Michael Diehl1, Jose´ Onuchic1, inhibitory effects on T4-prompted increase in blood pressure. Alternatively, 3 1 PTU significantly improved the in-vivo left ventricular dysfunction with no Biman Jana , Anatoly Kolomeisky . 1Rice University, Houston, TX, USA, 2University of Houston, Houston, TX, considerable effects on cardiac hypertrophy or ex-vivo right ventricular con- 3 tractile abnormalities subsequent to T4 treatment. Conversely, T4 treatment USA, Indian Association for the Cultivation of Science, Jadavpur, India. discontinuation showed significant improvements in all T4-provoked cardiac Cellular transport phenomena are supported motor proteins. Although the defects both in-vivo and ex-vivo. In conclusion, our data confirm the proposal cargo motion by a single motor protein has been fully investigated, the that hyperthyroidism-induced cardiovascular irregularities persevere even mechanism of the transportation by multiple motor proteins under external with active anti-thyroid treatment. Also, it demonstrates that the mechanisms loading forces remains unclear. Recent experimental studies on the motion responsible for these deformities are possibly due to mutual thyroid hormone of two interacting kinesins carrying a cargo indicate that they produce forces effects on certain molecular pathways, which require further exploration. that are less than expected form a cooperating team of molecular motors. Moreover, it reveals that full recovery of hyperthyroidism could promptly Using structure-based molecular dynamics simulations, we showed that ameliorate cardiac function and decrease the mortality hazard. However, it con- two kinesins are not able to share the loading forces equally unless they tradicts previous studies showing that the effect of recovery on T4-induced car- are spatially very close. Therefore, the trailing kinesin faces the challenge diovascular abnormalities appears to be model-dependent and should be of catching up to the leading one. However, the velocity of kinesin is weakly cautiously construed. sensitive to the loading forces. The distance between two interacting kine- sins remains large under these circumstances, leading to low cooperativity 2092-Pos Board B412 between them. We further found the microscopic origin of weak sensitivity. Functional Regulation of Small Conductance Calcium Activated It is due to the strong binding affinity between the neck linker and the motor Potassium Channel on Atrial Myocytes by Hydrogen Sulfide in Diabetes head which reduces the ATP releasing rate. Overall, our study provides a Mellitus structurally detailed mechanism that connects the intrinsic structural prop- Dai-Min Zhang, Shao-Liang Chen. erty of the kinesin motor protein to its response to external loads as well Department of Cardiology, Nanjing First Hospital of Nanjing Medical as to its collective behavior University, Nanjing, China. Previous studies established an essential role for Small Conductance Calcium 2095-Pos Board B415 Activated Potassium(SK) channels in cardiac dysrhythmias, especial in atrial Cargo Transport by Teams of Kinesin-1 Motors is Slowed Down by dysrhythmias. Hydrogen sulfide (H2S), an endogenously generated gaseous Macromolecular Crowding transmitter, has recently been suggested to regulate cardiovascular function. Guilherme Nettesheim1, Gabriel Jaffe1, Stephen J. King2, In the present study, we aim to investigate whether H S activity might affect George T. Shubeita3. 2 1 2 function and expression of SK channel via intrinsic signaling pathway with Physics, The University of Texas at Austin, Austin, TX, USA, Burnett or without high glucose induction. Using multiple molecular approaches and School of Biomedical Sciences, University of Central Florida, Orlando, FL, 3 patch clamping techniques, we investigated the mechanisms underlying cardiac USA, Physics Program, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates. protective effects of H2S on SK channel activity in atrial myocytes under dia- betic condition, as well as on SK channel expression. Our results showed that a The cytosol is crowded by a high concentration of macromolecules. Crowding reduction of SK channel expression level in atrial myocytes induced by high can alter protein conformation, binding rates, and reaction kinetics, yet it is not known how crowding affects cargo transport by molecular motors. Here, we glucose reversed by H2S donors in a dose-dependent way. Atrial dysrhythmias report on experiments in which we mimicked cellular crowding in vitro to induced by high glucose were abolished by H2S via activating SK channel. study its consequences on cargo transport by kinesin--1 motors. Surprisingly, These findings indicated that H2S mediate a protective effect by regulating SK channel activity. Therefore, SK channel may serve as a therapeutic target. we find that crowding significantly slows transport by teams of motors, while having a negligible effect on single motor run-length or velocity. To understand 2093-Pos Board B413 how this multiple-molecule behavior emerges from single motor properties, we TREK-1 Has a Protective Role against Global Ischemia-Reperfusion- applied controlled forces on single motors and motor ensembles using an opti- Induced Cardiac Injury cal trap. We find that the detachment kinetics of individual motors in response Cory Parks1, Andreas Schwingshackl2, Salvatore Mancarella1. to force depend on the presence of crowding agents. This change in motor 1Physiology, University of Tennessee Health Science Center, Memphis, TN, response not only explains the differences in the velocity of multiple-motor USA, 2Department of Pediatrics, Mattel Children’s Hospital, UCLA, CA, transport that we observe, but also makes predictions on how cargoes trans- USA. ported by motor ensembles should respond to force, which we confirm using þ TREK-1 (TWIK-related K channels) is a 2-pore domain potassium channel the optical trap. Finally, since in vivo transport is accomplished by multiple- that is activated in the physiological range of action potential. TREK1 is motors and in crowded conditions, our studies suggest that both aspects are thought to be important for the regulation of resting membrane potential, ac- necessary to understand kinesin function in the cell. Indeed, we find that trans- tion potential duration and cell excitability. TREK1 is also expressed in the port by ensembles of motors in living cells is impacted by crowding similarly to heart, but its role in the myocardial response to injury has not been examined. what we observe in vitro. This study assessed the role of TREK1 in the heart and in a murine model of ischemia-reperfusion (I-R) injury. Baseline ECG recordings were established 2096-Pos Board B416 by use of telemetry. Heart rates (beats per minute, bpm) were similar between Kinesin Processivity is Determined by a Kinetic Race from a Vulnerable the two groups: 580 5 12 bpm in WT mice and 587 5 24 bpm in TREK1 One-Head-Bound State knockout (KO) mice, p<0.05, n=10. Global myocardial I-R was performed Keith J. Mickolajczyk, William O. Hancock. on Langendorff perfused isolated hearts from TREK1 KO mice and controls Biomedical Engineering, Penn State University, University Park, PA, USA. (C57/6J). Hearts were subjected to 20 minutes global ischemia and functional Kinesin processivity, defined as the number of steps that occur on average per recovery was measured upon reperfusion (40 minutes). Area of total infarc- interaction with a microtubule, is an important biophysical property that tion was assessed in heart sections with 2,3,5 triphenyltetrazolium chloride underlies the motor’s mechanical and transport capabilities. Kinesin motor (TTC stain) and subsequent imaging analysis. TREK1 KO hearts showed proteins, representing 45 genes in 14 families in the human genome, have significantly larger infarctions compared with control mice given similar processivity values that vary over multiple orders of magnitude. These differ- areas at risk. Accordingly, cardiac function during reperfusion was signifi- ences in processivity are presumably responsible for enabling the vast diver- cantly worsened in TREK1 KO mice compared with WT mice. These data sity of tasks that kinesins carry out in the cell, which include vesicle transport, suggest that TREK1 has a protective role in murine myocardial ischemia- microtubule dynamics control, mitotic spindle alignment, and cytokinesis.

BPJ 7860_7863 Tuesday, February 14, 2017 427a

Despite its fundamental importance to understanding the molecular bases of 2099-Pos Board B419 these cellular processes, a quantitative and testable mechanochemical model Allostery Wiring Map for Kinesin Energy Transduction and its Evolution that describes how structural differences between kinesins alter kinetic steps Jessica Richard, Elizabeth D. Kim, Hoang Nguyen, Catherine D. Kim, in the ATPase cycle to produce functional changes in processivity is lacking. Sunyoung Kim. Here we use high resolution single-molecule microscopy to directly observe Biochemistry & Molecular Biology, LSU School of Medicine & Health the stepping behavior of kinesin-1 and 2 motors with different neck linker Sciences Center, New Orleans, LA, USA. lengths. We identify a one-head bound vulnerable state where a kinetic race How signals between the kinesin active- and cytoskeletal-binding sites are between attachment of the tethered head and detachment of the bound head transmitted is an open question and an allosteric question. By extracting occurs, and find that its duration is negatively correlated with processivity. correlated evolutionary changes within 700þ sequences, we built a model of Using a cross-family comparative approach, we map functional differences residues that are energetically coupled and that define molecular routes for back to structural differences to build an understanding of the design princi- signal transmission. Typically, these coupled residues are located at multiple ples underlying motor processivity. Overall, our results provide a quantitative distal sites and, thus, are predicted to form a complex, nonlinear network framework for understanding kinesin processivity both biophysically and as it that wires together different functional sites in the protein. Of note, our model pertains to the physiologically relevant emergent behaviors that result from its connected the site for ATP hydrolysis with sites that ultimately utilize its tuning. free energy, such as the microtubule-binding site, drug-binding loop-5, and necklinker. To confirm the calculated energetic connectivity between non- 2097-Pos Board B417 adjacent residues, double-mutant cycle analysis was conducted with 22 kinesin High Resolution Three-Dimensional Tracking with Optical Tweezers mutants. There was a direct correlation between thermodynamic coupling in Reveals Protofilament Switching of the Kinesin-8 KIP3 experiment and evolutionarily-derived energetic coupling. We conclude that Michael Bugiel, Erik Schaeffer. energy transduction is coordinated by multiple distal sites in the protein, rather Center for Plant Molecular Biology, University of Tuebingen, Tuebingen, than only being relayed through adjacent residues. Moreover, this allosteric Germany. map forecasts how energetic orchestration gives rise to different nanomotor The budding yeast Kinesin-8 Kip3 is a highly processive motor protein behaviors within the superfamily. that walks to the end of cytoskeletal microtubules and shortens them in a collective manner. How exactly Kip3 reaches the end is unclear. Microtu- 2100-Pos Board B420 bules usually consist of 12 to 16 circularly arranged tubulin polymer chains, Altered Mechanical Properties of Kinesins with Mutations that Cause called protofilaments. Left-handed rotations of microtubules in Kip3 gliding Hereditary Spastic Paraplegia assays indicated sideward motion of Kip3 perpendicular to the microtubule Chelsea Kelland, Khari Gilmore, Lauren Thornton, Liautaud Prophete, axis, i.e. a switching between single protofilaments. We used high resolution Thomas M. Huckaba. optical tweezers in a force-feedback mode to track the path of single Kip3 Biology, Xavier University of Louisiana, New Orleans, LA, USA. motors. Previous 2D assays with alternating sideward loads showed that Hereditary Spastic Paraplegias (HSPs) are a genetically and clinically hetero- Kip3 steps sideward in both directions consistent with a diffusive sideward geneous set of neurodegenerative diseases that all share an axonopathy of the motion of Kip3 on the microtubule lattice. Here, we topographically sus- corticospinal tract. To date, there are at least 70 distinct genetic loci that, pended microtubules such that Kip3-coated microspheres can freely rotate when mutated, cause HSP in humans. One of those loci is the neuronally en- around the microtubules in three dimensions. Tracking these single-motor riched kinesin transport motor, Kif5A. Of particular interest to this study is driven microspheres with a 3D, zero-load force-clamp showed that Kip3 that 22 of the 25 mutations in Kif5A that have been shown to cause HSP are switched protofilaments in discrete steps equally frequent in both directions. in the motor domain - the portion of the kinesin protein that has the dual respon- A statistical analysis confirmed a diffusive sideward motion of Kip3, con- sibility of interacting with the microtubule and carrying out the ATPase cycle. sistent with the 2D results. Interestingly, this sidewards diffusion was Perhaps unsurprisingly, most of the mutations cluster to either the microtubule bounded by the microtubule seam. The diffusive switching mechanism binding site or the nucleotide binding pocket. We have recombinantly- may enable Kip3 to bypass obstacles and reach the microtubule end for expressed Kif5A with each of the separate HSP-causing mutations and have length regulation. performed a series of well-characterized in vitro assays (ATPase, stopped flow, microtubule affinity, microtubule gliding, single molecule motility) to 2098-Pos Board B418 better understand the altered catalytic properties that result from each mutation. A Comparative Single-Molecule Study Between the Mechanical Stability In addition, we have performed molecular dynamics simulations to model the of Kinesin KIF16B Attachment to Lipid Membranes and to Microtubules altered structural properties of each of the mutant proteins. We find that all mu- Serapion Pyrpassopoulos, Henry Shuman, E. Michael Ostap. tations are loss-of-function mutations, and that there are separable functional Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, deficits for each mutation that limit kinesin’s ability to functionally transport PA, USA. cellular cargoes. KIF16B is a member of the highly processive kinesin-3 family that partici- pates in the trafficking and tubulation of early endosomes along microtubules. 2101-Pos Board B421 KIF16B attaches to lipid cargos via a PX motif at its C-terminus which has Eg5 Inhibitors have Contrasting Effects on Microtubule Stability and high affinity for bilayers containing phosphatidylinositol-3-phosphate Spindle Integrity Depending on their Modes of Action (PI3P). As the PX domain has been proposed to be a primary mechanical an- Geng-Yuan Chen1, You-Jung Kang1, A. Sophia Gayek2, Wiphu Youyen3, chor for the KIF16B-cargo attachment, we measured the adhesion forces and Erkan Tuzel€ 3, Ryoma Ohi2, William O. Hancock1. detachment kinetics of the PX domain as it interacts with membranes contain- 1Department of Biomedical Engineering, Pennsylvania State University, ing 2% PI3P and 98% DOPC. Using optical tweezers, we found the PX University Park, PA, USA, 2Department of Cell and Developmental Biology, detachment-rate to be loading rate dependent between 5 – 35 pN with rates Vanderbilt University, Nashville, TN, USA, 3Department of Physics, ranging from 0.32 5 0.11 s1 –175 3.3 s1, respectively. The rates of these Worcester Polytechnic Institute, Worcester, MA, USA. separation events were substantially slower than those measured when the PX The goal of this work was to investigate the mechanisms underlying different domain was adhered to the PI3P headgroup in the absence of a fluid bilayer, Eg5 (kinesin-5) inhibitors and to assess their effects on Eg5-mediated stabiliza- consistent with previous steady-state biochemical experiments that suggest tion of dynamic microtubules. Antimitotic Eg5 inhibitors were grouped into hydrophobic residues within the PX domain interact with the bilayer. We those that bind Loop-5 and produce an ADP-like state (monastrol, STLC, ispi- compared the PX membrane-attachment durations with the motile kinetics nesib and filanesib) and those that produce a rigor-like state (BRD9876). To and mechanics of a two-headed KIF16B-motor construct. In the presence of understand their contrasting effects on Eg5 mechanics, inhibitors were 1 mM MgATP, the most probable detachment force and dissociation rate of analyzed in mixed-motor gliding assays consisting of different ratios of a processing molecule are 5.2 5 1.4 pN and 0.19 5 0.041 s1, respectively. kinesin-1 and Eg5 motors, in which Eg5 ‘‘braking’’ dominates motility. The dissociation rate of a single KIF16B-PX domain from 2% PI3P at a Loop-5 inhibitors increased gliding speeds, consistent with the compounds similar force is 0.32 5 0.11 s1. KIF16B dimerizes upon binding to endo- inducing a weak-binding state in Eg5, whereas BRD9876 slowed gliding, somes, so we expect the membrane attachment duration to exceed the consistent with Eg5 being locked in a rigor state. Quantitative critical detach- microtubule interaction lifetime. Our results suggest that the membrane-PX ment force analysis (Arpag et al. Biophys J. 2014) indicates that L5 inhibitors interaction is a suitable anchor to support KIF16B motor activity, and pro- enhance Eg5 detachment whereas the rigor inhibitor BRD9876 generates an vide a biophysical framework for a better understanding of the mechanics Eg5 species with decreased susceptibility of detachment. Biochemical and of endosome transport. This work was supported by NIH grant GM087253 single-molecule assays demonstrated that BRD9876 acts as an ATP- and to EMO and HS. ADP-competitive inhibitor with a KI of 4 nM. Consistent with its recently

BPJ 7860_7863 428a Tuesday, February 14, 2017 reported microtubule polymerase activity (Chen and Hancock, Nature Comm. how noncanonical MT interaction of Cin8 establishes a mechanism for bidirec- 2015:8160) and its dominant two-head-bound strong-binding population (Chen tional motility of Cin8 along MTs. et al., JBC 2016:M116), Eg5 was shown to stabilize microtubules against depo- lymerization in vitro following taxol washout. This stabilization was eliminated 2104-Pos Board B424 in monastrol but was enhanced by BRD9876, consistent with strong binding of Exploring the Mechanism of Microtubule Depolymerization by the Eg5 to microtubules enhancing microtubule stability. Finally, in metaphase- Kinesin 13 KLP10A and it’s Phosphoregulation arrested RPE-1 cells, STLC promoted spindle collapse, whereas BRD9876 Matthieu P.M.H. Benoit, Daniel J. Diaz-Valencia, Ana B. Asenjo, did not. Thus, different Eg5 inhibitors inhibit spindle formation through con- Gary J. Gerfen, David J. Sharp, Hernando J. Sosa. trasting mechanisms and rigor inhibitors are predicted to paradoxically stabilize Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, NY, microtubule arrays in cells. USA. Kinesins-13s are non-motile molecular motors that regulate microtubule (MT) 2102-Pos Board B422 dynamics by promoting MT depolymerization activity at the MT tips. We have Regulation of Activity of the Kinesin-5 CIN8 used cryo-electron microscopy and functional assays to investigate the mecha- Larisa Gheber1, Alina Goldstein1, Ofer Shapira1, Ervin Valk2, Mart Loog3, nism of action and regulation of the Drosophila melanogaster kinesin-13 Nurit Siegler1, Darya Goldman1. KLP10A. Previous work has shown that phosphorylation of serine 573 in the 1Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel, KLP10A motor domain down regulates depolymerization activity. We have ob- 2Chemistry, University of Tartu, Tartu, Estonia, 3Institute of Technology, tained cryo-EM sub-nanometer resolution maps of constructs that include the University of Tartu, Tartu, Estonia. KLP10A motor and N-terminal neck domains bound to MTs and to tubulin The homoterameric bipolar kinesin-5 motors perform essential functions in depolymerization intermediates. Comparison of these structures revealed mitotic spindle dynamics by crosslinking and sliding apart antiparallel micro- key features of the depolymerization process. To study the down-regulation tubules. S. cerevisiae cells express two kinesin-5s Cin8 and Kip1, which mechanism by phosphorylation we have combined a variety of cell biology overlap in function. We have recently demonstrated that Cin8 and Kip1 are and biophysical methods and compared the behavior of phosphomutant minus-end directed on the single-molecule level and can switch directionality (S573E) and wild-type (WT) KLP10A. Surprisingly, we found that the binding under a number of conditions (Duselder et al., 2015; Fridman et al., 2013; affinity and ATPase activity stimulated by curved tubulin polymers (mimicking Gerson-Gurwitz et al., 2011). The mechanism of this directionality switch MT ends) of WT-KLP10A and S573E-KLP10A were relatively similar (kcat and its physiological significance remain unclear. We have also demonstrated of 1.8 s1 and 2.5 s1 respectively). In contrast, we found significant differ- 1 1 that Cin8 is differentially phosphorylated during late anaphase at three cyclin- ences in their MT-lattice stimulated ATPases (kcat of 0.56 s and 0.14 s dependent kinase 1 (Cdk1) sites located in its motor domain. This phosphory- respectively). We also found that the mutant S573E had shorter residence lation regulates Cin8 activity during anaphase (Avunie-Masala et al., 2011), time relative to WT (t of 5 s and 28 s respectively) while undergoing one- but its mechanism remains unclear. Here we examined the hierarchical regula- dimensional diffusion over the MT lattice. These results suggest a model in tion of the activity of Cin8 by in vitro kinase assay, live-cell imaging and in which phosphorylation of S573 reduce KLP10 MT depolymerization activity vitro motor motility assay. We also developed quantitative analyses to by decreasing the probability of reaching the MT ends by one dimensional compare the contributions of the three Cdk1 sites in the catalytic domain of diffusion. Consistent with this model we found by fluorescent live cell imaging Cin8 to regulation of its intracellular activity. We found that the three Cdk1 that KLP10A-S573E MRFP labeled constructs accumulate significantly less at sites undergo phosphorylation in vitro by Cdk1 kinase and that this phosphor- the tip of growing MTs than WT-KLP10A MRFP labeled constructs. ylation affects the motile properties of Cin8. Our analysis also reveals that each one of the Cdk1-specific sites in the catalytic domain of Cin8 control its local- 2105-Pos Board B425 ization to the anaphase spindles. Phospho-deficient mutation at the S277 site Analysis of the Motility Properties of the Kinesin-4 Familiy Members exerts the strongest effect, indicating that phosphorylation of this site primarily KIF7 and KIF27 by Cdk1, regulates the dynamics of Cin8 attachment to- and detachment from Yang Yue1, T. Lynne Blasius1, Stephanie Zhang2, Benjamin Walker2, the anaphase spindles and contributes to the regulation of its function. 1 Frid- Jared C. Cochran2, Kristen J. Verhey1. man, V. et al. J Cell Sci 126, 4147-4159, doi:10.1242/jcs.125153 (2013). 1Department of Cell and Developmental Biology, University of Michigan 2 Gerson-Gurwitz, A. et al. Embo J 30, 4942-4954 (2011). 3 Duselder, A. Medical School, Ann Arbor, MI, USA, 2Department of Molecular and et al. J Biol Chem 19, 620799 (2015). 4 Avunie-Masala, R. et al. J Cell Sci Cellular Biochemistry, Indiana University, Bloomington, IN, USA. 124, 873-878 (2011). The kinesins are a large superfamily of ATP-dependent motor proteins that can walk along microtubules to transport cargo. Some members of the kinesin-4 2103-Pos Board B423 family have been shown to play critical roles in Hedgehog signal transduction. Noncanonical Microtubule Interaction of Yeast Kinesin-5 CIN8 However, no motility has been observed for the Drosophila motor Costal2 Kayla Bell1, Hyo Keun Cha2, Ardian S. Wibowo1, Charles V. Sindelar2, (COS2) and its mammalian homologue KIF7 appears to lack the ability to Jared C. Cochran1. undergo processive motility. Thus, the motility and enzymatic properties, 1 Department of Molecular and Cellular Biochemistry, Indiana University, and thus the functional roles, of the COS2/KIF7/KIF27 subfamily remain 2 Bloomington, Bloomington, IN, USA, Department of Molecular Biophysics poorly understood. Using single-molecule imaging, we found that KIF27 is a and Biochemistry, Yale University, New Haven, CT, USA. very slowly-processive motor whereas KIF7 shows no motility in vitro. This Kinesin-5 motors play central roles in establishing and maintaining the mitotic is in contrast to other kinesin-4 family members, such as KIF4 and KIF21A, spindle during cell division. Cin8 is a homotetrameric kinesin-5 in Saccharo- which show processive motility. To test whether defective kinesin-4 motors myces cerevisiae that moves bidirectionally along microtubules (MTs), switch- become processive when working in teams, we carried out microtubule gliding ing directionality based on ionic strength, motor coupling and antiparallel MT assays in vitro and peroxisome transport assays in living cells. In both assays, binding. Here we have investigated MT interactions of the truncated Cin8 KIF7 failed to transport cargo whereas KIF27 showed slow motility, consistent ATPase motor domain under different ionic strength conditions using thermo- with their motility in single molecule assays. Using a microtubule pelleting dynamic, kinetic, and structural methodologies. Bacterially expressed and assay and ATPase activity assays, we found that the inability of KIF7 and purified Cin8 motors were predominantly monomeric in solution and showed KIF27 to show fast processive motion correlates with defective ATPase activ- MT-stimulated ATPase activity. Cosedimentation experiments showed tight, ity. Thus, we show that the COS2/KIF7/KIF27 subfamily exhibits processivity superstoichiometric Cin8 binding to MTs with approximately four motors defects that are evolutionarily conserved. We propose that these kinesins do not bound per ab-tubulin dimer in the MT lattice under various biochemical and function as transport motors but rather serve as scaffold proteins that link nucleotide conditions. Control reactions demonstrate defined and reversible Hedgehog signaling pathway components to microtubule filaments. Cin8 oligomerization on the MT. Competition assays with human kinesin-5 Eg5 showed Cin8 binding to both the canonical kinesin MT binding site as 2106-Pos Board B426 well as a hypothesized noncanonical MT binding site. Using a mutant Eg5 defi- Photo Regulation of Kinesin Motor Activity Utilizing Photochromic cient in ATPase activity, different Cin8 constructs demonstrated variable MT- Dronpa-Loop11 Fusion Protein stimulated ATPase activity via the noncanonical MT binding site. Removing Kohei Uchida, Shinsaku Maruta. the large loop L8 insert in Cin8 resulted in weakened MT binding with apparent Bioeng, Maruta Lab, Soka University, Hachioji, Tokyo, Japan. stoichiometric MT binding. We obtained insight into Cin8 interaction with the Kinesin is an ATP driven motor protein that moves along microtubules. Ki- canonical MT binding site from cryo-EM reconstructions at 7-8 A˚ resolution. nesin plays physiologically important roles of intracellular transport. Struc- Steady state kinetics of Cin8 ATPase showed tight MT and ATP binding, ture and energy transducing mechanism of kinesin have been well studied. with weakened MT binding at higher ionic strengths. We present a model for Previous crystallographic studies revealed that kinesin has several unique

BPJ 7860_7863 Tuesday, February 14, 2017 429a loops in its motor domain. Loop11 and Loop12 are located in the microtu- The giant sarcomeric protein titin (up to 3.7 MDa) spans the half-sarcomere and bule binding region of kinesin. They were considered to bind to microtubule has been regarded as blueprint for sarcomere assembly. Only little is known alternately during motility cycle with ATP hydrolysis. Previously we have about the kinetics of titin’s integration into the myofilament and its mechanistic demonstrated that incorporation of photochromic molecules into the micro- role in sarcomere assembly. We study sarcomere dynamics by recombining flu- tubules binding site of kinesin enabled photo-reversible regulation of kinesin orophores into the titin filament. In cardiomyocytes derived from knock-in ATPase activity. In this study, we designed the photochromic protein mice expressing a titin fusion protein with eGFP attached to M-Band exon 6, ‘‘Dronpa derivative 145K-145N’’ fused with kinesin microtubule binding FRAP experiments show an unexpected level of mobility of M-band titin site, ‘‘Loop11’’ and ‘‘Loop12’’ in order to control kinesin motor activity that is calcium dependent. To investigate titin’s role in sarcomere assembly photo-reversibly. First, the Dronpa 145K-L11-145N fusion protein was pre- and disassembly we inserted dsRed into titin’s Z-disc region. The insertion pared. 145K-L11-145N showed reversible absorption spectral changes upon of the fluorescent proteins at the N- and C-terminus of titin does not interfere 400 nm and 500 nm light irradiation in a manner consistent with original with normal cellular function and double heterozygous knock-in animals Dronpa tandem dimer 145K-145N. Ionic strength dependent microtubules have no obvious phenotype. The animals survive, are fertile, and have normal pelleting assay suggested that 145K-L11-145N associate with microtubules body and heart weights. Their myocytes allow us to simultaneously visualize electrostatic interaction. Bright state of 145K-L11-145N inhibited conven- titin’s N- and the C-terminus using live cell imaging. Towards elucidating ti- tional kinesin driven microtubules gliding in the in vitro motility assay. tin’s role in myofilament dynamics we follow titin expression as well as sarco- Moreover, Bright state of 145K-L11-145N inhibited microtubule dependent mere assembly and disassembly in differentiated mouse ES-cells, embryonic kinesin ATPase activity with the IC50 of 2 mM. We examined also dark state and neonatal myocytes, and satellite cells derived from titin-eGFP and -dsRed of 145K-L11-145N. double heterozygous mice. During myofibrillogenesis in cardiomyocytes and skeletal muscle cells, titin integrates simultaneously into the Z-disc and 2107-Pos Board B427 M-band of the sarcomere. Finally, we use our animal models to follow titin Characteristic Properties of Novel Photochromic Inhibitor of Kinesin Eg5 synthesis, transport and degradation in developing and mature myocytes. The Composed of Spiropyran Derivative combination of fluorescent proteins, mouse genetics, live imaging, and super Kei Sadakane, Mao Takaichi, Ryoma Yamamoto, Shinsaku Maruta. resolution microscopy provides a deeper understanding of sarcomere assembly Soka University, Tokyo, Japan. and dynamics. Kinesin Eg5 which is a kind of motor protein has an important role on the cell division. Eg5 tetramer crosslinks with anti-parallel microtubule and induce for- 2110-Pos Board B430 mation of bipolar spindle in M phase. It is known that kinesin Eg5 overexpress Myosin II-Dependent Suppression of Podosomes by an ARNO-ARF1 in some of cancer cells. Kinesin Eg5 has been considered as a target of anti- Signaling Axis cancer therapy. Several small molecules were recognized as potent inhibitors Nisha Mohd Rafiq1, Zi Zhao Lieu1, TingTing Jiang1, Cheng-han Yu1, of Eg5. Interestingly the inhibitors bind to the common pocket in Eg5 motor Paul Matsudaira1, Gareth Jones2, Alexander Bershadsky1. domain. STLC is one of the well-known potent inhibitor of Eg5. The crystallo- 1Mechanobiology Institute, Singapore, Singapore, 2King’s College London, graphic structure of Eg5-STLC complex revealed that STLC binds to the London, United Kingdom. pocket composed of a2, a3 helix and loop L5. The inhibitory mechanism of Podosomes represent a special class of integrin-mediated cell-matrix adhesions STLC has been well studied. Photochromic molecules such as azobenzene formed by migrating and matrix degrading cells. Here, we demonstrated that in and spiropyran derivatives, which change their structures and properties revers- macrophage-like THP1 cells and in fibroblasts stimulated to produce podo- ibly by light irradiation, are expected to be applicable to photo-switches of somes, down-regulation of ARF1 by siRNA, and by pharmacological inhibitors bionanomachines. Previously we have demonstrated that STLC analogues led to striking podosome elimination. Treatments that induced podosome composed of azobenzenen (ACTAB) or spiropyran (SP-APA) inhibit Eg5 formation increased the level of GTP-bound ARF1. Furthermore, siRNA ATPase activity and motor activity photo-reversibly upon UV and visible light knockdown of the ARF1-GEF ARNO also resulted in dramatic podosome irradiations. Moreover, HeLa cell division was photo-regulated with ACTAB. inhibition. ARNO was found to co-localize with the adhesive rings of podo- In this study, we have tried to study the optimum conditions to regulate the somes while ARF1 was localized to vesicular structures transiently contacting function of Eg5 with the photochromic inhibitors we have synthesized. In the podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels results, pH and ionic strength dependent effect of the inhibitors on the ATPase and triggered assembly of myosin-IIA filaments in THP1 cells that resemble activity and Eg5 driven microtubule gliding. At pH6.8, SP-APA showed signif- sarcomere-like organization typically observed in fibroblasts, whilst the sup- icant efficiency to control ATPase and motor activities as an inhibitor that has a pression of myosin-IIA rescued podosome formation despite ARF1 inhibition. photo-switching system. Finally, expression of constitutively active ARF1 in fibroblasts induced forma- tion of putative podosome precursors; actin-rich puncta that coincided with ma- 2108-Pos Board B428 trix degradation sites and containing proteins of the podosome core but not of Tight Coupling between the Heat Dissipation and Molecular Motor’s the adhesive ring. We conclude that ARNO-ARF1 regulates formation of podo- Transport Properties in Nonequilibrium Steady State somes by inhibition of Rho/myosin II and promotion of actin core assembly. Wonseok Hwang, Changbong Hyeon. Computational Sciences, Korea Institute for Advanced Study, Seoul, Korea, 2111-Pos Board B431 Republic of. Local Pulses of Rhoa Activation Assemble Polarized Network We report a theoretical analysis showing tight coupling between velocity V, Architectures for Efficient Actomyosin Contractility diffusion coefficient D, and the heat dissipation of kinesin-1 motor protein Franc¸ois B. Robin1, Jonathan M. Michaux2, Edwin M. Munro2. which is reminiscent of the recent surprising observation of enhanced diffu- 1Developmental Biology Department, Institute for Biology Paris-Seine, sivity of exothermic enzyme in solution by its own catalytic turnover (Riedel Paris, France, 2Molecular Genetics and Cell Biology, University of Chicago, et al., Nature, 2015). From the quantification of V, D, and heat dissipation in Chicago, IL, USA. terms of rate constants using periodic one-dimensional hopping model, we Spatiotemporal patterning of actomyosin contractility plays a key role in cell found: (i) D increases in the form of 3rd order polynomial of V when V is and tissue morphogenesis during early development. In embryonic cells, acto- augmented by the increase of ATP concentration; (ii) the increase of diffu- myosin arrays are highly dynamic structures that remodel on a time scale of 10s sivity with the heat production is a natural outcome of systems in non- of seconds, through a combination of local actomyosin turnover and rapid equilibrium steady states; (iii) the energetic cost for determining the position spatial redistribution of filaments and motors caused by myosin activity or actin of a kinesin-1 with a given precision is close to lower bound. polymerization. Because of these dynamic and active properties, contractility is complex and intrinsically self-organizing. Cytoskeletal Assemblies and Dynamics We used the C. elegans early embryo to understand how cells pattern force gen- eration through local modulation of self-organized contractility, focusing on 2109-Pos Board B429 pulsed contractility in the C. elegans embryo. From Ribosome to Sarcomere - Titin Dynamics in Striated Muscle Cells We combined two-color fluorescence imaging, live single-molecule imaging, Michael Gotthardt1, Franziska Rudolph1, Judith Huettemeister1, particle tracking, image analysis, and numerical modeling to tease apart the Katharina da Sliva Lopes1, Lily Yu2, Nora Bergmann1, Claudia Fink1, mechanisms of pulse initiation and termination. Our results demonstrate that Eva Wagner3, Stephan Lehnart3, Carol Gregorio2. the mechanical component (advection) played little role in pulse initiation or 1Max Delbrueck Center for Molecular Medicine, Berlin, Germany, termination, and that the process was mostly governed by Actin and Myosin 2University of Arizona, Tucson, AZ, USA, 3Goettingen University, turnover. In our system, autocatalytic RhoA activation/recruitment is respon- Goettingen, Germany. sible for pulse initiation, while the delayed recruitment of a RhoA inactivator

BPJ 7860_7863 430a Tuesday, February 14, 2017

(RGA-3/4) onto Actin filaments drives pulse termination. Using single- results highlight that variations in the localization and/or pharmacological molecule imaging, we further show that this mode of actin assembly drives mechanism of NM2 inhibition produce distinct effects on intracellular strain the formation of structural units displaying a characteristically polarized archi- and cellular morphogenesis. tecture with (1) a gradient of myosin accumulation, myosin being recruited at Supported by Hungarian Research and Innovation Fund (VKSZ_14-1-2015- the center of the pulses and (2) a transient anisotropic organization of the actin 0052). network, where filaments elongate from the instide of the pulse, barbed ends pointing outwards, as visualized by displacement of the actin-barbed-end- 2114-Pos Board B434 tracking formins that processively elongate actin filaments. An Potogenentic Toolkit for Reversible Labeling and Remote Based on these results, we propose here that pulsed contractions represent a Manipulation of Cytoskeleton In Situ mode of actomyosin assembly which structures the actomyosin network in Qian Zhang1,2, Lian He1, Guolin Ma1, Yubin Zhou1,3. 1 functional modules to drive efficient cell contractility. Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, Houston, TX, USA, 2Department of 2112-Pos Board B432 Infectious Diseases, Renmin Hospital of Wuhan University, Wuhan, China, Lifetime of Membrane-Cytoskeleton Bonds is Mediated by RHO-GTPases 3Department of Medical Physiology, College of Medicine, Texas A&M in a Cancer Cell University, Temple, TX, USA. Vivek Rajasekharan1, Varun K.A. Sreenivasan1, Jeffrey N. Myers2, Most of the current methodologies to visualize the cytoskeleton are irreversible Fred A. Pereira1, Brenda Farrell1. and can only provide a static picture of cytoskeleton. These methods often 1Baylor College of Medicine, Houston, TX, USA, 2The University of Texas involve the fixation of cells or permanently perturb the behaviors of cyto- MD Anderson Cancer Center, Houston, TX, USA. skeleton. To overcome this obstacle, we designed a set of optogenetic tools Cells especially cancer cells are capable of rapid cytoskeleton remodeling to reversibly label and manipulate cytoskeleton dynamics in living cells by and often membrane reorganization in response to environmental cues. The harnessing the power of light. These genetically-encoded small optical tools propensity to remodel is reflected in part by the lifetime t of the membrane- were demonstrated to i) instantly and reversibly photo-label actin, microtubule cytoskeleton bonds at the edges of cells; t increases as their number density and the plus-end of microtubule without perturbing their normal functions, and on-rate increases. We measure t by monitoring the time-course of the ii) manipulate the dynamics of microtubule following stimulation with light membrane peeling force Fp at the edge of a cancer cell. Experiments are con- emitting at varying wavelengths; and iii) drive the target protein or organelle ducted at the near-equilibrium region (cf. irreversible), where an optical twee- towards either ends of MTs to recruit effectors and control the downstream zers is used to apply a load with a handle which is bound to a slowly moving signaling pathways. These noninvasive optical tools provide new opportunities cell, and the handle displacement is detected at a resolution of 500 ms after to remotely monitor and perturb the cytoskeleton, and can also be applied in averaging. drug screening to identify potential adverse effects of drug candidates on cyto- R Under constant load, Fp increases monotonically with time; at force f and time skeleton. (Supported by the National Institutes of Health (GM112003) and the tR this slope abruptly changes indicating membrane-cytoskeleton bond rupture. Welch Foundation (BE-1913 to Y.Z.), and a China Scholarship Council award Repeating for many cells we find fR is not constant but increases with tR.We to Q.Z.) propose it represents the rupture of a cluster of bonds with larger clusters demonstrating greater rupture forces and lifetimes. This is in agreement with 2115-Pos Board B435 theory that calculates the lifetime of a cluster of bonds between two bodies Mechanics and Dynamics of Cation-Induced Actin Bundles (J.Chem.Phys.121:8997). Comparing experimental data with this theory, we Nicholas Castaneda1, Tianyu Zheng2, Hector Rivera-Jacquez1, Qun Huo2, find that molecular parameters are within expected ranges reported for biomol- Hyeran Kang1. ecular bonds in vitro withevidence that the bonds can re-bind. Cells treated with 1NanoScience Technology Center, University of Central Florida, Orlando, Rho-GTPase inhibitors possess membrane-cytoskeleton bonds with lower stiff- FL, USA, 2NanoScience Technology Center, Department of Chemistry, ness that show no rebinding on the timescale of the measurements (zero on- University of Central Florida, Orlando, FL, USA. rate). This is predictable since active Rho-GTPases form linkages between The assembly of actin filaments into bundles plays an essential role in mechan- the membrane and actin-effector proteins of the cytoskeleton. This measurable ical strength and dynamic reorganization of cytoskeleton. Divalent counterions change in bond properties provides a quantitative method for evaluating the at high concentrations promote bundle formation through electrostatic attrac- role of Rho-GTPases in dynamic cytoskeleton remodeling. This is relevant tion between charged filaments. Although it has been hypothesized that specific as Rho-GTPases are upregulated in many human cancers including the HN- cation interactions may contribute to salt-induced bundling, molecular mecha- 31 cell line used in this study. nisms of how salt modulates bundle assembly and mechanics are not well es- tablished. Here we determine the mechanical and dynamic properties of actin 2113-Pos Board B433 bundles at varying divalent cation concentrations. Using total internal reflection Subcellular Spatial Control of Non-Muscle Myosin 2 Redistribution and fluorescence (TIRF) microscopy, we measure the bending stiffness of actin Stress Fiber Strain by Molecular Tattoo bundles determined by persistence length analysis. We characterize real-time Adam I. Horvath, Boglarka H. Varkuti, Miklos Kepiro, Gyorgy Hegyi, formation of bundles by dynamic light scattering intensity and direct visualiza- Mihaly Kovacs, Andras Malnasi-Csizmadia. tion using TIRF microscopy. Our results show that divalent cations stiffen actin Department of Biochemistry, MTA-ELTE Molecular Biophysics Research bundles and modulate time-dependent average bundle size. The work suggests Group, Eotvos Lorand University, Budapest, Hungary. that cation interactions serve a regulatory function in actin bundle mechanics The cellular distribution of the motor protein non-muscle myosin 2 (NM2) and dynamics. leads to different forms of intracellular strain driving cell motility, cytoki- nesis and morphogenetic processes including axonal growth and retraction. 2116-Pos Board B436 However, it remains elusive how these cellular processes are governed by CaMKII Control of Actin Cytoskeletal Dynamics the dynamic changes in NM2 localization and supramolecular assembly. Shahid M. Khan1, Justin E. Molloy2. To address this problem, we determined the effect of different types of 1Macromolecular Dynamics, Molecular Biology Consortium, Berkeley, CA, NM2 inhibition on the dynamics of load-bearing stress fibers and unloaded USA, 2Mill Hill Laboratory, The Francis Crick Institute, London, United inner cytoplasmatic NM2 structures in live HeLa cells. We followed NM2 Kingdom. redistribution via FRAP, applied also in combination with our recently The calcium calmodulin dependent kinase (CaMKII) binds and organizes actin developed optopharmacological tool, Molecular Tattoo, which allows sub- Binding is abolished by calcium calmodulin, triggering cytoskeletal remodel- cellular confinement of drug effects via 2-photon induced photocrosslinking ing with important implications for synaptic transmission in dendritic spines; to targets. We found that the Rho-kinase inhibitor, Y-27632, dramatically CaMKII and actin-rich post-synaptic micro-compartments that expand within accelerates NM2 redistribution and induces stress fiber dissolution, due to one minute upon synaptic stimulation to initiate long-term changes associated NM2 filament disassembly resulting from myosin light chain dephosphory- with learning. CaMKII organizes static F-actin bundles in-vitro, but fluores- lation. When NM2 was inhibited by para-nitroblebbistatin (pNBleb) or cence microscopy single particle tracking (SPT) of GFP-tagged CaMKII holo- locally by tattooed azidoblebbistatin, in the stress fibers a significant accel- enzymes (GFP-CaMKII) in spines has not detected stimulus-dependent jumps eration and suppression of NM2 redistribution was detected at moderate and in mobility consistent with un-bundling. However, changes in binding affin- high inhibitor concentrations, respectively. The observed effects were local ities, in response to osmotic and mechanical forces, might provoke diverse and specific for load-bearing peripheral stress fibers, implying the role of cytoskeletal CaMKII-actin architectures in vivo. To look for these, we initially mechanical load in NM2 redistribution. Furthermore, in these tests stress fi- used SPT in live cells and found both rat neuronal CaMKII isoforms bound bers remained intact, contrary to that seen upon Rho-kinase inhibition. These RFP-actin labeled stress fibers weakly, comparable to G-actin, with strong

BPJ 7860_7863 Tuesday, February 14, 2017 431a binding to the fibers obtained only when multiple holoenzyme subunits are compartmental IQGAP1 initiates a disassembly mechanism involving rapid engaged (Khan et al. 2016. Biophys. J.111,395-408). We have now measured and unconstrained actin polymerization around the compartment and dispersal the binding of GFP-CaMKII molecules to rhodamine-phalloidin labeled, single of its vesicle contents. Together, these findings suggest IQGAP1 achieves this F-actin filaments and characterized the architectural transitions of the formed control over compartment dynamics by harnessing both stabilizing and antag- filament networks to mechano-osmotic stress. CaMKII induced formation of onistic interactions with actin. mechanically-resilient networks with aster-like nodes. Cryo-electron tomogra- phy showed CaMKII localized in small clusters (n < 5) at the nodes. 2119-Pos Board B439 Although nodes disappeared rapidly upon addition of calcium calmodulin, Component Turnover in the Cytokinetic Ring Maintains Organizational the network structure took several minutes to dissipate. Both C. elegans and hu- Homeostasis and Tension Production 1 1 2 2 man CaMKIIs formed networks at stoichiometric concentrations, revealing an Shuyuan Wang , Sathish Thiyagarajan , Ting G. Chew , Junqi Huang , 2 2 3 3 evolutionary-conserved interaction. At the micromolar concentrations investi- Saravanan Palani , Anton Kamnev , Ying Gu , Snezhana Oliferenko , Mohan Balasubramanian2, Ben O’Shaughnessy4. gated, CaMKII-actin bundles were sparsely distributed in the networks. Macro- 1 molecular crowding by addition of polyethylene glycol or force generated by Department of Physics, Columbia University, New York, NY, USA, 2Division of Biomedical Sciences, Warwick Medical School, University of addition of myosin motors plus ATP bundled the filaments; but CaMKII 3 retarded, rather than assisted, this transition. CaMKII also protected against Warwick, Coventry, United Kingdom, Randall Division of Cell and Molecular Biophysics, King’s College London, London, United Kingdom, filament fragmentation by motor forces. We propose that stimulation- 4 triggered disruption of spine CaMKII-actin interactions has two distinct effects, Department of Chemical Engineering, Columbia University, New York, formation of a compliant F-actin network primarily due to loss of nodal con- NY, USA. tacts and polymerization of the released G-actin. We are simulating these pro- Why do actin, myosin-II and other components turn over in the cytokinetic con- cesses in a 3-dimensional ‘‘virtual’’ spine to determine CaMKII, G and F-actin tractile ring? Turnover is a fundamental feature of the ring, an actomyosin ma- concentrations and affinities that best mimic the observed spine dynamics upon chine whose components are organized to generate tension and help divide the stimulation. cell, but systematically probing turnover is experimentally challenging. Per- meabilized fission yeast protoplasts (ghosts) offer an exciting solution, since 2117-Pos Board B437 the cytoplasm is absent so membrane-anchored contractile rings are isolated Modeling the Cooperativity of Tropomyosin Binding to Actin Filaments and turnover almost completely switched off (Mishra et al., 2013). The role Glen M. Hocky1, Jenna R. Christensen2, David R. Kovar3, Gregory A. Voth4. of turnover is on vivid display. 1James Franck Institute, University of Chicago, Chicago, IL, USA, Here we combined mathematical modeling and experiment to study contractile 2Department of Molecular Genetics and Cell Biology, University of Chicago, rings in fission yeast S. japonicus ghosts. On addition of ATP myosin-II aggre- Chicago, IL, USA, 3Department of Molecular Genetics and Cell Biology, gated dramatically and hierarchically, revealed by the increasingly punctate Department of Biochemistry and Molecular Biology, University of Chicago, Rlc1p-GFP distribution over time. Other components aggregated similarly. Chicago, IL, USA, 4Department of Chemistry, James Franck Institute, Some ring segments unanchored from the membrane and shortened, with Institute for Biophysical Dynamics, University of Chicago, Chicago, zero tension. IL, USA. These findings suggest turnover maintains component organization in the ring. Tropomyosins modulate the activity of actin binding proteins—including To examine this hypothesis, we developed a 3D, molecularly explicit mathe- myosin, cofilin, the Arp2/3 complex, and formins—by themselves binding matical model of the cytokinetic ring in S. japonicus ghosts. Simulations quan- and coating actin filaments. A single tropomyosin protein is a coiled-coil titatively reproduced key experimental observations: (i) Myosin-II aggregated capable of end-to-end association, and one segment of an actin filament is hierarchically with statistical properties close to experiment, (ii) Formin capable of simultaneously accommodating two tropomyosin cables. The Cdc12p at actin filament barbed ends aggregated, colocalized with myosin-II, coating of actin filaments by tropomyosin is known to be highly cooperative, (iii) Myosin-II aggregation was slower with jasplakinolide-stabilized actin, however the details of this process are not well understood. Using labeled actin (iv) Unanchored ring segments shortened, with vanishing tension. and labeled fission yeast tropomyosin, Cdc8, we can observe by TIRF micro- Importantly, simulated ring tensions continuously decreased due to the compo- scopy the formation of small tropomyosin Cdc8 ‘seeds’ that appear to initially nent aggregation. Overall, our simulations revealed that the observed myosin-II associate with actin filaments and then subsequently extend from both ends. aggregation originates in the inherent instability of contractile actomyosin Additionally, we are capable of observing two distinct tropomyosin cables structures: contractile elements in series tend to bunch up. In normal cells associated with a single actin filament. Our setup allows us to study in detail component turnover continuously restores the spatial distribution, protecting the interactions of tropomyosin Cdc8 with the actin filament and with other tension generation. Without turnover the bunching up is runaway and self- tropomyosin Cdc8 molecules. To interpret the experimental observations, we fueling, with ever more punctate contractile elements. Thus, component turn- have used statistical approaches, kinetic master equations, and a new kinetic over maintains organizational homeostasis in the contractile ring, a requirement Ising model. Theory and modeling lends insight into the mechanism underlying for tension production. Cdc8’s highly cooperative binding kinetics. Our results are consistent with a small amount of ‘‘face cooperativity,’’ that is, a slight preference for tropomy- 2120-Pos Board B440 osin Cdc8 molecules to associate with the actin filament opposite to an already What Silences the Spindle Checkpoint? A Single Particle Study 1 2 2 1 bound tropomyosin cable. These findings suggest that some tropomyosin Kwaku N. Opoku , Lori Koch , Susan Biggins , Charles L. Asbury . 1Physiology & Biophysics, University of Washington, Seattle, WA, USA, binding cooperativity is mediated through the actin filament, and not only 2 via end-to-end binding of Cdc8 molecules. Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. 2118-Pos Board B438 Accurate mitosis depends on a surveillance system called the spindle assembly Coordinating Role of IQGAP1 in the Regulation of Multivesicular Endo- checkpoint. The checkpoint acts at kinetochores, which attach chromosomes to somal Compartment Dynamics the dynamic tips of spindle microtubules. When a kinetochore is unattached or Volker Schweikhard, Edward Samson, David Tsao, Tyler McLaughlin, improperly attached, the checkpoint kinase Mps1 phosphorylates core kineto- Michael Diehl. chore components, catalyzing the generation of a diffusible ‘wait’ signal to Rice University, Houston, TX, USA. block anaphase chromosome segregation. When a kinetochore becomes prop- IQGAP1 is a large, multi-domain scaffold that helps orchestrate cell signaling erly attached, this signal is somehow silenced to allow anaphase to proceed. and cytoskeletal mechanics by controlling interactions among a spectrum of re- Two classes of models are proposed to explain how Mps1 is selectively ceptors, signaling intermediates, and cytoskeletal proteins. While this coordina- silenced at microtubule-attached kinetochores. In competitive binding models, tion is known to impact cell morphology, motility, cell adhesion, and vesicular Mps1 and microtubules compete for binding to the kinetochore, either directly traffic, among other functions, the spatiotemporal properties and regulatory or allosterically, such that Mps1 is released upon kinetochore-microtubule mechanisms of IQGAP1 have not been fully resolved. Herein, we describe a attachment. In switch models, kinetochore attachment to the end of a microtu- series of super-resolution and live-cell imaging analyses that identified a role bule inhibits Mps1 kinase activity without causing its release. By direct obser- for IQGAP1 in the regulation of an actin cytoskeletal shell surrounding a novel vation of single fluorescent kinetochores in vitro, we find that Mps1 levels are membranous compartment that localizes selectively to the basal cortex of not reduced when a kinetochore binds laterally to the side of a microtubule, nor polarized epithelial cells (MCF-10A). These largely immotile yet highly dy- when it tracks processively with a disassembling tip. Thus, association of Mps1 namic structures are identified as multivesicular membrane processing stations with a kinetochore is not strictly competitive with microtubule attachment. that participate in adherens junction protein traffic. We also show that IQGAP1 Incorporating a fluorescent kinase sensor should allow us to test the switch appears to both stabilize the actin coating and constrain its growth. Loss of models.

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2121-Pos Board B441 formation. We began with previously published data on fission-yeast spindle- Local Load-Bearing by Kinetochore-Fibers in the Mammalian Spindle pole-body size and microtubule number, kinesin-5 motors, kinesin-14 motors, Provides Mechanical Isolation and Redundancy and passive crosslinkers. Our model results agree quantitatively with our exper- Mary Williard Elting1, Dylan B. Udy1, Manu Prakash2, Sophie Dumont3. iments in fission yeast, thereby establishing a minimal system with which to 1Cell and Tissue Biology, UCSF, San Francisco, CA, USA, 2Bioengineering, interrogate collective self assembly. We identify a set of functions essential Stanford, Stanford, CA, USA, 3Cell and Tissue Biology, Cellular and for the generation and stability of spindle bipolarity. When kinesin-5 motors Molecular Pharmacology, UCSF, San Francisco, CA, USA. are present, their bidirectionality is essential, but spindles can form in the pres- When cells divide, microtubule bundles called kinetochore-fibers (k-fibers) ence of passive crosslinkers alone. We identify characteristic failed states of attach chromosomes to the mammalian spindle. Active forces generated at ki- spindle assembly, which are avoided by creation and maintenance of antipar- netochores move chromosomes, and the dynamic spindle must robustly anchor allel microtubule overlaps. k-fibers to bear this load. Where and how anchorage occurs are not understood. To spatially map load-bearing by k-fibers and determine its molecular basis, we 2124-Pos Board B444 cut k-fibers at different spindle locations and quantitatively measure residual Flagellar Length Control can be Achieved through a Simple Diffusion- load-bearing in different molecular backgrounds. The relaxation response Based Mechanism immediately after ablation indicates that k-fibers are anchored not only at their Nathan L. Hendel, Wallace F. Marshall. ends, but along their lengths. We find that the load of k-fiber anchorage is borne Biochemistry and Biophysics, University of California, San Francisco, very locally: longitudinally in their first few microns from kinetochores, far San Francisco, CA, USA. from poles; and laterally within 1-2 mm, without neighboring k-fibers sharing An important question in cell biology is how cells know how big to make their load. Depleting the microtubule crosslinker NuMA reduces local load- organelles. The eukaryotic flagellum is an ideal model for studying size control bearing anchorage in the spindle body, while inhibiting or depleting microtu- because its linear geometry makes it essentially one-dimensional, greatly bule motor Eg5 and crosslinker PRC1 do not. A simple viscoelastic model simplifying mathematical modeling. The assembly of flagella is regulated by suggests that elastic connections of k-fibers to the spindle bear the load of intraflagellar transport (IFT), in which trains of kinesin motors walk to the active kinetochore forces moving chromosomes, while centromere viscosity tip of the flagellum and deposit the cargo necessary for the flagellum to determines the timescale of relaxation after ablation removes load-bearing con- grow. The competing length control factor is a length-independent decay of nections. Together, the data indicate that the architecture of the dynamic the flagellum. In Chlamydomonas reinhardtii flagella, this process results in mammalian spindle provides k-fibers with mechanical isolation and load- initial rapid growth followed by convergence to a steady-state length. Curi- bearing redundancy well-suited for robust chromosome segregation. ously, the rate at which motors are recruited to begin transport is indirectly pro- portional to the length, implying some kind of communication between the base 2122-Pos Board B442 and the tip. We propose a model in which motors unbind after cargo delivery Integrated Model of Cytokinetic Ring Constriction and Septation in and diffuse back to the base, and are reused in IFT. In this model, the diffusion Fission Yeast Reproduces Experimental Values of Ring Tension time of the motors serves as a proxy for length measurement. To explore the Shuyuan Wang1, Sathish Thiyagarajan1, Ben O’Shaughnessy2. viability of this diffusion-based length control, we computationally built this 1Department of Physics, Columbia University, New York, NY, USA, model in three different ways. First, we built an agent-based model in which 2Department of Chemical Engineering, Columbia University, New York, we used object-oriented programming to explicitly model flagella and motors, NY, USA. including time dynamics. Second, we modeled the number density along the A major challenge in cytokinesis research is that contractile ring constriction flagellum as a vector, and built a stochastic matrix to simulate time dynamics couples to other processes such as flow of actomyosin cortex and cytoplasm, and determine a steady-state. Third, we used differential equations to directly membrane addition and, in cell-walled organisms like fission yeast, septation, solve for the steady-state length. In all three, we found that the diffusion model the inward growth of new cell wall in the wake of the constricting ring that en- can achieve steady-state length and an inverse relationship between length and closes the daughter cells. The ultimate goal is a quantitative model of ring recruitment rate. This is remarkable because this is perhaps the simplest expla- constriction coupled with these processes. nation of length control, giving it credence in light of evolution. We developed such a model for fission yeast, whose cytokinetic ring is partic- ularly well characterized. We integrated a 3D molecularly explicit simulation 2125-Pos Board B445 of the constricting ring, highly constrained by established amounts, biochem- The Flagella Beat of Chlamydomonas Has Distinctly Regulated Static and istry and organization of components, with a simulation describing septum Dynamic Components, which Accord with Curvature Controlled Dynein synthesis by beta glucan synthases (Bgs) in the plasma membrane. The Bgs- Activity 1 2 € 3 1 mediated septum growth was assumed mechanosensitive. To our knowledge, Veikko F. Geyer , Pablo Sartori , Frank Julicher , Jonathon Howard . 1Yale University, New Haven, CT, USA, 2Institute for Advanced Study, this is the first quantitative account of the total cytokinetic constriction process. 3 The ring is attached to the inward-growing septum edge whose curvature in- Princeton, NJ, USA, Max Planck Institute for the Physics of Complex creases as constriction progresses, influencing ring organization and tension. Systems, Dresden, Germany. In the reverse direction, local ring tensions affected the mechanosensitive The axoneme, the mechanical core of eukaryotic cilia and flagella, is a huge septum growth: growth at septum edge valleys (bumps) was accelerated (decel- macromolecular assembly of microtubules, cross-links and dynein motors. erated) by the centripetal force from tension, maintaining near-circularity of the The coordinated activity of the dyneins produces a diversity of flagellar wave- septum. We quantified deviations from circularity by roughness; simulated forms that drive fluid flows across tissue surfaces or propel micro-swimmers. septum edge roughness statistics reproduced statistics from edges in live cells While it is believed that the flagellar beat is a result of feedback - based on (Thiyagarajan et al., 2016). Ring tension suppressed roughness and enforced the ability of motors to produce and sense forces - the mechanism by which proper septum closure, furnishing daughter cells with sealed cell walls. motors are controlled remains unknown. Using a novel method we measured ring tension in live fission yeast protoplast To elucidate how molecular motors are involved in the generation of flagellar cells, which increased from ~400 to ~800 pN as constricted proceeded, and was waveforms, we studied the beat of axonemes isolated from Chlamydomonas reduced ~ 65% in myo2-E1 mutants. The model reproduced these tensions, and reinhardtii, a single cell green alga. Chlamydomonas flagella can exhibit showed that tension results from anchoring of actin filament barbed ends to the both of the most common waveforms found in nature: the asymmetrical membrane. breaststroke-like beat as well as the symmetrical sperm-like beat. Using high speed microscopy and image analysis we precisely determine 2123-Pos Board B443 axonemal shapes in space and time. A characterization of the waveform prop- Physical Determinants of Bipolar Mitotic Spindle Assembly and Stability erties at different ATP concentrations shows that the static asymmetry of the in Fission Yeast breaststroke - which has an approximately circular shape - can be separated Robert Blackwell, Christopher Edelmaier, Oliver Sweezy-Schindler, from the dynamic beat component. This analysis reveals that the breaststroke Adam Lamson, Zachary Gergely, Eileen O’Toole, Ammon Crapo, can essentially be viewed as a sperm-like beat traveling around a circular shape Loren Hough, Richard McIntosh, Matthew Glaser, Meredith Betterton. and allows us to examine both waveform components independently. Physics, University of Colorado at Boulder, Boulder, CO, USA. By comparing the dynamic component of experimentally measured waveforms Mitotic spindles use an elegant bipolar architecture to segregate duplicated to a mechanical model of the axoneme, we found that the shapes were consis- chromosomes with high fidelity. Bipolar spindles form from a monopolar initial tent with a model in which dynein motors respond to changes in axonemal cur- condition; this is the most fundamental construction problem that the spindle vature. We furthermore show that the static asymmetric shape underlying the must solve. Microtubules, motors, and crosslinkers are important for bipolarity, beat could also result from curvature controlled dynein activity. but the mechanisms necessary and sufficient for spindle assembly remain un- Together our findings present novel insights into how molecular motors shape known. Here we describe a physical model that exhibits de novo bipolar spindle the asymmetric waveform of Chlamydomonas flagella.

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Cell Mechanics, Mechanosensing and Motility III etry. The contribution of this work is to identify scaling laws for the region with graded material properties and a fanned geometry. Based on the observation that 2126-Pos Board B446 neonatal ligament tears often occur at the ligament-bone interface, while injuries As the Beating Heart Stiffens in Development, So Does the Nuclear Lamina in adults primarily occur mid-substance, we propose that the graded interface de- Sangkyun Cho1, Stephanie Majkut2, Amal Abbas1, Ken Vogel1, velops in response to the stress state experienced at dissimilar material interfaces. Jerome Irianto1, Christina Y. Chen3, Manorama Tewari1, Andrea Liu2, Benjamin Prosser3, Dennis E. Discher1. 2129-Pos Board B449 1University of Pennsylvania, Philadelphia, PA, USA, 2Department of Electron Microscopy of the Complex Formed by Heavy Meromyosin and Physics & Astronomy, University of Pennsylvania, Philadelphia, PA, USA, C-Protein 3Department of Physiology, University of Pennsylvania Perelman School of Charlotte Scarff1, Alba Fuentes Balaguer1, Donald Winkelmann2, Medicine, Philadelphia, PA, USA. John Trinick1. In the first organ that develops - the embryonic heart - coordinated beating of car- 1School of Molecular and Cellular Biology, Leeds University, Leeds, United diomyocytes is driven by contractile striations that couple to collagenous extra- Kingdom, 2Department of Pathology, Robert Wood Johnson Medical School, cellular matrix (ECM). However, it is unclear what impact beating has on the Rutgers University, New Brunswick, NJ, USA. large nucleus, especially on the levels of lamin-A/C (LMNA), which is a major Most studies of regulation of vertebrate striated muscle contraction have nuclear structural protein and disease gene. While Lmna-knockout mice exhibit focussed on activation of the thin filament via calcium binding to the ‘developmental defects of the heart’ and die shortly after birth, we first show by troponin-tropomyosin complex. There is accumulating evidence, however, quantitative mass spectrometry (MS) that lamin-A/C is expressed very early on that thick filament activation is an additional regulatory mechanism. Under re- in development, with levels paralleling the development of tissue stiffness and laxing conditions, the two heads of a myosin can interact to form an inactive tension in normal embryonic hearts. Lamin-B expression is comparatively con- asymmetric off-state, called the interacting-heads motif (IHM). The off-state stant, such that lamin-A:B stoichiometry scales with stiffness very similarly to inhibits cross-bridge formation and cycling by blocking actin binding in one that reported for normal adult tissues, which vary in stiffness by several orders head and ATPase activity in the other. Docking of the IHM onto the thick fila- of magnitude [Swift, Science 2013]. Within intact embryonic hearts and its 3D ment backbone results in the super-relaxed state (SRX) observed in muscle fi- arrangement of cardiomyocytes, nuclei are seen to beat in synchrony with tissue. bres, where the myosin ATPase is strongly inhibited. What regulates formation ‘Nuclear beating’ also occurs in vitro on collagen-I coated gels of controlled and disruption of the IHM is largely uncharacterised. Myosin binding protein stiffness, and exhibits an optimum on gels that match the stiffness of embryonic C, also known as C-protein, is a key sarcomere component, bound at sites hearts. Lamin-A:B in the embryonic cardiomyocytes exhibit an optimum at spaced by 43 nm in the cross-bridge regions of thick filaments. The arrange- somewhat higher matrix stiffness that corresponds surprisingly well to the stri- ment, mode of action, and interactions of C-protein with myosin are poorly un- ation order of cytoplasmic myofibrils (rather than peripheral pre-myofibrils). derstood. In the heart, phosphorylation of C-protein increases the rate of These observations are consistent with the Theory of Coherent Beating within contraction and its ablation disrupts the SRX. To investigate the interaction single cells that correctly predicts matrix-dependent contraction. Nuclear of human cardiac C-protein with b-cardiac heavy meromyosin (HMM), we lamin-A thus seems to be an embryonic sensor of coherent stress that mechani- have imaged the complex by electron microscopy and single particle averaging. cally couples to surrounding myofibrils and, in turn, to the collagenous matrix. Cardiac HMM in the IHM motif has the characteristic blocked and inhibited head conformations. The fraction of HMM in the IHM motif is increased 2127-Pos Board B447 when dephosphorylated C-protein is bound, suggesting it stabilises the IHM. Understanding the Role of Stiffness in Pathological Cardiac Fibroblast Characterization of the complex should provide insight into the regulatory Signaling and structural roles of the cardiac C-protein/myosin interaction in heart. Tova Christensen1, Kristi Anseth2, Leslie Leinwand1. 1Molecular, Cellular, and Developmental Biology, University of Colorado, 2130-Pos Board B450 Boulder, CO, USA, 2Chemical Engineering, University of Colorado, Mechanosensitive Adhesion Explains Stepping Motility in Amoeboid Cells Boulder, CO, USA. Calina A. Copos1, Robert D. Guy1, Sam Walcott1, Juan Carlos del Alamo2, Cardiac fibrosis is characterized by extracellular matrix buildup and tissue stiff- Alex Mogilner3. ening. Fibroblasts in the heart respond to pathological cues by activating to pro- 1Applied Mathematics, University of California Davis, Davis, CA, USA, liferate, upregulate expression of alpha smooth muscle actin, synthesize 2Mechanical and Aerospace Engineering, University of California San Diego, extracellular matrix protein, and secrete biochemical cues to surrounding cells. San Diego, CA, USA, 3Courant Institute and Department of Biology, New The cues that trigger this transformation remain incompletely resolved; how- York University, New York, NY, USA. ever, stiffness appears to play a critical role in this process. In vitro experiments Cell movement is required in many physiological and pathological processes using hydrogels with different stiffnesses show that fibroblasts activate when such as immune system response and cancer metastasis. The movement of the cultured on stiff hydrogels while they remain unactivated on soft hydrogels single-cell amoeba Dictyostelium discoideum, is characterized by cycles of as they do in the diseased and healthy heart, respectively. Understanding fibro- morphological expansion and contraction and highly coordinated mechanical blast activation through mechanosensitive signaling will provide new therapeu- forces on the substrate by means of transient cell-substrate adhesions. Despite tic targets for fibrosis treatment. recent intense studies, the mechanisms of rapid shape changes and how they lead to motility of amoeboid cells is still an open question. The existing paradigm 2128-Pos Board B448 for the migration of Dictyostelium has been proposed to be the result of complex Mechanobiology of the Ligament to Bone Insertion biochemical processes coupled to biomechanics. Here, we develop a model to Aisa Biria1, Shreyas Mandre2, Madhusudhan Venkadesan1. study the interplay of cellular mechanics, cell-substrate interaction, and the re- 1 Department of Mechanical Engineering and Materials Science, Yale sulting migration. The novelty of this work is that we demonstrate that a simple 2 University, New Haven, CT, USA, School of Engineering, Brown mechanical-only model can explain how amoeboid motility is achieved and University, Providence, RI, USA. robustly maintained to produce the complex and highly-coordinated features The interface between materials with mismatched elastic moduli is failure-prone of amoeboid motility. We explore several models for cell-substrate adhesion due to singular stress concentrations that could develop at the interface. Yet most and conclude that cyclic expansions and contractions in cell length together ligaments tear at mid-substance and not at the ligament-bone interface. Current with the experimentally measured spatio-temporal dynamics of traction forces physiological understanding of these interfaces centers around the graded mate- are emergent features of a mechano-sensitive interaction with the substrate. rial properties of the ligament as it nears the bone, with increasing mineralization Experiments of wild-type and mutant strains of Dictyostelium suggest of the ligament. Open questions remain on which features of the gradation, or different types of motility behavior. Our model shows how the different motility more generally of the ligament-bone interface, significantly improve the robust- modes arise based on surface properties but also how cells mediate the frequency ness and eliminate the stress singularity. To address these questions, we develop a of the motility cycle in order to continue migrating with the same stride length. 2D elasticity model of the interface and analyze it using a combination of numer- ical simulations and asymptotics. Increasing size of the gradation zone yields 2131-Pos Board B451 diminishing returns once the zone gets longer than the width of the ligament. A Computational Framework to Accurately Predict Enthalpy and Moreover, we apply a previous result in elasticity and show that the Configurational Entropy Landscapes of Multivalent Interactions of Cell frequently-observed fanned geometry of ligaments near the interface can also Mimetics eliminate the singularity. Previously, the functional advantage of the fanned ge- Aravind R. Rammohan1, Matthew Mckenzie1, Ravi Radhakrishnan2, ometry was thought simply to increase the cross-sectional area. However, our Natesan Ramakrishnan2. analysis reveals a different and more significant functional benefit to this geom- 1Corning Inc., Corning, NY, USA, 2UPENN, Philadephia, PA, USA.

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We present a multiscale modeling framework to predict the adhesion of a neutralized dexamethasone-induced reduction of cell mobility and cell cluster model particles from nano-to-micron scales of different shapes and compli- formation. Neither dexamethasone nor ketamine had strong effects on the den- ances that mimic functionalized nanoparticles to biological cells by virtue of sity and growth of neurites. In summary, given the complex actions of ketamine being decorated with receptors/antibodies in their surface. The model derives on morphodynamic parameters in cultured neurons, its mechanism as an anti- cell surface receptor interactions with functionalized peptides using atomistic depressant cannot be explained with its NMDA-antagonistic properties alone simulations. These interaction potentials are then fit to predefined functions but implies additional impact on the formation of communication networks which are used to characterize the interaction between the particle/cells with and morphology related pathways. substrates and to measure the free energy of adhesion/binding constants. Further, in this framework we also account for variations in the receptor ligand 2134-Pos Board B454 interactions due to the inherent compliance in the bilayer membranes on which Left-Right Symmetry Breaking during Early Development of C. elegans 1,2 1 € 1 2 the receptors are tethered. These calculations are performed using a standalone Anagha Datar , Saroj Nandi , Frank Julicher , Stephan Grill . 1Biological Physics, Max Planck Institute for the Physics of Complex framework for estimating effect of membrane shape/curvature on the flexibility 2 of the receptors. This membrane-compliance effect shows that decrease in Systems, Dresden, Germany, Biotechnologisches Zentrum, Dresden, membrane area results in increased receptor rigidity which ends up limiting Germany. the overall adhesion of the receptors to the functionalized surfaces or in other Body plan of most of the organisms is left-right (LR) symmetric on the exterior words much weaker dissociation constants (i.e. mM as opposed to microM). In but highly asymmetric in arrangement of the internal organs. This asymmetry is this work we show a computational tool where we couple chemistry of peptide, established during early stages of embryonic development and is crucial for a membrane fluctuations and cellular sizes to estimate the overall binding affin- proper cell fate determination and morphogenesis. Typically, the anterior- ities of these model cells to functionalized surfaces. We also showcase the po- posterior (AP) and dorsal-ventral (DV) symmetries are broken prior to the wer of this framework in simulating realistic heterogeneity in cell surface establishment of L-R axis. There are also definite cues responsible for symme- receptors by demonstrating effect of a combination of weak and strong recep- try breaking along AP and DV directions. For instance in C. elegans embryos, tors in modulating the overall binding affinity of the cell. The model as it stands entry point of a sperm becomes the posterior side while location of the EMS can be a tool for development of functionalized surfaces aimed at cell based cell becomes the ventral side. However, for LR symmetry breaking which hap- assays and or cell based therapies. pens at 4 to 6 cell transition in C. elegans, there are no obvious cues and the asymmetry is established by a directionally conserved skew or tilt of two cells. 2132-Pos Board B452 It is shown that the skew is strongly dependent upon counter-rotating chiral In Vitro Reconstitution of Tissue Homeostasis and Stochastic Cell Fate flows in the cell cortex (Naganathan et al. 2014). Although the theory of active Choice chiral fluids can explain the origin of such flows, it is not understood how do the Kyogo Kawaguchi, Allon M. Klein. flows affect the skew that breaks LR symmetry. Our work explores the hidden Department of Systems Biology, Harvard Medical School, Boston, asymmetries in the system, in terms of non-uniform distribution of force gen- MA, USA. erators, namely myosin motors in the cell cortex and asymmetric cell-cell con- Many adult tissues are dynamically sustained by the rapid turnover of cells. In tacts. We also present a theoretical model for explaining how such a system can recent years, statistical physics has provided a lens through which to study cell effectively give rise to an unbalanced torque that may generate the skew. turnover and self-renewal in adult tissue maintenance. Individual cell fate 2135-Pos Board B455 choices between cell division and differentiation were found to be stochastic, Robust Early Embryogenesis of Caenorhabditis Elegans due to Mechanical evidenced by the statistical properties of long-term clonal tracing experiments Cues and Proper Cell Division Timing in adult tissues. However, the mechanism of tissue homeostasis - how cell di- Rolf Fickentscher, Philipp Struntz, Matthias Weiss. vision and differentiation are precisely balanced in stochastic fate choice - is Experimental Physics 1, University of Bayreuth, Bayreuth, Germany. still unknown. Regulation mechanisms can involve intrinsic fate choice tuned Embryogenesis of the small nematode Caenorhabditis elegans is a remarkably at the single-cell level, or include extrinsic feedback from neighboring cells or robust and reproducible, but still poorly understood self-organization phenom- signaling molecules. Since different mechanisms, intrinsic or extrinsic, will enon. Using light sheet microscopy and modeling/simulation, we show that a result in different universality classes at the level of abstract models, analytical coupling of cellular volumes and cell cycle times in combination with a me- and numerical results of interacting particle systems can be useful in discrim- chanically guided arrangement process is key for a fail-safe embryogenesis inating the underlying rules in real tissues. Here we developed a system to of C. elegans. allow detailed study of tissue homeostasis using epithelial cells cultured in vi- In particular, we have monitored cell trajectories and cellular volumes in C. el- tro. Within two-dimensional cultures, we observe ongoing cell exclusions egans embryos over several hours by means of a custom-made lightsheet mi- compensated by cell division, resulting in a steady-state of the cell sheet that croscope. Length and time scales of the worm’s development were altered is maintained for weeks. We construct a phenomenological description on via RNAi methods and different ambient temperatures. Experimentally deter- the mechanism of feedback between cell division and differentiation through mined cell trajectories during early embryogenesis are accurately described a theoretical model, and address the length scale associated to the feedback by a simple mechanical framework, i.e. cells adopt positions that yield least through analyses on the fine time-scale data obtained from in vitro experiments. repulsive interactions among themselves and with the confining eggshell [1]. Our findings constrain possible mechanisms of homeostatic control. Consis- Cell migration velocities follow an Arrhenius scaling, indicating that the effec- tency of our findings with previous clonal experiments and recent in vivo tive driving forces are determined by internal biochemical processes, e.g. by mouse live imaging results will be discussed. remodeling of the cytoskeleton. Our experimental data also revealed an anticor- relation of cell volume and cell cycle duration, with significant differences be- 2133-Pos Board B453 tween somatic and germline precursor cells. Anticorrelated cell volumes and Ketamine Promotes Neurite Growth and Recovers Dexamethasone- cell cycle times can be rationalized via a simple model that invokes a limiting Induced Mobility Decrease in Cultured Hippocampal Cells molecular component that supports cell division, e.g. nuclear pore complexes. Andreas W. Henkel, Zoran B. Redzic, Mohammed S. Al-Qallaf. Integrating this aspect into the aforementioned mechanical framework, we Physiology, Kuwait University, Safat, Kuwait. observed that the different scaling of the germline precursor lineage is crucial The ‘‘plasticity hypothesis’’ proposes that major depression is caused by for a robust cellular arrangement process during early embryogenesis [2]. morphological and biochemical modifications in neurons and astrocytes and [1] R. Fickentscher, P. Struntz & M. Weiss, Biophys. J., 105 (2013) that the illness is at least partially associated with modifications of cellular com- [2] R. Fickentscher, P. Struntz & M. Weiss, Phys Rev Lett., in press (2016) munications between these cells. In this study we examined effects of Keta- mine, an NMDA antagonist that was conventionally used as an anesthetic, 2136-Pos Board B456 on dexamethasone-treated neurons. Several clinical trials have shown that Assessing Cell-Substrate Interaction with Dissipation Monitoring Function this drug has very potent anti-depressive properties, becoming effective within of the QCM-D a few hours. Ketamine has also been shown to have fast effect on the neuronal Jennifer Chen1, Lynn Penn1, Ning Xi2, Jun Xi1. plasticity, including synaptogenesis and dendrite formation. In this project, we 1Chemistry, Drexel University, Philadelphia, PA, USA, 2Department of studied its morphodynamic effects in hippocampal neuronal cultures by mo- Industrial and Manufacturing System Engineering, University of Hong Kong, tion-, neurite growth analysis and cluster formation. Time-lapse images taken Hong Kong, Hong Kong. over 48 hours and the resulting videos were analyzed by several newly devel- Cell adhesion is essential in cell communication, cell regulation, and the devel- oped complex algorithms for morphometry and cell mobility. Dexamethasone, opment and maintenance of tissues. The mechanical interactions between a cell a synthetic corticosteroid, was used to induce depression-associated changes and its extracellular matrix (ECM) can influence and control the function and in cultured hippocampal neurons. Results show that ketamine significantly the behavior of cells. Gaining a better understanding about the mechanical

BPJ 7864_7868 Tuesday, February 14, 2017 435a interaction of cell adhesion can provide advances in biomaterial for implants, pects hindering this process is associated to the analytical estimation of phys- potential drug treatments for improvement of cell adhesion of implants, and ical parameters belonging to single cells. The typical approach consists in fundamental understanding of signaling pathways related to cell adhesion. fitting a mathematical model to the experimental signals or, in other words, We have developed a non-invasive real time method using the quartz crystal in optimizing the parameters of a chosen functional. This strategy is fast and microbalance with dissipation monitoring (QCM-D) to quantitatively monitor effective for deterministic models, slightly affected by environmental noise, such cellular processes using the dissipation factor DD. In this study, we but a paradigm shift is required when dealing with nanoscale systems, intrinsi- have used this method to examine the adhesion of human epidermal keratino- cally described by a thermal-driven statistical distribution. The method pro- cytes to the QCM-D sensor surface coated with titanium, a common material posed in this work consists in extracting meaningful mechanical parameters for medical implant. The key results from this study have validated this method from experimental data based on solving the associated estimation problem. as an in-vitro approach for examining cell-implant interaction. An approach to cell mechanics investigation based on Sequential Monte Carlo algorithms have been implemented. Starting from a generalized state space rep- 2137-Pos Board B457 resentation, the experiment and its uncertainties and noise have been modelled The Role of Global and Local Mechanical Signals in Modulating Cell and the characterizing parameters have been identified through the exploitation Spreading of estimation algorithms. The analysis has been applied in a simulation envi- Magdalena Stolarska1, Aravind Rammohan2. 1 2 ronment, allowing to check the algorithm inference capability and to define Mathematics, University of St. Thomas, St. Paul, MN, USA, Mathematics, the optimal experimental protocol. Single cell mechanical estimation experi- Corning, Inc., Corning, NY, USA. ments have been designed to match the selected procedure, implementing the Cell spreading on a flat surface is controlled by the interaction of cell adhesion, estimation strategy in an embedded platform based on a single core dsPIC de- actin-based cellular deformations, and intracellular stresses. It has been shown vice. The effectiveness of the proposed approach has been tested on a cell line that many cell types obtain larger spread areas on stiffer substrates [1]. We pre- before and after treatment with cytoskeleton-disrupting drugs. Preliminary re- sent a 2D model and finite element simulations of a spreading cell interacting sults show that modern filtering theory can be effectively applied to extract sin- with a deformable substrate through focal adhesion complexes. Focal adhesion gle cell mechanical parameters in real-time, also providing a valuable guide to complexes are modeled by collections of linear springs that depend on local identify the most efficient experimental design. concentrations of a ligand-activated bound integrin and can form and break dynamically in a stress and strain dependent manner. The cell is treated as a 2140-Pos Board B460 hypoelastic material that undergoes active deformations that represent cell The Expression and Degradation of SM22-Alpha/Transgelin are Regu- spreading. By considering various formulations for the active deformation, lated by Mechanical Tension in the Cytoskeleton which is characterized by an active rate of deformation tensor field, we use Rong Liu, M. Moazzem Hossain, J.-P. Jin. this model to better understand whether the mechanism of cell spreading is Wayne State University, Detroit, MI, USA. localized or governed by a global signal. Specifically, we consider the ability SM22-alpha, also named transgelin, encoded by the gene TAGLN is a calponin- of a cell to integrate local signals and define an active rate of deformation related protein found in smooth muscle, fibroblast and cancer cells. SM22- that depends on the cell-level integration of signals arising from bound integrin alpha was discovered three decades ago but its biological function remains concentrations, which enhance cell spreading, and from intracellular tension, unclear. In addition to an application as a differentiation marker for smooth which inhibit cell spreading. We also consider a spatially inhomogeneous muscle cells, SM22-alpha has been reported to regulate the structure and dy- active rate of deformation field that depends on local values of bound integrin namics of actin cytoskeleton and cell motility in fibroblasts and cancer cells. concentrations and intracellular tension. We find that cell spread areas are gov- Here we report a novel finding that the expression and degradation of SM22- erned by a combination of global signal integration, which accounts for long- alpha/transgelin are both regulated by mechanical tension. Mass spectrometry range communication within a cell, and local integrin binding and tensile stress. detected that SM22-alpha was significantly decreased in mouse aortic rings It is this combination of signaling mechanisms that allows us to obtain exper- after incubation under low mechanical tension. Using specific monoclonal imentally observed cell spread area dependence on substrate stiffness. antibodies developed against chicken gizzard SM22-alpha, we found high [1] Yeung, T. et al. ‘‘Effects of substrate stiffness on cell morphology, cytoskel- levels of SM22-alpha in human fetal lung fibroblast cells line MRC-5 and pri- etal structure, and adhesion.’’ Cell Motility and the Cytoskeleton 60 (2005): mary neonatal mouse skin fibroblasts. Similar to that of calponin 2, the level of 24-34. SM22-alpha is positively dependent on the mechanical tension in the cytoskel- 2138-Pos Board B458 eton as determined by the stiffness of culture substrate. Quantitative RT-PCR Force Localization and Cell Shape in Epithelial Monolayers demonstrated a transcriptional regulation of TAGLN gene expression by me- Erik Schaumann1, Margaret Gardel2. chanical tension in the cytoskeleton. The cellular localization of SM22-alpha 1Department of Chemistry, University of Chicago, Chicago, IL, USA, overlaps with that of myosin IIA and blebbistatin inhibition of myosin motor 2Institute for Biophysical Dynamics, James Franck Institute, Department of decreased the expression of SM22-alpha. The level of SM22-alpha is decreased Physics, University of Chicago, Chicago, IL, USA. in skin fibroblasts isolated from calponin 2 knockout mice compared to that in Epithelial monolayers are a classic system of active matter, in which the calponin 2-positive wild type cells, suggesting their correlated functions. With component particles (cells) endogenously generate forces and change their the close phylogenetic relationship between TAGLN and the calponin genes, own shapes without external stimuli. The mechanics of single cells and circular SM22-alpha is identified as a calponin-like cytoskeleton regulatory protein. colonies of keratinocytes can be successfully described using a continuum These findings laid a groundwork for understanding the physiological function model, however, it is not known how general this model is. Using micropattern- of SM22-alpha in mechanoregulation of cytoskeleton and cell motility and its ing to constrain colonies of MDCK cells to reproducible geometries, the rela- relationship with calponins. tionship between the shapes of individual cells within the colonies and the mechanical output of otherwise similar colonies can be assessed. We find 2141-Pos Board B461 that wild-type cells are motile with respect to the colony borders and distribute Compressive Stress Stalls Growth and Decreases Cytoplasmic Diffion Morgan Delarue1, Greg Brittingham1, Oskar Hallatschek2, Liam Holt1. traction forces to localized hot spots that move over time, thus breaking from 1 2 the continuum model. ZO-1/ZO-2 knockdown cells, which display elevated NYU Langone Medical Center, New York, NY, USA, UC Berkeley / contractility and straighter cell-cell borders, are employed to examine the Stanley Hall, Berkeley, NY, USA. impact of these parameters on the magnitude and localization of traction forces. Any cell population growing in a limited space can generate mechanical compressive stresses. Tumors growing within tissues and microbes that are 2139-Pos Board B459 naturally confined by their environment both build up growth-induced pressure. Real-Time Identification of Cell Mechanical Properties While the effects of tensile mechanical stresses have been widely studied, much Alice Bartolozzi1, Alessandro Soloperto2, Gemma Palazzolo2, less is known about the effects of compressive mechanical stresses on cell phys- Michele Basso1, Francesco Difato2, Massimo Vassalli3. iology. We developed a microfluidic mechano-chemostat that enables a precise 1Information Engineering (DINFO), University of Florence, Florence, Italy, temporal control of mechanical and chemical conditions. We found that rate of 2Neuroscience and Brain Technologies, Italian Institute of Technology, cell growth is affected by compressive stress: Cell growth decreased exponen- Genova, Italy, 3IBF, CNR National Research Council, Genova, Italy. tially with pressure. In order to investigate the molecular origin for such a In the last years, the exploitation of nanotechnology to the study of biological growth decrease, we developed genetically encoded multimeric nanoparticles systems opened new avenues towards innovative clinical approaches based on (GEMs) to assess the regulation of cellular crowding and the effects of a me- single cell mechanical characterization. Nevertheless, due to the lack of chanical compressive stress on cell microrheology. GEMs are particles of throughput typical of high resolution tools, a complete translation of research 16nm or 35 nm in size naturally expressed by cells that enable direct particle findings to real life applications has not been accomplished yet. One of the as- tracking. We observe that the motion of GEMs is non-ergodic and subdiffusive,

BPJ 7864_7868 436a Tuesday, February 14, 2017 and that crowding is highly regulated in a cell by the regulation of ribosome boundary conditions, i.e., the tractions and displacements present at their mem- biogenesis. Moreover, similar to overall growth-rate, GEM diffusion follows branes. This assumption opens three mechanisms for pattern formation in large the same exponentially decreased with increasing compressive stress that cell populations: wavelike behavior, kinematic feedback in motility that leads growth rate. To conclude, we speculate that macromolecular diffusion becomes to sliding layered patterns, and directed migration during invasions. 1. Wave- rate limiting for growth under compressive stress. Intermediate mechanical like behavior among ameloblast cells during amelogenesis has been inferred stresses could drive changes in crowding and diffusion to regulate cell physi- from enamel microstructure, while strain waves in populations of epithelial ology, from metabolism to signaling. cell shave been observed in vitro. We show that ‘‘determination fronts’’, where cells transition into a new state, can be governed by tsunami-like wavefronts 2142-Pos Board B462 propagating across a cell population, a depiction that is supported by data for Cellular Durotaxis from Differentially Persistent Motility ameloblasts. 2. A kinematic feedback mechanism, ‘‘enhanced shear motility’’, 1 2 3 Elizaveta A. Novikova , Matthew Raab , Dennis E. Discher , accounts successfully for the spontaneous formation of layered patterns during Cornelis Storm4. 1 amelogenesis. 3. Directed migration is exemplified by a theory of invader cells CEA, Institut de Biologie en de Technologies de Saclay, Gif-sur-Yvette, that sense and respond to the strains they themselves create in the host popula- France, 2Institut Curie, Paris, France, 3University of Pennsylvania, 4 tion as they invade it: analysis shows that the strain fields contain positional in- Philadelphia, PA, USA, Eindhoven University of Technology, Eindhoven, formation that could aid the formation of network structures, stabilizing the Netherlands. slender geometry of branches and helping govern the frequency of branch Cells move differently on substrates with different elasticities. In particular, the bifurcation and branch coalescence (the formation of closed networks). In all persistence of their directionality is greater on substrates with a higher elastic cases, morphological outcomes are governed by the ratio of the rates of two modulus. We show that this behavior - without any further assumptions - competing processes, one a migration velocity and the other a relaxation veloc- will result in a net transport of cells directed up a soft-to-stiff gradient. Using ity related to the propagation of strain information. Relaxation velocities are simple random walk models with controlled persistence and stochastic simula- approximately constant for different species and organs, whereas cell migration tions, we characterize this propensity to move in terms of the durotactic index rates vary by three orders of magnitude. We conjecture that developmental pro- measured in experiments. A one-dimensional model captures the essential fea- cesses use rapid cell migration to achieve certain outcomes, and slow migration tures of this motion and highlights the competition between diffusive spreading to achieve others. We infer from analysis of host relaxation during network for- and linear, wavelike propagation. Since the directed motion is rooted in a non- mation that a transition exists in the mechanical response of a host cell from directional change in the behavior of individual cells, the motility is a kinesis animate to inanimate behavior when its strain changes at a rate that exceeds rather than a taxis. Persistence-driven durokinesis is generic and may be of 104 -103 s1. The transition has previously been observed in experiments use in the design of instructive environments for cells and other motile, mecha- conducted in vitro. In initially homogeneous populations, outcomes are nosensitive objects. strongly influenced by the presence of double-curvature in the cell sheet: the 2143-Pos Board B463 evolving metric of the sheet triggers strain-cues among cells, which break Characterization of the Frustrated Differentiation of Mesenchymal Stem symmetry. Cells Induced by Normadic Migration Between Stiff and Soft Region of Gel Matrix 2145-Pos Board B465 Satoru Kidoaki1, Hiroyuki Ebata1, Rumi Sawada2, Kousuke Moriyama1, Mitochondrial Fluctuations as a Measure of Biomechanical Properties of Thasaneeya Kuboki1, Yukie Tsuji1, Ken Kono2, Kazusa Tanaka2, Murine Cells 1 1 2 1,3 1 Wenlong Xu , Elaheh Alizadeh , Jordan Castle , Ashok Prasad . Saori Sasaki . 1 1IMCE, Kyushu University, Fukuoka, Japan, 2National Institute of Health Chem. & Biol. Engr., Colorado State University, Fort Collins, CO, USA, 2Dept. of Biology, Colorado State University, Fort Collins, CO, USA, Sciences, Tokyo, Japan. 3 Mesenchymal stem cells (MSCs) have been known to exhibit substrate School of Biomed. Engr., Colorado State University, Fort Collins, CO, USA. stiffness-dependent differentiation, and history of the mechanical dose from Introduction: The active mechanical properties of the cellular cytoskeleton culture environment to MSCs sensitively is found to alter its phenotype. A are important in a number of significant cellular processes from migration certain level of substrate stiffness and duration period on that determine the to cell division, and are altered in cancer. A non-invasive single-cell assay fate of MSCs. In relation to this, we have found before that microelastically- of these properties would be of significant value for understanding these patterned hydrogel with heterogeneous distribution of matrix stiffness allow cellular processes, would give insights into mechanical heterogeneity between MSCs to suppress fate determination into specific differentiation lineages, cells, and would be of therapeutic importance in cancer. A promising tech- and contribute to keep the undifferentiated state. We call such mode of nique is derived from passive particle microrheology, wherein fluorescent MSCs as ‘‘frustrated differentiation’’, which serves to construct culture sub- beads are inserted into the cytoplasm, their mean square displacement strate for MSCs to maintain their stemness in high-qualified state. The basis (MSD) measured, and mechanical parameters calculated using viscoelastic of this phenomenon is in the enforced oscillation of mechanical dose from envi- theory. The non-invasive version of this method uses mitochondrial fluctua- ronment to MSCs during the nomadic migration between stiff and soft region of tions towards the same goal which is potentially a very useful assay due to gel matrix, which eliminate the history of experience on a certain level of stiff- its relative simplicity. Methods: We carried out a detailed study of mitochon- ness. To design such heterogeneous microelastically-patterned gels, we have drial fluctuations in the C3H-10T1/2 cell line, a murine embryonic mesen- applied the photolithographic microelasticity patterning using photoculable chymal cell line, as well as two osteosarcoma cell lines with low and high gelatins. The emergence of frustrated mode of differentiation was previously metastatic potentials, DUNN and DLM8. Cells were cultured under standard confirmed by immunofluorescence and RT-PCR analysis for the expression growth condition for 24 hr, and then stained with MitoTracker Green FM markers, but more detailed and precise characterizations are of course required. (ThermoFisher Scientific). Random cells were selected for imaging using In this study, to fully characterize the frustrated differentiation of MSCs, we spinning disk confocal microscopy at a high temporal resolution (100 ms investigated oscillation of the mechanical dose and mechanical signal input per frame) for 100 s. We optimized a multi-step image processing protocol to MSCs employing the long-term traction force microscopy for MSCs culture to identify mitochondria with high fidelity. We selected the mitochondria on the microelastically-striped patterned gels. In addition, we performed cDNA with life-time spanning the entire imaging period and with no interaction microarray analysis for the MSCs culture in such mode of frustrated differen- with other mitochondria and calculated the MSD. Results: The MSD of iso- tiation. As the result, MSCs in normadic movement between stiff and soft re- lated mitochondria resembles that of a particle in a viscoelastic medium, in gion of gel surface were confirmed to exhibit characteristic transcriptome agreement with previous results. However comparison of MSD between con- and marked large fluctuation profile of traction forces compared with plain con- trols and cells treated with drugs that disrupt the actin and microtubule trol gels. network showed surprisingly small effects, while treatment with ATP synthe- sis inhibitors significantly decreased the MSD. Our results imply that mito- 2144-Pos Board B464 chondrial fluctuations are primarily driven by active fluctuations of the Cells as Strain-Cued Automata cytoskeleton, and are relatively unaffected by perturbing the actin or microtu- Brian Cox. bule network, at least in the cell lines in our study. Comparison of MSDs be- Arachne Consulting, Sherman Oaks, CA, USA. tween less invasive and more invasive murine osteosarcoma cells showed Autonomous cells can form patterns by responding to local variations in the significant differences at long time scales. Hence active fluctuations differ strain fields that arise from their individual or collective motions. Evidence significantly between different cell types. We also observed significant hetero- of cells acting as strain-cued automata have been inferred from patterns geneity among cells of the same type and even more heterogeneity in the mito- observed in nature and from experiments conducted in vitro. In simulations, chondria from a single cell. Our analysis suggests that mitochondrial cells are assumed to pass information among themselves solely via mechanical fluctuations reflect the active contractile properties of the cytoskeleton.

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2146-Pos Board B466 2149-Pos Board B469 Substrate Chemistry and Morphology Influence the Valvular Interstitial Focal Adhesion Kinase Regulatory Interactions Quantified by TIRF-FRET Cells Mechanobiology Taylor J. Zak, Allen Samarel, Seth Robia. Luisa Ulloa Severino1,2, Rosaria Santoro3, Maurizio Pesce3, Cell and Molecular Physiology, Loyola University, Chicago, IL, USA. Loredana Casalis2, Denis Scaini1,2. Focal adhesion kinase related non-kinase (FRNK) is an inhibitor of focal adhe- 1University of Trieste, Trieste, Italy, 2Elettra-Sincrotrone S.C.a.p., Trieste, sion kinase (FAK)-dependent cell growth, survival, and migration pathways. In Italy, 3Centro Cardiologico Monzino, Milano, Italy. vascular smooth muscle cells, both FAK and FRNK are upregulated after Calcific Aortic Valve Disease (CAVD) is the most common form of valve dis- vascular injury, suggesting balanced stimulation and inhibition of growth dur- ease in the Western world and represents a major healthcare burden. The pri- ing recovery. Here we propose a novel mechanism by which FRNK can inhibit mary driver for valvular calcification is the differentiation of valvular FAK: direct binding of FRNK to FAK forming an inhibitory complex within interstitial cells (VICs) into a diseased phenotype, the osteoblastic-like cells. focal adhesions. This hypothesis is supported by co-immunoprecipitation of Another feature of this disease is the significant change in the organization, FAK and FRNK. In addition, total internal reflection fluorescence (TIRF)- composition and mechanical properties of the extracellular matrix (ECM) mode fluorescence resonance energy transfer (FRET) measurements suggest that beside being the result of the dysfunction of the valve cells, seems to that FRNK binds directly to FAK within focal adhesions of live vascular contribute to the progression of the pathology altering cellular molecular smooth muscle cells. We hypothesized that this interaction might be regulated signaling. Here, we studied the mechanical, morphological and molecular prop- by serine phosphorylation of FRNK S217, a site we found to be basally highly erties of VICs grown on substrated with different surface chemistry and/or me- phosphorylated at S217 by extracellular signal-regulated kinase 1/2 (ERK). To chanical properties, which mimic the valve ECM structure, to highlight the determine the significance of S217 phosphorylation, we generated a non- matrix effect on VICs behavior and the role played in calcified tissue formation. phosphorylatable mutant FRNK by mutating S217 to alanine. S217A-FRNK Using atomic force microscopy (AFM) and immunofluorescence microscopy exhibited significantly higher amounts of FRET compared to wild type we assessed the biomechanics, morphology and the pathway involved in the FRNK, suggesting increased interaction with FAK. Progressive acceptor mechanical stress and in onset of pathology of porcine and human VICs grown photobleaching analysis revealed that the FRNK-FAK complex consisted of on polyacrylamide gels of different stiffness and on variable-geometry carpets FRNK and multiple FAK proteins in a high order hetero-oligomer. To deter- of vertically aligned carbon nanotubes. Our results will allow to identify a suit- mine the functional role of the proposed regulatory site we performed an able material for engineering aortic valve artificial scaffolds. apoptosis assay using flow cytometry. S217A FRNK was a much more potent inducer of apoptosis in vascular smooth muscle cells compared to wild type 2147-Pos Board B467 FRNK. Additionally, we determined that S217A FRNK was a more potent in- Hydration Control in Brain Extracellular Matrix:Astrocytes, Perycites, hibitor of FAK signaling by examining FAK Y397 phosphorylation. We and Fibroblasts Adjust their Contractile Responses to Local Water conclude that dephosphorylation of FRNK at S217 during response to injury Activity results in increased FRNK-FAK complex formation, thereby inhibiting Maria P. McGee, Mary Kearns, Michael Morykwas, Louis Argenta. FAK signaling. Plastic and Reconstructive Surgery, Wake-Forest University Medical School, Winston-Salem, NC, USA. Mitochondria in Cell Life and Death II Efficient brain function requires maintenance of optimal tissue hydration. For example, ex vivo, in mouse hippocampal slices, local brain interstitial dehydration 2150-Pos Board B470 impairs neuronal function, and mounting evidence indicates that, first, brain inter- Population Dynamics of Mitochondria in Mammalian Cells stitial cells regulate transcapillary fluid exchange and, second, water flows in and Kellianne Kornick, Moumita Das. out of brain tissue at speeds proportional to the aw (water activity) in the explant Physics and Astronomy, Rochester Institute of Technology, Rochester, culture media, as expected from passive physical principles. We measured the re- NY, USA. sponses of brain interstitial cells to changes in aw to find a common signal linking Mitochondria are organelles located inside eukaryotic cells that play several the passive adjustments to physiologic responses mediated by genetically pro- key cellular roles, including providing energy (i.e., ATP), participating in grammed cell action. We obtained three types of primary cell cultures from cell signaling, triggering cell differentiation, and initiating apoptosis. All human brain (Sciencell Research Laboratories), astrocytes, pericytes, and peri- organisms are believed to have low levels of variation in mitochondrial DNA vascular fibroblasts, expressing growth-factor astrocyte protein, a-smooth mus- (mtDNA), and repeated mitotic segregation and clonal expansion can enable cle actin, and fibronectin, respectively, and incorporated them into fully a mitochondrion to eventually dominate the mtDNA pool. Alterations in hydrated, 3D collagen gels floating in culture medium. We progressively mtDNA are connected to a range of human health conditions, including: epi- decreased the cultures’ aw by adjusting the dissolved colloids to produce pressures lepsy, heart failure, Parkinson’s disease, diabetes, and multiple sclerosis. of 4, 42, 100, and 200 mmHg, (corresponding to <5% plasma osmotic pressure or Therefore, understanding how changes in mtDNA accumulate over time and a <10 mM shift in NaCl concentration). The colloids fully penetrated the gels; no are correlated to changes in mitochondrial function and cell properties can volume change was observed in gels without cells. Over time, the cells contracted have a profound impact on our understanding of fundamental mammalian the gel and expelled fluid in proportion to the pressure, giving linear regression cell biology and the origins of some human diseases. Motivated by this, we coefficients > 0.9. The slopes of fitted regression lines were -0.99 5 .02 for as- develop and study a mathematical model to determine which cellular parame- trocytes; 1.33 5 0.23 for fibroblasts; and 1.90 5 0 .19 ml/h/mmHg for peri- ters have the largest impact on mtDNA population dynamics. The model con- cytes (Means 5 SE, n = 4-5). Rate increases with aw were significantly larger for sists of coupled differential equations to describe subpopulations of healthy and pericytes than fibroblasts (p = 0.043) and astrocytes (p = 0.0048). These results dysfunctional mitochondria subject to mitochondrial fission, fusion, autophagy, show that brain interstitial cells can detect and respond to minute changes in hy- and mutation, and varying levels of cellular ATP, which depend on ATP gen- dration, adjusting contractile forces to prevent excessive hydration/dehydration eration during fusion and energy dissipation by fission and other cellular mech- in alignment with passive fluid-transfer along aw gradients. anisms. We study the time evolution of each sub-population under specific selection biases and pressures by tuning specific terms in our model and obtain 2148-Pos Board B468 a phase diagram in the parameter space of the ratio of the rates of fusion, Growing Up or Growing Old? mutation, and autophagy of the healthy and dysfunctional populations. The Nash D. Rochman. study complements experiments, where it is often difficult to tune one param- Mechanical Engineering, Johns Hopkins, Baltimore, MD, USA. eter at a time. Our results may provide insights into how sub-populations Growth rate, proliferation, aging, and division are of central importance for un- of mitochondria survive and evolve under different selection pressures and derstanding single-cell dynamics as well as homeostasis and pathogenesis in with time. multicellular systems; however, in the absence of a unifying framework, exper- imental investigation and modelling of the regulatory mechanisms for any one 2151-Pos Board B471 of these properties in isolation results in a confusing landscape. Here we discuss MGR2 Depletion Affects Protein Import and Mitochondrial Metabolism the interplay between growth and proliferation with a specific focus on the YAP Kevin Damri, Oygul Mirzalieva, Ruth Hartke, Pablo M. Peixoto. and mTOR pathways largely motivated by recent work elucidating cell volume Natural Sciences, Baruch College - CUNY, NEW YORK, NY, USA. regulation. This motivates our discussion about the importance of age- Despite containing their own genome, mitochondria are not autonomous organ- dependent cooperation/competition relationships for the establishment of elles. Over 1,146 of the known mitochondrial proteins are encoded in nuclear optimal growth strategies in both single and multicellular contexts. Further, DNA and therefore must be imported from the cytosol. The process has been by mapping optimal growth strategies to their corresponding age distributions, extensively studied and is known to involve several protein complexes spanning we investigate the impact of asymmetric division on robustness. the outer and the inner mitochondrial membranes. However, a new subunit of the

BPJ 7864_7868 438a Tuesday, February 14, 2017 import complex TIM23 was just recently discovered: Mgr2. Our initial analysis Medicine, Miami, FL, USA, 4Brown University, Providence, RI, USA, of protein sequence showed that, like other TIM23 subunits, Mgr2 contained 5Brown University, Providence, RI, USA. GXXXG motifs in certain trans membrane domains. These motifs were Mammalian cytochrome c (Cytc) is a small globular protein and functions as a implicated in subunit oligomerization and pore stability. We hypothesized that mobile electron carrier in the electron transport chain (ETC), shuttling elec- Mgr2 was a structural component of the insertion pore, and that its deletion trons from bc1 complex to cytochrome c oxidase (COX). In addition, Cytc par- would impair peptide import and mitochondrial functioning. We analyzed ticipates in type II apoptosis, when it is released from the mitochondria. Such a protein import in Mgr2 deficient strains using fluorescein labeled synthetic molecule, central to life and death decisions of the cell, should be tightly import peptides. We also tested the effect of Mgr2 deletion on mitochondrial controlled. We previously showed that Cytc is regulated by cell signaling path- function by measuring accumulation of fluorescent membrane potential ways in a highly tissue-specific manner and that Cytc isolated from heart and indicators and by determining respiratory activity using a Clark-type oxygen liver is phosphorylated on Tyr97 and Tyr48, respectively. Here we add another electrode. Moreover, TIM23 function was also examined in isolated mito- phosphorylation site and show that Cytc isolated from kidney is phosphorylated chondria by patch clamp electrophysiology. The results suggested that on Thr28. This phosphorylation is likely mediated by AMP kinase. Phosphor- Mgr2 depletion affected the basal membrane potential and the response to ylated and Thr28Glu phosphomimetic Cytc cause partial inhibition in the reac- depolarizing and hyperpolarizing agents. Interestingly, patch clamp analysis tion with COX supporting our model that under healthy conditions ETC suggested a decrease in pore size and gating properties. Further investigation proteins are phosphorylated to limit electron flux in the electron transport chain, will target the role of GXXXG motifs present in Mgr2 and other TIM23 which in turn prevents a hyperpolarization of the mitochondrial membrane po- components. tential, a known cause of reactive oxygen species (ROS) production and trigger of apoptosis. To test our model we generated Cytc knockout cells expressing 2152-Pos Board B472 Thr28Glu phosphomimetic Cytc. We observed reduction of intact cellular GDAP1 Regulation by Oxidative Stress respiration, mitochondrial membrane potential, and ROS. Finally, high resolu- Sreeram Ravi, Andrew P. VanDemark, Kirill Kiselyov. tion crystal structure analyses and molecular dynamics simulations suggest that University of Pittsburgh, Pittsburgh, PA, USA. the Thr28-containing epitope is the most flexible internal part of Cytc, which is GDAP1 or Ganglioside Induced Differentiation Associated Protein 1 is a in close proximity to key residues on COX and thus suggests a mechanism for poorly understood protein involved in mitochondrial function and several sub- the altered enzyme kinetics. types of Charcot-Marie-Tooth disease. Its function is presently unclear, although a role in mitochondrial fragmentation of redox regulation has been 2155-Pos Board B475 discussed. GDAP1 expression was originally proposed to respond to ganglio- Bcl-2 or Bcl-xL Overexpression Stimulates Lactic Fermentation without side GM3 buildup. Our data show that similar to its relatives glutathione Affecting Whole Cell Respiration S-transferases (GST) GDAP1 transcription is activated by oxidative stress in Laurent Dejean, Nawras Samaan, Ali Abed, Bushra Mahmood, a manner involving transcription factor Nrf2. Acute GDAP loss increases the Jessica Wilson, Preet Kaur, Hooi Chong. sensitivity of cells to oxidative stress induced by ethanol and exogenous lipids. Chemistry, California State University of Fresno, Fresno, CA, USA. These data support the idea that GDAP1 is a component of cytoprotective ma- It is now well established that shifts in energy metabolism are associated with chinery actively responding to and regulating oxidative stress. cancer development and progression. The most studied of these phenomena is 2153-Pos Board B473 the Warburg effect, which corresponds to an increase of anaerobic glycolysis Investigation of the Interactions of the SS-31 Peptides with Cardiolipin vs mitochondrial oxidative phosphorylation to produce energy for cellular pro- Variants: A Potential Therapeutic for Barth Syndrome cesses. However, the mechanisms related to these metabolic switches are still Murugappan Sathappa1, Wayne Mitchell1, Adrian Coscia1, Kevin Boyd1, a matter of debate. Bcl-2 family proteins contain both pro- (e.g. Bax), and Eric May1, Hazel H. Szeto2, Nathan N. Alder1. anti-apoptotic (e.g. Bcl-2 and Bcl-xL) members which are respectively encoded 1Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA, by tumor suppressors and proto-oncogenes. Up-regulation of the anti-apoptotic 2Mitochondrial Therapeutics Consulting, New York, NY, USA. proteins Bcl-2 and Bcl-xL have been associated with Non-Hodgkin’s lym- Szeto-Schiller (SS) peptides are synthetic tetrapeptides containing an alternating phoma; and certain studies indicate that Bcl-2 family proteins play a role in aromatic-cationic motif. They are readily taken up by cells and are targeted to the the regulation of energy metabolism. However, the molecular players involved mitochondrial inner membrane. Among them, SS-31 has been shown to bind to in this regulation are still to be defined. We recently observed that Bcl-2 or Bcl- bilayers in a cardiolipin-dependent manner via electrostatic and hydrophobic in- xL (Bcl-2/xL) overexpression led to a significant increase of lactate production teractions. As a therapeutic agent, SS-31 leads to clinical improvements in many rates in a mouse pro-lymphocyte B cell line. However, both lactate production complex diseases, likely linked to its selective interaction with the cardiolipin- and glucose consumption fluxes are stimulated by Bcl-2/xL overexpression. rich inner membrane, protection of mitochondrial ultrastructure, curtailment Also, basal, uncoupled and non-coupled whole cell respiration remain similar of ROS, and restoration of oxidative phosphorylation. SS-31 represents a first- in the 3 cell lines. Taken together, these results suggest that the fermentative in-class cardiolipin-protective therapeutic; however, its molecular mechanism component, but not the oxidative component, of carbohydrate metabolism is of action remains largely unknown. In this study we have explored the use of stimulated in these genetic contexts. This phenomenon is associated with an in- SS-31 as a potential therapeutic for Barth syndrome (BTHS), a multisystem dis- crease of glycolytic/fermentative (LDH and GAPDH enzyme activity) and mito- order characterized by cardiac and skeletal myopathy, neutropenia, growth retar- chondrial (Citrate synthase activity) markers; but only when Bcl-xL (and not dation, and premature death in young males. BTHS is caused by mutations in the Bcl-2) is overexpressed. These data suggest that Bcl-2 and Bcl-xL expression gene that encodes the transacylase tafazzin, involved in acyl chain remodeling of levels may play an active role in the stimulation of lactic fermentation commonly cardiolipin following its biosynthesis. Dysfunctional tafazzin results in observed in blood cancer cells; and that Bcl-2 and Bcl-xL-mediated stimulation decreased levels of cardiolipin with abnormal fatty acid tail profiles, and an accu- of lactate production are the results of different molecular mechanisms. mulation of the remodeling intermediate monolysocardiolipin. Using model membrane systems and in organello studies, we show that the binding affinities 2156-Pos Board B476 Wolbachia of SS-31 to bilayers containing cardiolipin and monolysocardiolipin are similar. : Interactions with Mitochondria Inside the Cell Moreover, the presence of SS-31 appears to modulate the interaction of the elec- Cristina Uribe-Alvarez, Natalia Chiquete-Fe´lix, Antonio Pen˜a, tron carrier cytochrome c with bilayers containing cardiolipin and its variants. Salvador Uribe-Carvajal. Our results suggest that SS-31 may act in part by altering membrane phase Instituto de Fisiologı´a Celular, Universidad Nacional Auto´noma de Me´xico, behavior. Our empirical results are complemented by computational approaches Me´xico, Mexico. that address the nature of the SS-31 bilayer interaction. Our results point to the Wolbachia pipientis is an intracellular obligate endoparasite/endosymbiont pre- potential use of SS peptides as therapeutics for BTHS and give key mechanistic sent in 70 percent of all insects in the world. Permanence within the host in- insights into the nature of their bilayer interactions. cludes strategies such as feminization, cytoplasmic incompatibility and complementation of metabolic needs. When outside the cell, Wolbachia sur- 2154-Pos Board B474 vives less than a week, remaining capable of infecting new cells, but unable Regulation of Cytochrome C by Phosphorylation: Mitochondrial Respira- to divide. Some hosts, such as filariae are unable to function properly when bac- tion and Apoptosis teria are killed. In these cases different host mitochondrion-related proteins are Maik Huttemann€ 1, Gargi Mahapatra1, Icksoo Lee2, Lawrence I. Grossman1, overexpressed, suggesting that there is a close Wolbachia-mitochondrion inter- Asmita Vaishnav1, Carlos T. Moraes3, Qinqin Ji4, Arthur R. Salomon5, action. In contrast, there are reports that Wolbachia is highly sensitive to ROS. Brian F.P. Edwards1. The metabolism of isolated Wolbachia has not been studied probably due to the 1Wayne State University, Detroit, MI, USA, 2Dankook University, difficulty in obtaining large biomass yields. In cells containing Wolbachia, Chungcheongnam-do, Korea, Republic of, 3University of Miami School of mitochondrial oxidative phosphorylation lifetime is extended, suggesting that

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Wolbachia are determinant for longer mitochondrial survival. This is reflected Inorganic polyphosphate (polyP) is a ubiquitous biological polymer that is in conservation of high concentration of cytochromes b, c, and a, in high rates well-conserved throughout the evolution and various species. Isoenergetic of oxygen consumption and in high respiratory control during aging. with ATP, polyP is composed of multiple subunits of orthophosphate, linked together by phosphoanhydride bonds. PolyP plays divergent roles in mamma- 2157-Pos Board B477 lian cells. In mitochondria, polyP is involved in the induction of the calcium- Quantitative Modeling of Pyruvate Dehydrogenase and its Impact in Sub- activated mitochondrial permeability transition pore (mPTP), as well as in strate Selection, Mitochondrial Respiration and Redox the stimulation of the calcium-induced cell death. Paradoxically, cells with Sonia Cortassa, Steven J. Sollott, Miguel A. Aon. reduced amounts of mitochondrial polyP showed higher levels of cell death NIH/NIA/IRP, Baltimore, MD, USA. in response to increased ROS. Here we show that decreased amount of polyP The pyruvate dehydrogenase (PDH) complex plays a key role in substrate selec- also decreases cells resistance to rotenone and heat-shock induced cytotoxicity. tion, primarily glucose and fatty acids (FAs). In mammals, substrate selection We also demonstrate that the presence of polyP in mammalian cells prevents underlies metabolic remodeling and flexibility, and takes place in all organs the aggregation of the C-subunit of ATP synthase. These findings are in agree- although at different degrees depending on nutritional, health or disease condi- ment with the recently proposed role of polyP as a molecular chaperone, tions. Using a computational model encompassing carbohydrate and lipid catab- involved in cell stress response. We hypothesize that polyP plays a dual role olism (b-oxidation), we analyzed the role of PDH in substrate selection by in the mitochondria-related cell death pathway: during the initial stress it pro- mitochondria, accounting for regulation via multiple effectors (Acetyl CoA tects mitochondria by preventing protein aggregation, while excessive stress 2þ [AcCoA], CoA, NADH, NAD, ATP, ADP, Ca , pyruvate) which target specific leads to the mitochondrial calcium overload and, eventually, to the polyP- kinases and phosphatases rendering phosphorylated (inactive) or dephosphory- mediated activation of mPTP. lated (active) the enzymatic complex. Mitochondrial selection between glucose and FAs was analyzed at different levels of both substrates through its impact on 2160-Pos Board B480 respiration, ROS emission, effector levels, and PDH and b-oxidation fluxes. In The Outer Membrane Potential Generation by the Electrogenic Phos- an integrated computational model of central catabolism, when cells are degrad- phoryl Group Transfer in Mitochondria ing glucose and FAs, AcCoA/CoA and NADH/NAD ratios modulation of PDH Victor V. Lemeshko. activity becomes crucial in substrate selection, in agreement with experimental Escuela de Fisica, Universidad Nacional de Colombia, Sede Medellin, data. Relative contributions to the AcCoA mitochondrial pool from glucose and Medellin, Colombia. FA catabolism were estimated at different ratios between pyruvate (Pyr) and pal- Significant part of ATP produced in mitochondria is transported into the mitoyl CoA (PCoA). When Pyr increased at constant PCoA, the flux through cytosol by transferring phosphoryl groups from the mitochondrial matrix PDH increased 3.6-fold whereas that of lipid transport decreased 15%. In ATP to either creatine or glucose through the electrogenic complexes formed contrast, increasing PCoA at constant Pyr produced ~40% increase in lipid con- by the adenine nucleotide antiporter (ANT), creatine kinase and voltage- sumption as compared to ~11% decrease through PDH. Respiration and H2O2 dependent anion channel (VDAC), or by ANT, VDAC and hexokinase. In emission both increased as a function of PCoA/Pyr ratio, suggesting that ROS addition, direct transfer of negatively charged phosphoryl groups from ATP generation is matched by ROS scavenging. Results show that under conditions of the mitochondrial intermembrane space to the cytosol might be realized of different nutrient availability, the higher sensitivity of PDH toward by various electrogenic VDAC-kinase complexes functioning as simplest bio- glucose-derived substrates is in agreement with the crucial role played by this logical generators of the transmembrane voltage. In this work we performed enzymatic complex in preserving glucose stores. the simplest thermodynamic analysis of the mentioned systems of the phos- phoryl group transfer through the mitochondrial membranes. The performed 2158-Pos Board B478 computational estimations demonstrated a high probability of generation of a Alternate Pathways for Inorganic Phosphate Uptake into Mitochondria positive outer membrane potential (OMP) by the hexokinase-mediated mech- Erin Seifert, Valentina Debattisti, Gyorgy Hajnoczky. anisms. Also, generation of positive or negative OMP is predicted by the cre- Thomas Jefferson University, Philadelphia, PA, USA. atine kinase-mediated mechanisms, depending on the ATP phosphorylation The mitochondrial phosphate carrier (PiC), encoded by the nuclear gene potential of mitochondria with respect to the creatine phosphate phosphoryla- SLC25A3, was purified more than 30 years ago. It is widely believed that tion potential of the cytosol. OMP of higher values may be generated in the PiC serves as the primary means of inorganic phosphate (Pi) uptake into mito- presence of various modulators of VDAC’s voltage sensitivity and conduc- chondria for oxidative phosphorylation (oxphos) and for buffering the vast tance. The presented computational models of OMP generation, as a mecha- amount of calcium that mitochondria can take up. Yet, the recent discovery nism of the electrical feedback control of the mitochondrial energy flux, of mutations in human PiC, and the development of a PiC floxed mouse only demonstrated a possible physiological role of the voltage-gating properties now allow PiC function to be directly studied in vivo. Human SLC25A3 muta- of various VDAC’s isoforms and of their ‘‘molecular corking up’’ by tubulin tions produce a severe clinical phenotype at birth and with striated muscle as a and similar factors. key affected tissue, and mice with cardiac-specific PiC loss eventually develop abnormal cardiac function. Yet, evidence of ample ATP and near normal func- 2161-Pos Board B481 tion despite PiC depletion in humans and mice with PiC depletion suggest that Using Engineered Recombinant Tubulin Sotypes to Study VDAC PiC expression far exceeds Pi needs and/or that functional compensation can be Regulation substantial. Here we test the hypothesis that Pi enters mitochondria by alternate Philip A. Gurnev1, David P. Hoogerheide2, Minhajuddin Sirajuddin3, route(s), allowing substantial mitochondrial ATP production despite near Lucie Bergdoll4, Jeff Abramson4, Tatiana K. Rostovtseva1, absence of PiC. We generated mice with Tamoxifen(Tam)-inducible skeletal Sergey M. Bezrukov1. 1 muscle (Skm) PiC knockdown (TamþCreþ); 3 weeks after Tam, PiC protein Section on Molecular Transport, Eunice Kennedy Shriver National Institute 2 was <5% of control in Skm mitochondria. Despite minimal PiC, TamþCreþ of Child Health and Human Development, Bethesda, MD, USA, Center for mice gained weight normally, performed as control mice on a Rotarod and a Neutron Research, National Institute of Standards and Technology, 3 treadmill at slow speed, though TamþCreþ mice fatigued earlier at higher Gaithersburg, MD, USA, Institute for Stem Cell Biology and Regenerative 4 speeds. Bioenergetics analysis in isolated Skm mitochondria revealed some re- Medicine, Bangalore, India, Department of Physiology, David Geffen sidual oxphos despite >95% PiC loss; maximal oxphos was ~35% of control School of Medicine, UCLA, Los Angeles, CA, USA. with pyruvateþmalate as substrate and rose to ~50% of control with fatty Highly variable anionic C-terminal tails (CTTs) of tubulin had been implicated acidþmalate. In contrast, maximal oxphos in TamþCreþ mitochondria was in regulation of permeability of the mitochondrial voltage-dependent anion abolished with succinate as substrate. Together these observations suggest channel (VDAC). Tubulin, a structural protein of microtubules, is a hetero- that, in the context of PiC loss, compensatory Pi uptake may be occur via the dimer comprising a/b subunits. In cells tubulin possesses significant variability dicarboxylate carrier, which exchanges succinate or malate for Pi. Whether due to the expression of several tubulin genes. Seven a- and eight b-tubulin iso- this occurs in vivo requires investigation. forms in the human tubulin gene family vary mostly in the length and primary sequence of the CTTs. This inherent non-homogeneity of the endogenous 2159-Pos Board B479 tubulin CTTs hindered the analysis of their interaction with VDAC reconsti- Dual Role of Inorganic Polyphosphate (POLYP) in the Regulation of tuted into planar lipid bilayers. A recent study (Sheldon et al., 2015) with Mitochondria-Dependent Cell Death peptide-protein chimeras evaluated the frequency of the VDAC blockage Maria de la Encarnacion Solesio Torregrosa1, Mitchell Marta-Ariza2, events to demonstrate that the on-rate is highly dependent on the tail chemical Fernando Goni2, Evgeny V. Pavlov1. composition. In that study CTT-like peptides were grafted to albumin, a protein 1Basic Sciences, NYU-College of Dentistry, NYC, NY, USA, 2Neurology, that does not bind to the membrane surface, though it is now conclusively es- NYU-School of Medicine, NYC, NY, USA. tablished that membrane binding of the tubulin body is an essential step in

BPJ 7864_7868 440a Tuesday, February 14, 2017 tubulin/VDAC interaction. Here we took an advantage of recently generated re- 2164-Pos Board B484 combinant yeast a/b-tubulin constructs with CTTs from various human tubulin Mitochondrial Permeability Transition Pore and Number of Openings isotypes (Sirajuddin et al., 2014). The effects of each of the engineered con- Nasrin Afzal1, W. Jonathan Lederer2, M. Saleet Jafri1. structs on the reconstituted VDAC were compared to those of w.t. yeast tubulin 1Krasnow Institute, George Mason University, Fairfax, VA, USA, 2School of and recombinant tubulin without CTTs. Single-molecule studies of the tubulin- Medicine, University of Maryland, Baltimore, MD, USA. VDAC interaction showed that length and total anionic charge of the individual Mitochondria are double membrane intracellular organelles that provide the CTTs correlate strongly with the average duration of the blockage. In partic- ATP to meet the energy demands of the cell. Mitochondrial dysfunction has ular, at 30-mV applied voltage the characteristic time of VDAC blockage by been implicated in diverse disease including neurodegenerative pathologies, the longer and more highly charged b3-CTT is up to hundredfold greater heart diseases and many more. In rat cardiac ventricular myocytes, due to than that observed for the a1-CTT, with the apparent gating charge doubled. the large energy demand of a beating heart, mitochondria comprise 30-40% These results suggest novel implications in VDAC regulation by the diversity of cell volume and number approximately 20,000 per cell. Within each mito- of tubulin isotypes found in vertebrates. chondrion, the inner mitochondrial membrane (IMM) provide a base for elec- tron transport chain (ETC) and ATP Synthase proteins. The ETC establishes 2162-Pos Board B482 an electrical and pH gradient with the matrix potential of approximately Mitochondrial VDAC as an Electromechanical Probe of Lipid-Dependent 180 mV. However, depolarizations of the IMM can occur following eleva- 2þ Membrane Binding of Alpha-Synuclein tions of reactive oxygen species (ROS) and/or elevations of matrix [Ca ]m Tatiana K. Rostovtseva1, Daniel Jacobs1, David P. Hoogerheide2, and under other conditions. These depolarizations are largely attributed Amandine Rovini1, Sergey M. Bezrukov1. to the opening of a putative mitochondrial permeability transition pore 1 2 SMT, NICHD, NIH, Bethesda, MD, USA, Center for Neutron Research, (mPTP). The exact molecular composition of the mPTP is controversial and NIST, Gaithersburg, MD, USA. over the years has been attributed to many inner and outer membrane protein Alpha-synuclein (a-syn) is a small, intrinsically disordered cytoplasmic pro- components as well as other proteins. Recently, the ATP synthase dimers have tein highly expressed in the central nervous system and implicated in Parkin- been implicated as key components of the mPTP as well. Contributions of son disease. a-Syn was found to specifically bind to native mitochondrial mPTP to the regulation of matrix Ca2þ, mitochondrial volume and pro- membranes in live cells and is known to be involved in neurodegenerative grammed cell death (apoptosis) have been suggested. The model suggests mitochondrial dysfunction and in neuroapoptosis. Thus, understanding the that in order to simulate experimental data the ion fluxes that pass through molecular mechanisms of a-syn interaction with model membranes mPTP are mainly Naþ and Kþ as these are the most abundant ions present. mimicking the mitochondrial outer membrane (MOM) is of particular impor- Furthermore, it takes the opening of only one mPTP for a transient depolari- tance. Here we study the a-syn-membrane interaction on a single-molecule zation or sustained depolarization with a current of 30 pA and 26 pA respec- level using the voltage dependent anion channel (VDAC) to probe the kinetic tively. Constraints on the protein identities and their behaviors are suggested and structural rearrangements of a-syn at the lipid membrane. We find that by these models. Importantly, this work suggests that specific experiments be both the frequency and duration of the VDAC blockage by a-syn depend carried out to test candidate proteins and protein-combinations for possible strongly on lipid composition. The highest interaction frequency of a-syn involvement in mPTP. with VDAC is observed in anionic lipids; it drops by 100-fold in cationic lipids. The interaction frequency also increases with nonlamellar phosphati- 2165-Pos Board B485 dylethanolamine content in neutral membranes. The duration of blockage is Direct Modulation of the Mitochondrial Permeability Transition Pore by also affected by lipid composition, particularly the onset of a-syn transloca- Oligomeric Alpha-Synuclein Causes Toxicity in PD tion through the VDAC pore. These results support a model in which Marthe Ludtmann1, Plamena Angelova1, Minee-Liane Choi2, a-syn’s N-terminus binding to the membrane is followed by C-terminal Mathew Horrocks3, Artyom Baev4, Daniel Little5, Michael Devine6, blockage of the VDAC pore. Comparison of electrophysiological data with Paul Gissen5, Evgeny Pavlov7, David Klenerman3, Andrey Abramov2, macroscopic measurements of a-syn-lipid binding using Fluorescence Corre- Sonia Gandhi2. lation Spectroscopy and Bilayer Overtone Analysis methods suggests that 1Molecular Neuroscience, UCL, Institute of Neurology, London, United three domains of a-syn play different roles in membrane interactions and, Kingdom, 2UCL, Institute of Neurology, London, United Kingdom, consequently, with VDAC pore. Our results establish VDAC as a suitable 3University of Cambridge, Cambridge, United Kingdom, 4Molecular electromechanical probe to report on monomeric a-syn membrane binding ki- Neuroscience, National University of Uzbekistan, Tashkent, Uzbekistan, netics and dynamic conformational adaptations to the lipid charges and lipid 5MRC Laboratory for Molecular Cell Biology, London, United Kingdom, packing stress. This system will facilitate further exploration of the well- 6Molecular Neuroscience, UCL, London, United Kingdom, 7Molecular recognized, but not well understood, regulatory roles of mitochondrial lipids Neuroscience, NYU College of Dentistry, New York, NY, USA. in bioenergetics and cell metabolism. Alpha-synuclein aggregation and mitochondrial dysfunction are central to the pathogenesis of Parkinson’s disease (PD). This study investigates the structure 2163-Pos Board B483 specific effects of a-synuclein on mitochondrial function and demonstrates the New Insights into the Molecular Structure and Regulation of the Mito- mechanism by which oligomerisation of the protein results in a toxic gain of chondrial Permeability Transition Pore function within mitochondria. Nelli Mnatsakanyan, Han-A Park, Jing Wu, Paige Miranda, We have previously shown that monomeric a-synuclein enters mitochondria Elizabeth A. Jonas. where it interacts with ATP synthase, and aids its efficiency. Employing a Internal Medicine, Yale University, New Haven, CT, USA. proximity ligation assay, we show an interaction between oligomers and The mitochondrial permeabilty transition (mPT) is believed to be a major cell ATP synthase, a proposed key component of the mPTP. We demonstrate death pathway during neurodegenerative diseases, traumatic brain injury and that beta sheet rich a-synuclein oligomers uniquely induce opening of the mito- stroke. Excessive release of the excitatory neurotransmitter glutamate in the chondrial permeability transition pore (mPTP) in whole cells and isolated mito- þ brain triggers intracellular Ca 2 overload and leads to the opening of the mito- chondria. We report that oligomers, but not monomers, generate reactive chondrial permability transition pore (mPTP). We have recently characterized a oxygen species and mitochondrial membrane lipid peroxidation, and these novel large conductance ion channel located in the membrane embedded c-ring trigger mPTP opening. This oligomer-induced effects lead to neuronal cell of F1F0 ATP synthase, which we suggest forms the pore of mPT. The F1F0 ATP death, which could be abrogated by preincubation of the cells with the synthase is a multi-subunit enzymatic complex that couples proton transloca- mPTP inhibitor Cyclosporin A. tion across the inner membrane to ATP synthesis. The c-ring of ATP synthase We validated our findings in an iPS derived neuronal model with an a-synu- F0 domain is connected to the F1 catalytic domain through the central and pe- clein triplication in which increased levels of aggregated a-synuclein cause ripheral stalks. We show now that the ATP synthase F1 subdomain detaches early onset PD. In this model we report that oligomers also have a strong inter- from F0 under glutamate-induced excitotoxicity and cell death. In patch clamp action with the ATP synthase. Furthermore, we demonstrate a low threshold for recordings we find that the F1 catalytic portion of the ATP synthase gates the mPTP opening on exposure to high laser and calcium. mPTP opening in this c-subunit channel. We also find that mutations of specific amino acid residues model led to cell death which could again be prevented by mPTP inhibition. within the channel reduce its conductance and protect neurons from glutamate- We were thus able to conclude that the mitochondrial abnormalities seen in induced excitotoxic death.We are currently investigating further the molecular the iPS cells with high levels of a-synuclein were likely to be mediated by mechanisms of c-subunit channel conductance, its regulation and its role in cell the beta-sheet oligomeric form of the protein. life and death. These findings will lead to a structure-based drug design of spe- This study provides evidence of a direct effect of oligomers on mitochondria cific therapeutic compounds targeting mPTP for the treatment of neurological and is the first to specifically link oligomeric a-synuclein to ATP synthase lead- disorders and stroke. ing to neuronal death.

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Light Energy Harvesting, Trapping, and MALDI-TOF spectrometry and were analyzed by absorption, fluorescence emission and circular dichroism spectra. Transfer Our results show that both protein complexes possess equal absorption and fluorescence emission spectra and fluorescence lifetime. The circular dichrosim 2166-Pos Board B486 spectra are basically the same for both proteins. The main difference between Slow and Fast Fluorescence Quenching of LHCII in Chlamydomonas these proteins is the thermal stability of ApcR, which has a Tm 5 C degree Reinhardtii Cells lower than ApcN. Roberta Croce, Lijin Tian, Emine Dinc, Laura M. Roy. VU University Amsterdam, Amsterdam, Netherlands. 2168-Pos Board B488 In this work we have studied the process of non-photochemical quenching Orange Carotenoid Protein Picosecond Dynamics Changes with Photo and (NPQ) in vivo in the model green alga C. reinhardtii. NPQ is the process Chemical Activation that protects algae and plants from high light damage by dissipating large Yanting Deng1, Mengyang Xu1, Haijun Liu2,3, Robert E. Blankenship2,3, part of the absorbed energy as heat and which is activated by the low lumenal Andrea G. Markelz1. pH. Despite a large research effort the NPQ mechanism remains debated. The 1Department of Physics, State University of New York-Buffalo, buffalo, NY, main obstacle to understand NPQ resides in the gap between in vitro and in vivo USA, 2Department of Biology, Washington University in St. Louis, St. Louis, studies. On the one hand, the complexity of the thylakoid membrane makes it MO, USA, 3Department of Chemistry, Washington University in St. Louis, very difficult to obtain molecular information from in vivo experiments. On the St. Louis, MO, USA. other hand, a good in vitro system for the study of the quenching is not avail- Orange carotenoid protein (OCP) is the only photosensory protein described able. This has generated to a series of contrasting models that cannot be vali- to date having a carotenoid as the active chromophore. Upon strong green- dated. We have developed a ‘‘minimal NPQ cell’’ which allows us to study blue (or white) illumination, OCP goes from the orange inactive state the effect of the individual NPQ players in the membrane of living cells. We (OCPO) to the red active state (OCPR) [1]. OCPR interacts with the phycobi- show that LHCII, the main antenna complexes of algae and plants, exists in lisome (PB), assisting in thermal energy dissipation. This dissipation dimin- different quenching states in the membrane depending on the growth condi- ishes the formation of reactive oxygen which damages the photosynthetic tions. This difference is suggested to be due to a difference in crowding. How- apparatus and leads to cell death [2]. OCP has a large conformational change ever, we also show that LHCII is not able to switch to the quenched as it goes from OCPO to OCPR, and this structural change is thought to be conformation in response to pH. Instead, the presence of a very small amount essential to the energy quenching interaction with the PB. However, the of the protein LHCSR1 is sufficient to induce a large quenching (50%) in a conversion from inactive to active state has been recently shown to also membrane that contains only LHCII when the pH drops to 5.5. The quenching occur with high chaotrope concentrations. Specifically high concentration is very fast and completely reversible. Time-resolved fluorescence measure- NaSCN produces a long lived red state in the absence of photoexcitation ments at room and low temperature show that the quenching occurs in [3]. We suggest that both the susceptibility of OCP to large conformational 360 ps and is not induced by additional clustering of LHCII in the membrane. change and its interaction with PB are associated with changes in the long Based on these results we propose a new model that links NPQ to long term range picosecond structural flexibility. We measure OCP protein flexibility acclimation responses. changes with photoactivation and chemical activation using terahertz absorp- tion spectroscopy. Temperature dependent terahertz time domain spectros- 2167-Pos Board B487 copy is measured in the 100 - 290 K range on OCP solutions as a function Allophycocyanin from Gracilaria Chilensis and its Recombinant. A of illumination and NaSCN concentration. A rapid increase in the THz absor- Comparative Biophysical and Spectroscopic Study bance is observed in the 180-220 K range. We quantify the picosecond struc- Jorge A. Dagnino-Leone, Marta Bunster, Jose Martinez-Oyanedel, tural flexibility by both the net THz absorption and the dynamical transition Maria Victoria Hinrichs. temperature, which scales with structural stability. R.E.B acknowledges Bioquimica y Biologia Molecular, Universidad de Concepcion, Concepcion, DOE award DE-FG02-07ER15902 and A.G.M acknowleges NSF awards Chile. DBI 1556359 and MCB 1616529, and DOE award DE-SC0016317 for sup- Phycobilisomes (PBS) are auxiliary photosynthetic complexes that allow cya- port of the work. nobacteria and red algae to enhance the energy uptake in the range of 490- 1. Wilson, A. et al. Proc. Natl. Acad. Sci. U. S. A. 105:12075-12080(2008). 680 nm. 2. Wilson, A. et al. Plant Cell, 18:992-1007(2006). In Gracilaria chilensis, an eukaryotic red algae, PBS is composed of Phycoer- 3. King, J.D. et al. FEBS Lett, 588:4561-4565(2014). ythrin (PE), Phycocyanin (PC) and Allophycocyanin (APC); these proteins possess chromophores which capture energy and then transfer it to 2169-Pos Board B489 photosytems. Light-Harvesting Complex II in Controlled Architectures: Visualizing PBPs are oligomers of a ab heterodimer; it oligomerizes into a trimer (ab)3, Changes in Fluorescence Lifetimes 1 2 2 2 which is associated in hexamers (ab)6. Several of this hexamers form Peter G. Adams , Cvetelin Vasilev , C. Neil Hunter , Matthew P. Johnson . cylinder-like structures. 1School of Physics and Astronomy, University of Leeds, Leeds, United PBS has 2 components: antennas and core. The antennas are composed of PE Kingdom, 2Department of Molecular Biology and Biotechnology, University and PC, whose function is to capture energy between 490-570 and 590-625 nm of Sheffield, Sheffield, United Kingdom. respectively and transfer it to the core. The core is formed by APC, which can In the first stages of photosynthesis, light-harvesting membrane protein absorb energy in the 620-650 nm range. APC emission allows transferring en- complexes form an interconnected network, absorbing photons and ergy to the photosystems with high efficiency. PBS are also composed by linker transferring energy as electronic excited states with high efficiency. Light proteins which allow their correct assembly at the membrane and possibly regu- Harvesting Complex II (LHCII) has several important roles, including: (i) late the energy transfer. acting as an antenna complex for Photosystem II (PSII), (ii) activating Non- APC is also formed by a and b subunits, which possess a phycocyanobilin Photochemical Quenching (NPQ) and (iii) promoting multi-layer stacking (PCB) molecule, an open-chain tetrapyrrol molecule covalently attached to a of thylakoid membranes. In this study, we investigated how the optical prop- conserved cysteine residue. In the biosynthesis of the chromophore two en- erties of LHCII depend upon protein-protein and lipid-protein interactions. zymes are required heme-oxygenase and phycocyanibilin oxidoreductase. Microscale array patterns of either single- or multi-layers of LHCII were The attachment of PCB molecule to APC is mediated by the activity of a res- generated on solid substrates, and studied by atomic force microscopy and idue specific heterodimeric lyase, which is responsible for the final maturation fluorescence microscopy with spectral and lifetime imaging. Fluorescence of APC. spectra confirmed that the native chromophore organization of LHCII was The objective of the present work is to compare the functional properties maintained. Interestingly, LHCII had lower fluorescence lifetimes in multi- of native APC (ApcN) and recombinant APC (ApcR), and to achieve these layers, suggesting that increased LHCII-LHCII interactions promote the goal we have used molecular biology, biochemistry and spectroscopic quenched state. In other experiments, LHCII was deposited onto solid techniques. supports at varying protein density and in the presence or absence of a We purified apcN from Gracilaria chilensis using ammonium sulphate precip- surrounding lipid membrane. LHCII fluorescence was strongly enhanced itation and ionic exchange, hydroxyapatite and gel filtration chromatography. when lipids were added, potentially due to decreased self-quenching as the To express ApcR, we set up a three bi-cistronic vector system, which were co- lipids effectively dilute the LHCII membrane concentration, decreasing transformed in E coli and proteins expression were induced with IPTG. ApcR LHCII-LHCII interactions. These experiments reveal switching between was purified using ammonium sulphate precipitation, ionic exchange, IMAC quenched and unquenched states depending on LHCII-LHCII and LHCII- and gel filtration chromatography. ApcN and ApcR, were verificated by lipid interactions.

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2170-Pos Board B490 tures of porphyrin singlet and triplet states. Only a small fraction of photosensi- The Picosecond Kinetics of Non-Photochemical Quenching in Leaves in the tizer excited state energy is retained in the CS state suggesting a competing Presence of Open and Closed Reaction Centers energy transfer mechanism. Unbound fraction of ZnTPPS shows triplet quench- Herbert van Amerongen, Shazia Farooq. ing as a second order process with similar low CS state yields for both cyt c and Laboratory of Biophysics, Wageningen University, Wageningen, PpcA. Heteronuclear NMR and all-atom molecular dynamics simulations reveal Netherlands. the likely surface binding sites for ZnTPPS. However, small angle X-ray scat- When the photosynthetic apparatus of plants becomes saturated in high light, tering measurements demonstrate propensity of ZnTPPS to induce formation a protective mechanism, called non-photochemical quenching (NPQ), is of protein multimers under experimental conditions typical for our experiments. switched on. This is particularly important when the reaction centers (RCs) There results demonstrate an unexpected complexity of ZnTPPS binding inter- of photosystem II (PSII) are closed because in that case enhanced chlorophyll actions and photochemistry with c-type cytochromes and suggest the presence of triplet formation might occur, leading to the production of singlet oxygen, a competing dominant energy transfer process. which can lead to severe damage or even death of the plant. NPQ provides an additional decay channel for chlorophyll excited states, thereby shortening 2173-Pos Board B493 the excited-state lifetime and lowering the probability that excitations end up Optical and Electrochemical Properties of Synthetic Chlorophyll Oligo- in a closed RC. However, the exact mechanism of NPQ is still unknown and mers Connected with Amide Bond also the rate of NPQ has never been determined. It is therefore not known Tomohiro Tatebe, Hitoshi Tamiaki. whether this rate depends on the state of the RCs, i.e. whether they are Ritsumeikan University, Kusatsu, Japan. open or closed. We have designed a new setup on which it is now possible Chlorophylls are photosynthetically active pigments and frequently form to measure spectrally-resolved picosecond fluorescence on leaves with either various dimers in photosynthetic apparatuses. To mimic such supermolecules, open or closed RCs in the presence and absence of NPQ. We have measured we have already synthesized chlorophyll dyads linked with an amide bond and remarkable differences for leaves with open and closed RCs, which are in here report their optical and electrochemical properties in a solution. The Soret disagreement with the current views on NPQ and the results will be and Qy bands of the synthetic dimers were broader and shifted to a longer presented. wavelength, respectively, in CH2Cl2 than those of the corresponding monomer. In addition, new CD peaks were observed at the red-sides of these absorption 2171-Pos Board B491 bands in the dimers. The oxidation potentials were reduced by the dimerization. Mimicking Natural Photosynthsis: Ultrafast Charge Transfer in PpcA These changes were invisible in amino-linked chlorophyll dimers and ascrib- able to the intermolecularly stacking chlorin p-systems in the present dimers. Ru(bpy)3 Complexes Matthew O’Malley. Similar observation in chlorophyll trimers will be discussed. James Madison University, Harrisonburg, VA, USA. 2174-Pos Board B494 Converting light energy into its electrochemical equivalent requires precise Dephasing Times in the Phycobiliprotein Antenna Complexes PE545 and control and fine tuning of relevant kinetic and thermodynamic parameters, PE555 including primary charge separation. To this end, we developed a series Maria I. Mallus, Suryanarayanan Chandrasekaran, Ulrich Kleinekatho¨fer. of 22 cysteine mutants of PpcA, a 3-heme cytochrome from Geobacter Dept. of Physics & Earth Sciences, Jacobs University Bremen, Bremen, sulfurreducens as a model system to study short distance photo-induced Germany. electron transfer. These proteins were successfully expressed in E.coli and Since long-lived quantum coherence have not only been observed in the Fenna- isolated for covalent labeling with Ru(bpy)2(bpy-Br). Protein purity and Matthews-Olson (FMO) complex of green sulfur bacteria but also in the phyco- successful posttranslational modifications were confirmed with HPLC-MS. erythrin 545 (PE545) photosynthetic antenna system, the interest in these Time-resolved nanosecond and ultrafast transient absorbance characteriza- systems has been increased significantly. Moreover, similar results have been tion was performed at Argonne National Laboratory (ANL) and identified found for the PE555 complex in which the two alpha-beta monomers are rotated 6 constructs with apparent photo-induced charge transfer time constants of compared to the PE545 structure leading to a water filled channel. By now, all 20 ps or faster, including 2 constructs with 1-2 ps time constants. The three complexes have been investigated by a sequential combination of molecu- latter is a significant result as up to this point only natural photosynthetic lar dynamics (MD) simulations, calculations of vertical excitation energies along systems demonstrated such a fast initial charge separation, while all artificial the trajectory and quantum dynamics for the exciton transfer [1-3]. A comparison covalent constructs exhibited charge transfer rates 3 or more orders of of the obtained spectral densities and dynamics will be provided. An interesting magnitude slower. To understand molecular principles responsible for such property for the systems is the dephasing time which can give an estimate for how a dramatic acceleration of electron transfer rates, we conducted small- and long quantum coherences might survive in the respective system. In a first step, wide angle X-ray scattering data collection at the Advanced Photon Source the relationship between dephasing time and energy gap fluctuations of the indi- at ANL. Further, we are currently attempting to obtain X-ray crystallo- vidual pigments has been determined [4]. For the above mentioned complex but graphic and NMR structures of the ultrafast constructs. Finally, we per- also additional exciton and charge transfer systems it can be confirmed that that formed triplicate 250-300 ns all-atom molecular dynamics simulations of an inverse proportionality exists between dephasing time and average gap energy all 6 ultrafast constructs. Based on the obtained results we conclude that fluctuation. Interestingly, an interestingly very similar behavior has been found that photo-induced ultrafast charge transfer requires van der Waals contact for all these systems. In a subsequent step, the relationship between dephasing between heme vinyl groups and photosensitizers while contacts with propi- time and excitonic energy gap fluctuations for entire complexes including the onates or short covalent donor-acceptor distances play a much less signifi- respective inter-molecular couplings has been studied. cant role. [1] C. Olbrich, J. Strumpfer,€ K. Schulten, U. Kleinekatho¨fer, J. Phys. Chem. Lett. 2, 1771 (2011). 2172-Pos Board B492 [2] M. Aghtar, J. Strumpfer,€ C. Olbrich, K. Schulten, U. Kleinekatho¨fer, J. Photoinduced Charge and Energy Transfer in Complexes of C-Type Cyto- Phys. Chem. Lett. 5, 3131 (2014). chromes with Water-Soluble Porphyrins [3] S. Chandrasekaran, K. R. Pothula, U. Kleinekatho¨fer, J. Phys. Chem. B, Oleksandr Kokhan, Daniel R. Marzolf, C. Alexander Hudson, DOI: 10.1021/acs.jpcb.6b05803 (2016). Aidan M. McKenzie. [4] M. I. Mallus, M. Aghtar, S. Chandrasekaran, G. Ludemann,€ M. Elstner, U. Chemsitry and Biochemistry, James Madison University, Harrisonburg, VA, Kleinekatho¨fer, J. Phys. Chem. Lett. 7, 1102 (2016). USA. Due to their rich and tunable spectral and redox properties as well as relatively 2175-Pos Board B495 simple synthesis protocols, water-soluble porphyrins are promising candidates Using a Light-Driven Proton Pump Protein to Develop a Reduction- as photosensitizers for artificial photosynthesis. Quenching of porphyrin static Oxidation Reaction Cell fluorescence by monoheme horse heart cytochrome (cyt) c is a well-known phe- U-Ting Chiu, Ling Chao. nomenon. It is attributed to the fast charge separation (CS), though the CS state National Taiwan University, Taipei, Taiwan. has not been observed. Similarly, we found quenching of static fluorescence of Bacteriorhodopsin (BR) is a membrane protein found in the halophilic archaea porphyrins in the presence of PpcA, a 3-heme c-type cytochrome from the cyt c7 Halobacterium salinarum, which is characterized as a light-driven proton family from Geobacter sulfurreducens. In this report using ultrafast visible tran- pump. BR has a wide range of absorption wavelength in visible light, high ther- sient absorbance spectroscopy we demonstrate that the excited state of protein- mal stability and a broad range of pH tolerability, which render the possibility bound zinc tetraphenyl sulfonated porphyrin (ZnTPPS) decays with an apparent of using it for bioelectronic applications. Here, we developed a novel device time constant of about 200 ps in the presence of cyt c and approximately 20 ps in utilizing BR as a photoactive agent to generate steady currents through the the presence of PpcA. However, the spectral changes are dominated by the fea- continuous reduction-oxidation reaction of proton under light illumination.

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We inserted BRs originally protected by detergents into liposomes by dialysis lamic neurons. The addition of a glucose concentration at which GCK is sen- and used the charges of liposomes and pH to control the insertion orientation of sitive (and standard hexokinase is saturated) leads to an increase in cellular BR into the liposomes in order to increase the later proton pumping efficiency. metabolism, as measured by NADH autofluorescence; but only in the presence We deposited the liposomes with BRs on a porous support to form a supported of isoproterenol, a beta-adrenergic receptor agonist which increases intracel- lipid bilayer (SLB) with BRs. This porous support-SLB-BR structure, which lular calcium to activate neuronal nitric oxide synthase and facilitate S-nitrosy- separated platinum cathode from anode, allowed the BRs to pump proton lation. We also use a FRET-based GCK sensor to measure GCK activation and accumulate electrical potential in a specific direction in response to sun- under low and high glucose concentrations and in the presence of isoproterenol. light. The differences of the proton concentration and electrical potential across These studies reveal the presence of a functional GCK and the sensitivity of the porous support-SLB-BR structure could drive the reduction-oxidation reac- GCK to post-translational/signaling cascade modulation. Further, the lack of tion at the electrodes, and therefore generated an electrical current. metabolic response to glucose alone may be linked to high basal activity of GCK in this serum-grown cell line. These results point to a functional, Molecular and Cellular Neuroscience receptor-potentiated glucose sensing mechanism in neurons that is mediated through post-translational activation of GCK. 2176-Pos Board B496 Mechanisms of Fractional Calcium Currents through TRPV1 Channels in 2179-Pos Board B499 Primary Sensory Neurons Receptor Level Dissection of Common Versus Discrete Vesicle Release Zhuan Zhou. Pathways from Primary Vagal Afferent Terminals Institue of Molecular Medicine, Peking University, Beijing, China. James H. Peters. TRPV1 and TRPV2 channels are important molecular sensors on the plasma Dept. of Integrative Physiology and Neuroscience, Washington State membrane in mammalian physiology (temperature) and diseases (pain) in sen- University, Pullman, WA, USA. 2þ þ þ sory neurons. These nonselective cation channels permit Ca ,Na and K in- Primary vagal afferent neurons form strong excitatory glutamatergic synapses 2þ fluxes simultaneously through its pores to regulate intracellular Ca onto second-order neurons in the nucleus of the solitary tract and initiate key 2þ 2þ homeostasis and Ca -dependent neural transmitter release. The Ca influx autonomic and homeostatic reflex pathways. Vesicle fusion and release from 2þ can be determined by the fractional Ca current (Pf) through a cation channel vagal central terminals occurs via three distinct pathways including synchro- (Burnashev et al., 1995; Schneggenburger et al., 1993; Yu et al., 2004; Zhou nous, asynchronous, and spontaneous. While synchronous and asynchronous 2þ and Neher, 1993). Here, we report (1) Opposite Ca permeability was found release mechanisms require presynaptic action-potential depolarization; spon- as determined by the classic ‘‘Goldman-Hodgkin-Katz equation’’ of PCa/Na taneous release is ongoing and independent of action potentials. Evidence sug- (TRPV1 = 7.6 > TRPV2 = 2.8) under non-physiological intra- and extracellular gests these forms of release can be controlled independently indicating discrete solutions(Caterina et al., 1999; Caterina et al., 1997), or by the ‘‘fractional and potentially separate vesicle pools and release sites. However, the presence 2þ Ca -current’’ of TRPV1 vs. TRPV2 (Pf = 5.5% vs. 22%) under physiological and activation of transient receptor potential (TRP) channels (including solutions; (2) The selective filter ‘‘GMGX’’ is similar in TRPV1 and TRPV2, TRPV1) at the central terminals dramatically increases the rate of spontaneous except ‘‘X’’ (X = D for TRPV1 and E for V2). In TRPV1, switching native D to vesicle release and eventually diminish action-potential driven synchronous E of TRPV2, Pf(V1, D646E) was greatly increased toward Pf(V2) (from 5.5% release; consistent with a common releasable pool. Confounding this interpre- to 13%), and vice versa (from 22% to 5.0%); (3) Mutations of two sites outside tation is the observation that TRP channel activation also diminishes voltage of the ‘‘GMGX’’, reduced Pf by half; (4) In native neurons replacing TRPV1- activated sodium channel signaling. As such the decrease in synchronous 2þ WT (Pf = 5.5%) with TRPV1-D646E (Pf = 13%), the release mode of Ca - release may be a result of action potential failure, rather than depleted vesicle dependent single vesicle events was dramatically altered from partial (kiss- pools. Given that synchronous and spontaneously release glutamate activates and-run) to full release as determined by TIRF-imaging, implicating a physi- both AMPA and NMDA receptors postsynaptically we utilized ligand depen- logical relevance of Pf(TRP) studies. Taken together, TRPV1-D646 (or dent post-synaptic NMDA receptor blockade (MK-801) to overcome this 2þ TRPV2-E604) is the dominant site determining fractional Ca -influx through confound. We found that treatment with MK-801 during exclusively sponta- thermal sensitive TRP channels—a novel mechanism of TRPV channels in pre- neous release blocked the NMDA component of spontaneous events and sub- synaptic neural transmitter release for temperature and pain sensation. sequent action-potential driven synchronous release. This result demonstrates that glutamate released via spontaneous vesicle release is activating the same 2177-Pos Board B497 population of NMDA receptors as glutamate released synchronously. This A Benefit of Randomness in Synaptic Vesicle Release finding is consistent with a common release site for synchronous and sponta- Calvin Zhang1, Charles S. Peskin2. neous release at vagal afferent terminals. 1Department of Mathematics, University of Arizona, Tucson, AZ, USA, 2Courant Institute of Mathematical Sciences, New York University, New York, NY, USA. 2180-Pos Board B500 Noise is not only a source of disturbance, but it also can be beneficial for Associative Memory Cells are Recruited to Encode Triple Sensory Signals neuronal information processing. The release of neurotransmitter vesicles in via Synapse Formation synapses is an unreliable process, especially in the central nervous system. Jin H. Wang, Jing Feng, Wei Lu. Here we show that the probabilistic nature of neurotransmitter release directly Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. influences the functional role of a synapse, and that a small probability of Associative learning is a common form of information acquisition, and associa- release per docked vesicle helps reduce the error in the reconstruction of tive memory is essential for logical reasoning and associative thinking. In addi- desired signals from the time series of vesicle release events. tion to memory cells for two associated signals, here we report the recruitment of associative memory cells that encode triple sensory signals. Paired mouse 2178-Pos Board B498 whisker, olfaction and tail stimulations lead to odorant-induced and tail- Glucokinase Mediated Glucosensing in Hypothalamic Neurons induced whisker motions. In the mice of showing cross-modal reflexes, barrel Jennifer McFarland, Kendra Seckinger, Mark Rizzo. cortical neurons and astrocytes become to encode odor and tail signals along- University of Maryland, Baltimore, Baltimore, MD, USA. side whisker signal. Such neurons receive the new synapse innervations from Glucosensing is the ability of specialized cells to detect changes in extracellular the axons of the piriform and S1-tail cortical neurons, in addition to the innate glucose concentration and convert this information into a change in membrane synapse innervations from the axons of the thalamic neurons. The formation of potential; this allows glucose levels to influence the activity of cells that regu- new synapse innervations and the recruitment of associative memory cells late glucose-altering or glucose-dependent physiological processes. Two major require the upregulations of miRNA-324-5p and miRNA-133a-3p that downre- glucosensing cellular populations are pancreatic and hypothalamic cells; the gulate tau-tubulin kinase-1 (Ttbk1) and methylcytosine dioxygenase Tet3. The mechanism of glucosensing in pancreatic beta cells is established, but that of associated activations of the sensory cortices elicit their mutual synapse inner- hypothalamic neurons remains controversial. Beta cells glucosense by express- vations that recruit memory cells to store triple associated signals via epigenetic ing a specialized type of hexokinase (the rate-limiting enzyme in glucose meta- processes. Furthermore, after the extinction of cross-modal reflexes, the refine- bolism) called glucokinase (GCK). GCK, unlike standard hexokinase, is not ments of the synapses and memory cells in the barrel cortex are well main- saturated at physiological levels of glucose, allowing glucose metabolism in tained, while plasticity in the M1-motor cortex decreases. These results cells expressing GCK to increase when the extracellular glucose concentration indicate that the acquired signals remain stored in the sensory cortices and is raised. This increase in metabolism is then converted into a membrane depo- the decay of memory retrieval is due to the decreased refinement of their down- larization. Furthermore, S-nitrosylation can enhance the activity of GCK, and stream cortical regions in neural circuits from the sensory cortices to the therefore enhance glucose metabolism and glucosensing. Here we use GT1-7 memory-presentation cortices. In terms of the significance of our discoveries, cells to explore whether a similar mechanism of glucosensing exists in hypotha- the storages of multiple signals in individual neurons may expand memory

BPJ 7864_7868 444a Tuesday, February 14, 2017 volumes, strengthen cognition capabilities and facilitate creative inspiration dictive model of the circuit underlying the ecdysis sequence with the general productions. Information storages in the sensory cortices confer them to signify goal of understanding how nervous systems orchestrate complex motor se- the sources of the retrieved memory signals. This study is supported by quences in response to input stimuli. National Basic Research Program (2013CB531304 and 2016YFC1307100) and Natural Science Foundation China (81671071 and 81471123) to JHW. 2183-Pos Board B503 Structural Polymorphism of Amyloid Fibrils in Alzheimer’s Disease 1 2 3 3 2181-Pos Board B501 Liu Jiliang , John Badger , Biel Roig Solvas , Lee Makowski . 1Brookhaven National Lab, Upton, NY, USA, 2Delta G Technologies, San Bilobalide Protects against Focal Cerebral Ischemia Reperfusion Injury 3 by Inhibiting of Cell Death Pathways and Promoting of Angiogenesis Diego, CA, USA, Northeastern University, Boston, MA, USA. Yongqiu Zheng1, Mingjiang Yao1, Frank Yi2, Bin Yang1, Xiaodi Fan1, X-ray microdiffraction of histological sections of human tissue has been used to Jianxun Liu1, Matthew Orange3, Hua Zhu2. map variations in the wide-angle scattering from amyloid fibrils in human brain 1Xiyuan Hospital, Beijing, China, 2The Ohio State University, Columbus, in Alzheimer’s Disease (AD). These studies indicate that tissue derived from OH, USA, 3Department of Physical Education and Human Performance, subjects with different clinical histories may contain different ensembles of Central Connecticut State University, New Britain, CT, USA. fibrillar structures; plaques harboring distinct amyloid structures can coexist Ischemic stroke is a leading cause of long-term disability. Although previous within a single tissue section; and within individual plaques there may be a studies have identified some promising neuroprotective agents, such as cinnamo- gradient of fibrillar structure from core to margins. The significance of these philin, EGCG, and oxyresveratrol, their protective effects are still very limited. polymorphisms is uncertain. One possibility is that amyloid fibrils with Thus, identification of novel neuroprotective agents is highly desired for treat- different structures may contribute differentially to disease. However, testing ment of this disease. Previous studies have shown that Ginkgo biloba extract this hypothesis will require significantly more information about the structure has protective effects against neurodegenerative disorders such as cerebral insuf- and structural variation of amyloid. Here we report initial results of modeling ficiency, Alzheimer’s disease, and dementia. In this present study, we sought to amyloid fibril structures to explain the observed variations in x-ray scattering. test the potential protective role of an active component of Ginkgo biloba extract, By characterizing the polymorphisms in fibril structure and their distribution Bilobalide, in a rat model of middle cerebral artery occlusion (MCAO). We throughout the brain, we seek to better understand the nucleation and matura- found that intraperitoneal (IP) delivery of various Bilobalide doses during tion of plaques, the relationship between fibril structure and packing within the ischemia can protect against brain injury, as evidenced by reduced infarct size plaques and the contribution of these processes to the inception and inexorable and improved neurological scores after surgery. Histochemical analysis revealed progression of neurodegeneration and disease. that treatment with Bilobalide can significantly reduce apoptosis, autophagy, and 2184-Pos Board B504 promote angiogeneis following ischemia/reperfusion injury to the brain. In order Modulation of Amyloid Peptide Oligomerization and Toxicity by Extracel- to determine the molecular mechanisms underlying Bilobalide mediated neuro- lular Hsp70 protective effects, biochemical assays were performed, and the results suggested Antonio De Maio1, Isabel Rivera2, David M. Cauvi3, Nelson Arispe4. that Bilobalide can activate Akt prosurvival and eNOS pathways to promote cell 1Department of Surgery, Department of Neurosciences, School of Medicine, survival and angiogenesis, respectively. To confirm these biochemical observa- University of California, San Diego, La Jolla, CA, USA, 2Initiative for tions, we cotreated animals with a combination of Bilobalide and inhibitors of Maximizing Student Development (IMSD), University of California, San Akt and/or eNOS pathways. We found that treatment with inhibitors can Diego, La Jolla, CA, USA, 3Department of Surgery, University of California, partially abolish the beneficial effects of Bilobalide, indicating that Bilobalide San Diego, La Jolla, CA, USA, 4Department of Anatomy, Pysiology and indeed provides protection from neuronal injury through activation of these Genetics, Uniformed Services University, Bethesda, MD, USA. signaling pathways. Taken together, our results suggested that Bilobalide bene- Alzheimer’s disease (AD) is a progressive neurodegenerative disorder leading to fits stroke symptoms by reducing cell death pathways (apoptosis and autophagy) dementia due to advanced neuronal dysfunction and death. Treatment for AD is and promoting angiogenesis. As such, Bilobalide may be a potential agent for a major burden for the health care system in the U.S. and the disease has a large improving self-repair after ischemic stroke. emotional, social, and economic impact on the family members. Currently, the predominant theory for AD is the ‘‘amyloid hypothesis,’’ which states that 2182-Pos Board B502 abnormally increased levels of amyloid b (Ab) peptides results in the production Using Light-Sheet Microscopy to Understand Evoked Motor Sequence of a variety of Ab aggregates that are neurotoxic. The specific mechanism(s) for Generation Ab-induced cytotoxicity has not been completely elucidated yet. However, 1 2 2 2 Amicia D. Elliott , Feici Diao , Sarav Shah , Daniel Yasoshima , since the majority of Ab is released in the extracellular milieu, it is reasonable 3 3 2 Yicong Wu , Hari Shroff , Benjamin White . to assume that toxicity begins outside the cells and makes its way inside where 1NIGMS, National Institutes of Health, Bethesda, MD, USA, 2NIMH, 3 it disrupts the basic cellular process that results in cell stress and death. Cells National Institutes of Health, Bethesda, MD, USA, NIBIB, National exposed to stressful conditions respond by the expression of heat shock proteins Institutes of Health, Bethesda, MD, USA. (hsps) that are involved in processes to preserve cell viability. Hsps are molec- Motor sequences are critical elements of everyday behavior, but how they are ular chaperones that play a major role in protein folding and the solubilization of produced by central neural circuits is poorly understood. The complexity and misfolded aggregated polypeptides. Recently, it has been demonstrated that scale of the circuitry involved makes motor sequence generation very difficult hsps, in particular Hsp70, are exported into the extracellular milieu by an active to study at the cellular level in large, mammalian brains. However, the neural export mechanism independent of cell death. Therefore, both Ab peptides and circuits of the fruitfly also drive complex motor sequences, and are small Hsp70 may coexist in a common extracellular pool during pathological condi- enough to investigate at a brain-wide scale via emerging methods in micro- tions. We observed that incubation of extracellular Hsp70 affected the Ab scopy. A critical behavioral sequence for the fruitfly, called ecdysis, is required assembling process preventing oligomer formation in vitro. Moreover, the com- for molting at each developmental stage and consists of three serially executed, bination of Hsp70 and Ab peptides reduced the cytotoxicity of the peptide on stereotyped behavioral programs at the pupal stage. The neural circuit control- cultured neuron (N2A) cells. These results suggest a potential mechanism to ling the pupal ecdysis sequence includes approximately 300 peptidergic neu- reduce the detrimental effects of Ab peptides in AD. rons that express the Ecdysis Triggering Hormone receptor (ETHR) and are activated by peripheral release of Ecdysis Triggering Hormone (ETH). The 2185-Pos Board B505 activation of these neurons leads directly to the three phases of associated mo- Calcium Calmodulin Regulates Zinc Mediated Changes in the Structure, tor neuron activity that mediate the ecdysis sequence, and existing data indicate Self-Association, and Activity of CaMKII that specific subpopulations are required for each behavioral phase of ecdysis. Laurel Hoffman1, Lin Li2, Emil Alexov2, M. Neal Waxham1, However, the identities of the individual neurons that control each behavioral Hugo Sanabria2. phase remain largely unknown, as do the mechanisms by which they regulate 1The University of Texas Health Science Center, Houston, TX, USA, motor output. To achieve a detailed cellular-level understanding of the ecdysis 2Clemson University, Clemson, SC, USA. circuit, we have built a light-sheet microscope that is capable of imaging the Ca2þ-Calmodulin-dependent protein kinase II (CaMKII) is an interpreter of Drosophila pupal CNS rapidly at high resolution. We are currently using cal- Ca2þ signaling and has been shown to be a critical component of learning cium biosensors to monitor the neural activity in ETHR-expressing neurons and memory. This dodecameric oligomer also functions in part as a scaffolding and motor neurons of excised brains in response to ETH. Single-cell activity molecule, serving to promote the formation of higher order signaling com- imaging on the light-sheet microscope confirms that individual neurons plexes within synapses. We set to study the effect that divalent cations, known respond to ETH with varying onset times and distinct activity profiles that sug- to be critically important in neuronal signaling, have on the structure and func- gest the generation of fictive ecdysis behavior. Analysis of these data, and data tion CaMKII. First, we computed the electrostatic surface potential of the do- collected from other populations of neurons, are being used to generate a pre- decameric complex as a means to predict possible binding sites for divalent

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cations. A discrete set of high probability binding sites were identified at the both accommodation and IKCNQ, with no further reduction in response bath interface between catalytic and association domains of neighboring subunits, application of the KCNQ channel antagonist XE-991. Furthermore, pharmaco- 2þ far from the Mg binding sites required to coordinate ATP in the catalytic logical stabilization of KCNQ channels with retigabine restored IKCNQ in slices cleft. Next, we experimentally characterized the effects of divalent cations from cocaine-SA rats and attenuated cue-induced reinstatement when microin- on CaMKII structure using fluorescence, light scattering and electron micro- jected into the PFC. Thus, dopamine mediated changes in inhibitory KCNQ scopy. We found that some, but not all, divalent ions including Zn2þ promote channels may contribute to PFC hyperexcitability following cocaine-SA. the reversible formation of self-assembled CaMKII fibers that can extend for several mm. When associated in fibers, CaMKII activity is compromised; how- 2188-Pos Board B508 ever, Ca2þ/CaM activation of the enzyme disassembles fibers and the loss of Small Fluorescent Probes Show iGluRs are in the Synapses of Transfected activity is reversed. Interestingly, fiber assembly does not interfere with the Neurons under Basal Conditions 1 1 1 1 1 autophosphorylation of CaMKII at T286, an autoregulatory modification, Sang Hak Lee , En Cai , Chaoyi Jin , Pinghua Ge , Yuji Ishitsuka , 1 1 1 1 which confers autonomous activity in monodisperse CaMKII. We compile Kai Wen Teng , Andre A. de Thomaz , Duncan Lee Nall , Murat Baday , 2 3 3 our results with structural and mechanistic data from literature to propose a Okunola Jeyifous , Daniel Demonte , Christopher M. Dundas , 3 2 1 model of Zn2þ-mediated CaMKII fiber formation, where Ca2þ/CaM regulates Sheldon Park , Willian Green , Paul R. Selvin . 1Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA, fiber formation through allosteric modification of a divalent ion site identified 2 in our electrostatic map. Department of Neurobiology, University of Chicago, Chicago, IL, USA, 3Chemical and Biological Engineering, The State University of New York, Buffalo, Buffalo, NY, USA. 2186-Pos Board B506 We imaged transiently transfected live glutamate receptors (iGluRs), namely Genetic Rescue of Mitochondrial Calcium Efflux in Alzheimer’s Disease Preserves Mitochondrial Function and Protects against Neuronal Cell AMPAR and NMDAR, with super-resolution in three-dimension, labeled with Death differently sized fluorophores. We used small organic fluorescent dyes (~ 4 Pooja Jadiya, Alyssa A. Lombardi, Jonathan P. Lambert, Timothy S. Luongo, nm), small quantum dots (sQD, ~10 nm in diameter), or big (commercial) quan- Jin Chu, Domenico Pratico`, John W. Elrod. tum dots (bQD, ~ 20 nm in diameter). The iGluRs were imaged along with a syn- Center for Translational Medicine, Lewis Katz School of Medicine, Temple aptic protein, Homer1c, using fiducial markers that eliminate stage drift, and under conditions where the receptor cross-linking is monitored or eliminated. University, Philadelphia, PA, USA. With small probes under basal conditions, we find that both AMPAR and Background. Alzheimer’s disease (AD) is characterized by neurodegeneration, specifically the progressive loss of neuronal populations in the frontal cortex and NMDAR are predominantly within the synapse (~84-95%), in contrast to hippocampus. Numerous studies have shown that neuronal cell death and meta- bQDs, which show only 5-10% within the synapse. The results can likely be bolic dysregulation are fundamental cellular mechanisms driving the progres- explained by cross-linking and inhibited mobility of the iGluRs labeled with sion of AD and other dementia-related diseases. Previous studies have bQDs, but not with sQDs or organic fluorophores. Hence, this data indicates 2þ that there is not a highly diffusible pool of extrasynaptic iGluRs in the plasma suggested numerous mechanisms whereby intracellular Ca load is increased 2þ membrane, as has been widely reported. In addition, within a synapse, the dis- in AD and thereby likely significantly impacts mCa signaling. Methods. To 2þ tribution is non-homogeneous, perhaps due to the non-homogeneous distribu- discern if mCa signaling is causative in the progression of AD, human AD brain samples, an AD mutant mouse model (3xTg) and an AD-mutant cell tion of glutamate. þ line (N2a/APPswe) were examined for mitochondrial alterations in: Ca2 EPR and NMR: Spectroscopy and Imaging handling, ROS generation, membrane potential (DJ), permeability transition pore activation, OxPhos, APP metabolism and cell death. Results. 3xTg-AD 2189-Pos Board B509 mice and human AD brain samples displayed significant alterations in the 2þ Saturation-Recovery EPR Spin-Labeling Method for Quantification of expression of key mCa exchange genes, most notably a reduction in Lipids in Domains of Biological Membranes the expression of the mitochondrial Naþ/Ca2þ exchanger (mNCX, SLC8B1), 2þ Laxman Mainali, Witold Subczynski. the major efflux pathway in excitable cells. We discovered that mCa efflux 2þ Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA. and mCa retention capacity was severely impaired in N2a/APPswe cells. 2þ The presence of integral membrane proteins in biological membranes induces Rescue of mCa extrusion, via adenoviral expression of mNCX, enhanced 2þ the formation of distinct domains in the lipid bilayer portion of these mem- the clearance of pathogenic mCa , recovered (DJ), enhanced OxPhos, reduced branes. We were able to discriminate the existence of three lipid environments extracellular Ab1-40 levels and protected from ionomycin-, glutamate- and ROS- 2þ in intact fiber cell plasma membranes of the eye lens, namely bulk, boundary, induced cell death. Conclusions. Our data suggest that impaired mCa and trapped lipids. However, our qualitative approach did not allow quantifica- exchange is a central contributor to neuronal cell death in AD and that mNCX tion of lipids in each domain. Recently, based on the continuous wave (CW) represents a new therapeutic target to inhibit or reverse AD progression. electron paramagnetic resonance (EPR) spectra of phospholipid analog and cholesterol analog spin labels, we developed a method that allows evaluation 2187-Pos Board B507 of the relative amount of phospholipids and cholesterol in the bulk lipid domain Chronic Cocaine Self-Administration Potentiates the Dopamine-Induced and in the boundary plus trapped lipid domain [M. Raguz, L. Mainali,W. J. Hyperexcitability Gated by Inhibition of KCNQ/Kv7 Channels O’Brien and W. K. Subczynski, (2015), Exp. Eye Res., 140:179-186]. Here, Priyodarshan Goswamee, Jeffrey Parrilla-Carrero, William Buchta, we will present a new approach that, based on saturation-recovery (SR) EPR, Peter W. Kalivas, Arthur C. Riegel. allows evaluation of the relative amount of phospholipids and cholesterol in Neurosciences, Medical University of South Carolina, Charleston, SC, USA. the bulk plus boundary lipid domain and in the trapped lipid domain. Spectrom- Cocaine addiction is a chronic, unremitting brain disorder. In a rodent eter conditions for the successful application of this method to quantify lipids in extinction-reinstatement model of cocaine self-administration (SA), the ability membrane domains are described. These two methods clearly demonstrate that of drug-paired cues to precipitate relapse to drug-seeking requires stimulation the method’s time window allows the separation of results from the different of dopamine receptors in the prefrontal cortex (PFC) and activation of glutama- domains. CW EPR mixes results from the boundary and trapped lipids, while tergic transmission in the PFC to the nucleus accumbens core pathway. Here, SR EPR mixes results from the bulk and boundary lipids. These two methods we tested the hypothesis that spike frequency adaptation (i.e., accommodation) complement each other, providing a more complete picture of lipid lateral or- in PFC neurons is regulated via a dopamine-mediated closure of inhibitory ganization in intact membranes. The abilities of these methods are illustrated in voltage-gated potassium channels KCNQ/Kv7 and chronic cocaine-SA causes the intact fiber cell plasma membranes from porcine eye lenses. an enduring overactivation of this mechanism. To investigate this, we prepared Acknowledgments: This work was supported by grants EY015526, EB001980, brain slices from animals with a history of chronic cocaine-SA, extinction and EY001931 from the National Institutes of Health. training or cue-reinstatement testing and recorded glutamatergic pyramidal cells (L5) in the current clamp and voltage clamp configurations. At 24-hours 2190-Pos Board B510 after completion of operant training, all sampled cells in tissue from control Simulating Electron Paramagnetic Resonance Spectra of Slow-Motion (yoked-saline and yoked-cocaine) rats displayed accommodation. In contrast, Systems in the Time Domain in tissue from cocaine-SA rats at 24-hours after completion of operant training, Peter Martin1, Stefan Stoll2, David Thomas1. 84% of cells lacked accommodation. This neuroadaptation was enduring and 1Department of Biochemistry, Molecular Biology and Biophysics, University evident following 14d of extinction (60% cells) or re-exposure to drug predic- of Minnesota, Minneapolis, MN, USA, 2Department of Chemistry, tive cues (75% cells). In similar experiments in slices from transgenic cFos- University of Washington, Seattle, WA, USA. GFP rats, the adaptation was correlated with induction of cFos. In control Electron paramagnetic resonance (EPR) spectroscopy has proven to be an (but not cocaine-SA) tissue, bath applied dopamine (10 micromolar) inhibited excellent tool for probing local structure and dynamics in biological systems.

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However, the corresponding spectra can be complex and difficult to interpret. suggest a 1:1 ratio for the LPS:LptA complex and an affinity gradient from In order to accurately interpret experimental data, there has been a correspond- one end of the protein to the other based on Bmax and Kd values quantitatively ing active development of computational tools and user-friendly programs to determined by EPR spectroscopy. simulate continuous-wave (CW) EPR spectra, especially those in the slow- motion regime (dynamical time scales of z10-100 ns for nitroxides at 9-10 2193-Pos Board B513 GHz). Though the standard method has been to numerically solve the stochastic Antifreeze Mechanism Study of Ice Growth Inhibition by Spin Labeled Ice Liouville equation in the frequency domain, more recently, simulating CW Binding Proteins EPR spectra in the time domain has become increasingly popular. Existing Adiel Perez, Antonia Flores, Yong Ba. works have applied this method by simulating spin relaxation due to rotational Chemistry and Biochemistry, California State University, Los Angeles, Los diffusion, either by generating stochastic or molecular dynamics trajectories. Angeles, CA, USA. Other significant spin relaxation mechanisms that affect CW EPR spectral line- Ice binding proteins (IBPs) were found to play critical role for various organ- shapes have been treated in a phenomenological manner. In order to bridge this isms to survive in extreme cold climates. We carried out the study on spin gap in computational complexity and more accurately simulate EPR spectra ob- labeled type 1 IBPs whose sequence is from that of the Pseudopleuronectes tained in experiments, we extend previous treatments by explicitly including americanus (winter flounder). The spin labeled sites involved in our study other spin relaxation mechanisms and explore additional motional models. In have undergone a manipulation on different directions along the a-helical addition, our programs are implemented in the widely used Matlab software structure. Site directed spin labeling technique was used to make the spin environment. labeled peptides. Combination of the EPR spectra at low temperatures and observation of their inhibition to the growth of ice crystals gives us insight 2191-Pos Board B511 into their antifreeze mechanism on both molecular scale and microscopic scale. Molecular Breakdown of Double Electron-Electron Resonance Data with This poster will focus on presenting the experimental result of ice crystal inhi- Atomistic Simulations bition, while another poster of my fellow student researchers will focus on the Fabrizio Marinelli1, Jose’ Faraldo-Gomez2. EPR result. Osmometer-microscopy technique was used to observe the ice 1National Heart, Lung, and Blood Institute, Rockville, MD, USA, crystals. The changes in antifreeze activities, kinetics of ice crystals’ growths 2Theoretical Molecular Biophysics, National Heart, Lung, and Blood and shapes of the ice crystals by these spin labeled IBPs compared with the Institute, Bethesda, MD, USA. wild type IBP were observed and will be discussed in this poster. Results Double Electron-Electron Resonance (DEER) spectroscopy has become a land- include observations of IBP binding on both sides of the basal plane and prism mark technique to investigate the structure and dynamics of membrane pro- faces of ice seed crystals. Significant differences between thermal hysteresis teins, in different functional states and physiological conditions. From DEER and crystal structured due to the binding of the Spin labeled IBPs were observed experiments it is possible to obtain any given number of probability distribu- which is correlated to the EPR spectra at molecular level. Single cystal growth tions for distances between spin labels attached to biomolecules. In contrast on the horizontal axis is observed within the hysteresis gap of the spin labeled to diffraction methods or NMR spectroscopy, DEER is neither limited by the IBPs while the wild type IBP showed no crystal growth during the hysteresis need of crystallization nor by the protein size. This notwithstanding, it is often gap. There were various differences between thermal hysteresis values between not straightforward to interpret DEER data, as it reflects a plethora of protein the different spin labeled IBPs. conformations and rotameric states of the spin labels. Several strategies to 2194-Pos Board B514 disentangle this variability and to derive a clear structural interpretation have Antifreeze Mechanistic Study of Ice Binding Proteins (IBPS) through VT been put forward recently. Some of them entail the calculation of distance Epr Study on Spin Labeled IBPs distributions from approximate protein models and rotamer libraries for the Justin Quon, Antonia Flores, Adiel Perez, Yong Ba. spin-labels. Other more rigorous approaches, such as Ensemble-Biased Chemistry and Biochemistry, California State University Los Angeles, Los MetaDynamics (EBMetaD), are based on atomistic simulation methods, specif- Angeles, CA, USA. ically designed to reproduce the DEER distributions exactly and with a Ice binding proteins (IBPs) bind to the surfaces of seed ice crystals to inhibit minimal bias. Both kinds of approaches, however, rely on the probability dis- their growths and recrystallizations which prevents cell damages in IBP pro- tributions that are inferred from the actual measured data, and do not take into ducing organisms. We have studied the mechanism of ice growth inhibition account the experimental noise. Here, we present a powerful and simple by VT EPR techniques on spin labeled type I IBPs at different sites along its approach to minimally bias an atomistic simulation to sample a conformational a-helical structure. The spin labeled IBPs were achieved through method of ensemble that directly reproduces the DEER time-signal within the experi- cysteine-substitution mutagenesis and alteration of the sulfhydryl groups mental uncertainty. The method is based on the maximum-entropy principle with nitroxide spin labels. Combination of the EPR spectra at low temperatures and extends the EBMetaD approach. We assess the performance of the method and observation of their inhibitions to the growths of ice crystals gives us using spin-labeled T4 lysozyme in explicit water. The results demonstrate the insight into their antifreeze mechanism on both a molecular scale and micro- accuracy and efficiency of the method. In summary, we propose a novel, scopic scale. This poster will focus on presenting the EPR experimental results, rigorous technique to directly interpret experimental data from DEER measure- while another poster of my research lab will focus on the observation on ice ments in terms of physically realistic conformational ensembles. crystals inhibited by the spin labeled IBPs. Categorization of molecular motion regimes of the spin labeling groups among fast, intermediate, slow and aniso- 2192-Pos Board B512 tropic tumblings were determined through the VT EPR spectra which in turn LPS Binding to LptA reveal the water/ice surroundings along the different sites of the spin labeled Kathryn M. Schultz, Tanner J. Lundquist, Matthew A. Fischer, IBPs at various low temperatures. Candice S. Klug. Dept Biophysics, Med Col Wisconsin, Milwaukee, WI, USA. 2195-Pos Board B515 Gram-negative bacteria, such as Escherichia coli, have an asymmetric outer EPR Structural Dynamics of Calmodulin-RyR Peptide Complex membrane (OM) with an outer leaflet rich in lipopolysaccharide (LPS). LPS Cheng Her, Jesse E. McCaffrey, Christine B. Karim, David D. Thomas. is a large lipid essential for survival in Gram-negative bacteria and a major fac- Department of Biochemistry, Molecular Biology and Biophysics, University tor in the protection of bacteria from adverse environmental stresses. LPS is of Minnesota, Minneapolis, MN, USA. transported across the inner membrane by MsbA and through the periplasm We have used a combination of site-directed spin labeling and EPR spectroscopy to the outer leaflet of the OM by the LPS transport (Lpt) proteins LptA, to probe the interaction of calmodulin (CaM) with a peptide (RyRp) correspond- LptC, LptDE, and LptFGB2, the majority of which are essential to Gram- ing to the CaM-binding site on the ryanodine receptor, the muscle calcium negative bacterial survival. LptA is a periplasmic protein that has been shown release channel. A TOAC spin label, which is bifunctionally and stereospecifi- to form concentration dependent oligomers that stack end-to-end. The LptA cally coupled to the alpha-carbon and directly reports peptide backbone dy- protein fold is speculated to bind the LPS acyl chains in the central pocket of namics, was used to label RyRp at positions 5, 12, 18, or 25. CW-EPR showed the protein, to protect the hydrophobic region as the molecule crosses the sol- that RyRp alone exhibits a gradient of rotational dynamics (mobility), with the uble periplasm. In the studies presented here, the binding of LPS at individual N-terminal region having greater mobility than the C-terminal region. Addition sites throughout the LptA protein is characterized using site-directed spin label- of CaM restricted mobility at all four positions, while preserving the mobility ing (SDSL) electron paramagnetic resonance (EPR) spectroscopy. To deter- gradient. In the presence of saturating Ca, the immobilization of TOAC-RyRp mine the location of LPS binding on LptA, fifteen positions located in and by CaM is further increased. A bifunctional spin label (BSL) attached stereospe- around the hypothesized LPS binding pocket on LptA were mutated to cifically at Cys sites i and iþ4 was used to label CaM at positions 34 and 38 in the cysteine, purified, spin labeled at the unique introduced cysteine, and tested N-lobe and 106 and 110 in the C-lobe. Interspin distances measured by DEER for mobility changes due to the presence of excess exogenous LPS. Our data indicated that CaM, in the absence of peptide and Ca, exists in a closed

BPJ 7864_7868 Tuesday, February 14, 2017 447a conformation, while Ca induces a shift in equilibrium toward two additional con- 2198-Pos Board B518 formations, one more open and another more closed (‘‘compact’’). Addition of Structural Dimerization Analysis of the G44V CrgA Mutant from the RyRp caused the two lobes of CaM to collapse toward each other (compact M. Tuberculosis Divisome conformation), while exhibiting a broad distribution, indicating high flexibility. Joshua A. Taylor1, Haujun Qin2, Yisuel Shin1, Krishna Sarva3, Removal of Ca from the complex caused increased distance between the spin la- Malini Rajagopalan3, Timothy Cross1,2. bels, indicating greater lobe separation. Distances measured between BSL (on 1Department of Chemistry and Biochemistry, Florida State University, CaM) and TOAC (on RyRp) confirmed the compact but flexible conformation Tallahassee, FL, USA, 2NHMFL FSU, Tallahassee, FL, USA, 3Health of the complex. These results provide insight into the structural dynamics of Science Center, University of Texas, Tyler, TX, USA. the Ca-dependent regulation of RyR by CaM. CrgA is a 93 amino acid residue protein with two transmembrane helices. It is thought that CrgA plays an important role in the formation of the divisome com- 2196-Pos Board B516 plex for Mycobacterium tuberculosis by recruiting other proteins to the divi- High-Resolution Structural Dynamics of Bifunctionally Spin-Labeled some. Previous research has suggested that CrgA strongly interacts with FtsZ, Myosin by EPR of Oriented Muscle Fibers FtsQ, CwsA, and Penicillin-binding proteins, PBPA and FtsI. In particular, Yahor Savich, Benjamin P. Binder, Peter D. Martin, Andrew R. Thompson, CrgA is likely to play a key role in the activation and maintenance of FtsI in David D. Thomas. the divisome. While the functional role of CrgA with these other proteins is still Department of Biochemistry, Molecular Biology and Biophysics, University being investigated, efforts to structurally characterize this protein has led to the of Minnesota, Minneapolis, MN, USA. discovery that CrgA appears to be functioning as a dimer based on 2-hybrid as- We have performed electron paramagnetic resonance (EPR) on actin-bound says. Mutagenesis was conducted on the CrgA sequence, leading to the develop- myosin labeled with a bifunctional spin label (BSL), in oriented muscle fibers. ment of several new constructs. Among these, two mutants are of special interest Our goal was to determine the effect of known small-molecule effectors on the with the mutation of Glycine-44 to Valine and Alanine-78 to Valine resulting in structure of the complex of the myosin II catalytic domain (CD) bound to actin. increased stability of the dimer. Based on SDS-PAGE gels, the G44V mutant was The use of BSL in our EPR experiments greatly enhances the resolution of seen as the most dramatically stabilized dimer. Usually, glycine residues, when orientation and interspin distance measurements, due to the stereospecific exposed to the fatty acyl environment of a lipid bilayer, facilitates binding. bifunctional attachment to the protein backbone at two engineered Cys resi- Consequently, this mutant is of particular interest, not only for its role in Mtb dues. We used Dictyostelium myosin II with Cys labeling sites as a model. but also pertaining to membrane protein biophysics. This structure is now Three pairs of Cys residues were engineered on our construct, which were being characterized through solid state nuclear magnetic resonance (ssNMR) located on relay helix, K-helix (upper 50 kDa domain), and W-helix (lower spectroscopy. In particular, Magic Angle Spinning (MAS) spectroscopy is 50 kDa domain). Skinned muscle fiber bundles (actin filaments), decorated being employed to measure inter-helical and inter-monomer distance restraints with spin-labeled myosin in the absence of nucleotide (‘‘rigor’’), were oriented with the protein in liquid crystalline liposomes of POPC and POPG with parallel to the spectrometer’s applied magnetic field. This procedure enables differentially-13C labeled monomers. The results of these experiments will be measurement of the orientation of BSL with respect to actin, which in turn described. gives us the orientation of individual myosin II structural elements. We tested several known myosin effectors, including arachidonic acid (AA). We 2199-Pos Board B519 observed a significant change in the structure of actin-bound myosin induced Molecular Insights into Biomolecular Structure and Dynamics by 14N NMR by AA, consistent with a change in the actin-activated ATPase activity of the Maria Concistre1, James A. Jarvis2, Ibraheem M. Haies1, Ilya Kuprov1, spin-labeled construct. Our goal is to understand the structural changes in the Marina Carravetta1, Philip T.F. Williamson2. myosin II CD in the presence of activators and inhibitors of actomyosin. 1Chemistry Department, University of Southampton, Southampton, United This work was supported by NIH grant AR32961 to DDT. Kingdom, 2Biological Sciences, University of Southampton, Southampton, United Kingdom. 2197-Pos Board B517 The structural and dynamic analysis of proteins by NMR in the solid-state has The Alpha-Synuclein Fibril Fold - Comparing Models from Electron Para- typically required extensive isotope labelling. Here we report our progress using magnetic Resonance and NMR the naturally occurring isotopes 1H and 14N to probe the structure and dynamics Pravin Kumar1, Maryam Hashemi Shabestari1, Nathalie Schilderink2, of biomolecules. Employing indirect-detection methods, we have previously Ine M.J. Segers-Nolten2, Mireille M.A.E. Claessens2, Vinod Subramaniam3, demonstrated the feasibility of characterising the quadrupolar interaction present Martina Huber1. at the naturally occurring 14N sites within proteins using 13C as a ‘spy’ nucleus 1Leiden University, Department of Physics, Huygens-Kamerlingh Onnes (Jarvis, 2013). These methods demonstrated that in well folded proteins, limited Lab, Leiden, Netherlands, 2University of Twente, Nanobiophysics, MESAþ dynamic averaging of the quadrupolar interaction occurred and the magnitude Institute for Nanotechnology & MIRA Institute for Biomedical Technology of the quadrupolar interaction reflected the nature of the hydrogen bonding and Technical Medicine, Enschede, Netherlands, 3FOM Institute AMOLF & experienced by the nitrogen, with differences of up to 200 kHz measured between Vrije Universiteit Amsterdam, Nanobiophysics, MESAþ Institute for amides in alpha helices and beta sheets - allowing a detailed analysis of the elec- Nanotechnology, University of Twente & FOM Institute AMOLF, tronic environment along the protein backbone. We have extended this method- Amsterdam, & Vrije Universiteit of Amsterdam, Amsterdam, Netherlands. ology to exploit protons as a spy nucleus, where significant enhancements in Amyloid fibrils and plaques are hallmarks of neurodegenerative diseases. In sensitivity are realised. These advances have opened up the possibility of con- Parkinson’s disease, plaques (Lewy bodies) consist predominantly of the ducting a molecular analysis of unlabelled biomolecules such as ex-vivo or envi- a-synuclein (aS) protein. To understand aggregation and elucidate the role ronmental samples. Currently, we are applying these methods to amyloid fibres, of mutants in the disease, the molecular architecture of aS fibrils needs to be to provide insights into their backbone conformation and polymorphism. known. By double electron-electron paramagnetic resonance (DEER), nm- Jarvis, J.A., Haies, I.M., et al., An efficient NMR method for the characterisa- distance constraints can be determined. This is done by DEER on fibrils of tion of 14N sites through indirect 13C detection. Phys Chem Chem Phys, 2013. doubly spin-labeled aS-variants, diamagnetically diluted with wild-type aS 15(20): p. 7613-20. to suppress intermolecular interactions. Intramolecular distances in three pairs (56/69, 56/90 and 69/90) are reported. An approach to derive a model for the 2200-Pos Board B520 fibril-fold from sparse distance data assuming parallel b-sheets is described. Structural Determination of a Peptide Residue of a Protein of Immunolog- Using the DEER distances as input, a model was derived with three strands, ical Interest NMR comprising residues 56 to 90, in which the strands consist of eight to twelve Diego M. Lo´pez1, Adriana J. Bermudez2, Yuly E. Sa´nchez1. [1] residues each. Details are described in , where also the viability of such a sim- 1Physics, National University of Colombia, Bogota, Colombia, ple model is discussed. 2Fundacio´n Instituto Inmunologico DE Colombia, Bogota, Colombia. [1] Comparison to structural models in the literature given in is augmented in the The X-ray difraction and the nuclear magnetic recsonance (NMR) are the most present contribution by the discussion of the recently published NMR structure comun methos for the determination of the tridimentional structure of peptides [2] of aS fibrils. This structure enables us to compare our constraints directly and proteins. The nmr allows to know the structure of the molecule simulating with the NMR-derived structure, using the MMM model for spin-label their natural environment, which does not allow X-ray difraction, it gives us a [3] attachment. more accurate idea of the molecular estructure, because these structures show a [1] Hashemi Shabestari et al. Appl Magn Reson 46, 369 (2015) DOI 10.1007/ dinamic due to the flexibility of the lateral chains. This paper aims show s00723-014-0622-7 the process of determining the structure of a peptide residue of the Myelin [2] Tuttle et al. Nat Struct Mol Biol. 23, 409 (2016) basic protein (MBP) simulating their natural environment, through the use [3] Polyhach et al. Physical Chemistry Chemical Physics 13 2356 (2011) of NMR.

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Molecular Dynamics III the electrostatic interactions that are required for a lambda-dynamics treatment of the interconversion between the different site forms during the simulation. 2201-Pos Board B521 The tree data structure used by the FMM allows one to include alternative Increasing the Performance and Extensibility of Collective Variable charge distributions of the different protonation forms without requiring redun- Simulations dant computations. Therefore, our FMM enables efficient computation of elec- Giacomo Fiorin1, Je´roˆme He´nin2. trostatic forces and interaction energies between large numbers of titratable 1Institute for Computational Molecular Science, Temple University, sites with a small, nearly constant computational overhead. For taking full Philadelphia, PA, USA, 2Laboratoire de Biochimie The´orique, IBPC, CNRS, advantage of GPUs, the FMM implementation ensures that the computational Paris, France. work is evenly distributed among the large number of GPU processing units. To The collective variables module (Colvars) is a software library that is tightly this aim, our parallel GPU-implementation optimally matches hardware and integrated with molecular simulation and analysis programs (Fiorin et al, algorithmic requirements, resulting in very good scaling properties. Mol Phys, 2013). It is freely available in community programs for molecular 2204-Pos Board B524 dynamics such as NAMD and LAMMPS, and the visualization and analysis Towards Dynamic Pharmacophore Models by Coarse Grain Molecular program VMD. Colvars implements reusable functions to reduce the dimen- Dynamics sionality of complex chemical and biophysical systems, and a set of algorithms Nicholas Michelarakis1, Zara A. Sands2, Mark S.P. Sansom1, for enhanced sampling and statistical analysis. Recent updates have brought Phillip J. Stansfeld1. performance improvements for large systems and costly methods, a more flex- 1Biochemistry, University of Oxford, Oxford, United Kingdom, 2UCB ible scripting interface, and the addition of more stable free energy estimators. Pharma, Braine-l’Alleud, Belgium. We illustrate each advance through examples from biophysics, chemistry and Pharmacophore models play a key role in computer aided drug discovery e.g. in materials science. virtual screening of chemical databases, de novo drug design, and lead optimi- zation. Structure-based methods for developing pharmacophore models are of 2202-Pos Board B522 particular importance, and there have been a number of studies combining such Making Classical and Hybrid (QM/MM) Molecular Dynamics Easy and methods with the use of molecular dynamics (MD) simulations to model pro- Fast with QwikMD tein flexibility. At the same time, ongoing developments in multi-scale simula- Joa˜o V. Ribeiro1, Rafael C. Bernardi1, Till Rudack1, Klaus Schulten1,2. tions, in which atomistic and CG MD simulations are combined in a sequential 1Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL, 2 fashion, have been successfully used to explore the interactions of proteins with USA, Department of Physics, University of Illinois at Urbana-Champaign, membranes and their lipids [1,2]. Here we describe the use of CG-MD simula- Urbana, IL, USA. tions to explore the interactions of CG-particle probes with proteins, combined ‘‘Everything that living things do can be understood in terms of jigglings and with cavity detection methods, in order to identify potential protein binding wigglings of atoms.’’ Richard Feynman’s remarks in the early 1960’s summa- sites. Using cyclin dependent kinase 2 (CDK2) as a test case, we demonstrate rize what is today widely accepted, namely, that molecular processes can be the potential of this approach for identification of ligand binding sites, and as described by the dynamics of biological molecules, therefore connecting pro- part of an overall process of structure-based development of pharmacophore tein function with protein dynamics. Molecular dynamics (MD) simulation, models. in this regard, is the major methodology employed in structural biology to explore the dynamical behavior of macromolecules. Although the use of MD 2205-Pos Board B525 simulations has consistently increased over the last decades, the barrier Couplings between Local and Global Conformational Changes in Proton- imposed by the initial learning curve of the MD packages is still high. To assist Coupled Oligopeptide Transporters new users in overcoming this barrier, and to help the more advanced users to Mahmoud Moradi, Kalyan C. Immadisetty, Jeevapani Hettige. speed up tedious preparation steps, we developed QwikMD(1). This user- Department of Chemistry and Biochemistry, University of Arkansas, friendly program connects the widely used molecular graphics program Fayetteville, AR, USA. VMD to the widely adopted MD program NAMD. Employing QwikMD, a Here we report on an extensive set of equilibrium and nonequilibrium all-atom user is able to setup an MD simulation in just a few minutes, allowing quick molecular dynamics (MD) simulations of a bacterial proton-coupled oligopep- studies of point mutations, partial deletions or even atomic force microscopy tide transporter (POT), namely GkPOT, in an explicit membrane/water envi- experiments. Within the different modules of QwikMD, one can adopt a myriad ronment. Using several microseconds of unbiased MD trajectories, we have of simulation protocols, from single protein in vacuum to hybrid QM/MM sim- characterized both the local and global conformational dynamics of the trans- ulations. QwikMD makes it easy for a new user to perform MD simulations, porter upon the proton and/or substrate binding, within the simulation time- while it also serves as a learning tool. Many ‘‘info buttons’’ provide the theo- scales. Our results reveal a distinct behavior for local conformational retical background underlying the MD procedures carried out in modern MD dynamics in the absence and presence of the proton at the putative proton bind- simulations. ing site. Particularly, we find that the substrate binding conformation is drasti- 1. J. V Ribeiro et al., QwikMD — Integrative Molecular Dynamics Toolkit for cally different in the two conditions; the substrate binds to the protein in an Novices and Experts. Sci. Rep. 6, 26536 (2016). either lateral or vertical manner, in the presence or absence of the proton, respectively. This behavior is consistently observed in multiple sets of indepen- 2203-Pos Board B523 dent simulations for different substrates. On the other hand, we do not observe A Flexible, GPU - Powered Fast Multipole Method for Realistic Biomole- any statistically significant distinctive behavior in terms of the global confor- cular Simulations in Gromacs mational changes under different simulation conditions. On the other hand, Bartosz Kohnke1, R. Thomas Ullmann1, Carsten Kutzner1, the linear regression analysis of quantities associated with the global conforma- Andreas Beckmann2, David Haensel2, Ivo Kabadshow2, Holger Dachsel2, tional fluctuations provides evidence for a mechanism involving the concerted Berk Hess3, Helmut Grubmuller€ 1. motion of the transmembrane helices, consistent with the rocker-switch mech- 1Department of Theoretical and Computational Biophysics, Max Planck anism proposed for major facilitator superfamily transporters. Employing a Institute for Biophysical Chemistry, Goettingen, Germany, 2Institute for novel biasing scheme based on extrapolating the global rotational fluctuations Advanced Simulation (IAS), Forschungszentrum Julich€ GmbH, Julich,€ of the transmembrane helices, we have reconstructed the inward- to outward- Germany, 3KTH Royal Institute of Technology, Stockholm, Germany. facing conformational transition of the GkPOT transporter in the presence The calculation of electrostatic interactions is typically the computational and absence of the substrate/proton. Unlike the unbiased equilibrium simula- bottleneck of molecular dynamics (MD) simulations and thus decisive for tions, a strong correlation is observed between the local and global conforma- the overall simulation performance. Further, biomolecules typically contain tional changes observed in our nonequilibrium simulations. These observations many sites whose electrostatic charge distribution changes over time, e.g. provide evidence for a strong coupling between the protein global conforma- due to uptake and release of protons or tautomerism at protonatable sites, or tional changes and local binding site conformational changes. However, these electron transfer between redox-active cofactors. Besides computational effi- couplings are not observed, in a statistically sound manner, when only the un- ciency, a physically accurate electrostatics treatment therefore has to account biased equilibrium simulations are considered, even on the microsecond time- for this variability, too, thus aggravating the bottleneck. Taking advantage of scale. Our results, therefore, call into question the implicit assumption behind the computational power of GPUs through innovative algorithms, high- MD studies that use short simulations to speculate on long-timescale behavior throughput simulations of large systems become feasible. To that aim, we of the membrane transporters, which often function on timescales much longer have developed a fast multipole method (FMM) for the rapid computation of than those currently accessible to unbiased MD.

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2206-Pos Board B526 [2] to enhance the sampling in configuration space. We here use temperature Systematic Parameterization of Lignin for the Charmm Force Field accelerated MD (TAMD) with several order parameters [3], and explore the Josh V. Vermaas1, Loukas Petridis2, Gregg T. Beckham1, free energy landscape for the isomerization process of Pin1. Combining with Michael F. Crowley1. the string method [4], we will clarify the reaction pathways, and also examine 1National Renewable Energy Laboratory, Golden, CO, USA, 2Oak Ridge the reaction rate using milestoning [5] or non-Markov type analysis of trajec- National Laboratory, Oak Ridge, TN, USA. tories [6]. Plant cell walls have three primary components, cellulose, hemicellulose, and [1] Ning Xu et al. Biochemistry, 53, 5568-5578 (2014). lignin, the latter of which is a recalcitrant, aromatic heteropolymer that pro- [2] H.A. Velazquez and D. Hamelberg, J. Phys. Chem. B 117, 11509-11517 vides structure to plants, water and nutrient transport through plant tissues, (2013). and a highly effective defense against pathogens. Overcoming the recalcitrance [3] H. Fujisaki, K. Moritsugu, Y. Matsunaga, T. Morishita, L. Maragliano, of lignin is key to effective biomass deconstruction, which would in turn enable Front. Bioeng. and Biotechnol. 3: 125, Doi: 10.3389/fbioe.2015.00125. the use of biomass as a feedstock for industrial processes. Our understanding of [4] L. Maragliano and E. Vanden-Eijnden, Chem. Phys. Lett. 446, 182-190 lignin structure in the plant cell wall is hampered by the limitations of the avail- (2007). able lignin forcefields, which currently only account for a single linkage be- [5] L. Maragliano, E. Vanden-Eijnden and B. Roux, J. Chem. Theory Comp. 5, tween lignins and lack explicit parameterization for emerging lignin 2589-2594 (2009). structures both from natural variants and engineered lignin structures. Since [6] E. Suarez, J.L. Adelman, and D.M. Zuckerman, J. Chem. Theor. Comput. polymerization of lignin occurs via radical intermediates, multiple C-O and DOI:10.1021/acs.jctc.6b00339. C-C linkages have been isolated, and the current force field only represents a small subset of lignin the diverse lignin structures found in plants. 2209-Pos Board B529 In order to take into account the wide range of lignin polymerization chemis- Mechanics of Cadherin Unbinding using Coarse-Grained Models tries, monomers and dimer combinations of C-, H-, G-, and S-lignins as well Lahiru N. Wimalasena. as with hydroxycinnamic acid linkages were subjected to extensive quantum Chemistry and Biochemistry, The Ohio State University, Columbus, OH, mechanical calculations to establish target data from which to build a complete USA. molecular mechanics force field tuned specifically for diverse lignins. This was Cell-cell adhesion is mediated by calcium-dependent proteins called cadherins, carried out in a GPU-accelerated global optimization process, whereby all mol- which are important in neuronal connectivity and tissue integrity. Cadherins are ecules were parameterized simultaneously using the same internal parameter modular proteins with large extracellular domains that have typically been set. By parameterizing lignin specifically, we are able to more accurately repre- modeled using all-atom molecular dynamics (MD) simulations. However, these sent the interactions and conformations of lignin monomers and dimers relative simulations are computationally expensive and most of them only include small to a general force field. This new force field will enables computational re- fragments of these cadherin extracellular domains. To overcome these limita- searchers to study the effects of different linkages on the structure of lignin, tions, we used a coarse-grained (CG) model with the MARTINI force field as well as construct more accurate plant cell wall models based on observed sta- to study large cadherin complexes over long time scales. All-atom MD simu- tistical distributions of lignin that differ between disparate feedstocks, and lations were used to find optimal parameters for an elastic network model guide further lignin engineering efforts. that stabilized the protein secondary structure. The CG model allowed for a 5x increase in timestep and a 10-fold reduction of system sizes. Using this 2207-Pos Board B527 model we studied the dynamics and elastic response of classical cadherins Simulated Forced Unbinding of Clustered Protocadherins and clustered protocadherins. In these simulations, the extracellular domains Sanket P. Walujkar, Raul Araya-Sechhi, Marcos Sotomayor. of classical cadherins straightened before unbinding, while protocadherins slip- Department of Chemistry and Biochemistry, The Ohio State University, ped past each other during unbinding. Overall, our results confirm that our Columbus, OH, USA. model is an effective simulation tool for studying of the mechanics of cadherin Clustered protocadherins belong to the cadherin superfamily of adhesion pro- complexes. teins and are involved in neuronal connectivity and self-recognition. Clustered protocadherins have six extracellular cadherin (EC) repeats with about 100 res- idues each. Recent crystallographic structures suggest an antiparallel homo- 2210-Pos Board B530 philic binding interface that involves overlapped EC1 to EC4 repeats. Here, Influence of an Ionic Liquid on TRP-Cage Structure and Xaa-Pro we present steered molecular dynamics simulations of a, b, and g clustered pro- Dipeptide Conformational Sampling Joseph L. Baker1, Alexanndra Heyert2, Susan Knox3, Gerrick E. Lindberg2. tocadherins homodimers exploring the forced unbinding of their adhesive inter- 1 face. Constant velocity stretching simulations were performed on these Department of Chemistry, The Collegeof New Jersey, Ewing, NJ, USA, 2Department of Chemistry and Biochemistry, Northern Arizona University, homodimers at 10 nm/s, 1 nm/ns, 0.5 nm/ns, and 0.1 nm/ns. All protocadherins 3 show some unfolding at the highest stretching speed, whereas simulations at Flagstaff, AZ, USA, Department of Chemistry, Yale University, New slower speeds revealed unbinding pathways for the complex without unfolding. Haven, CT, USA. Force-extension profiles show broad force peaks that reflect the extended anti- Room temperature ionic liquids (RTILs) demonstrate great promise for the se- parallel interface of protocadherins, as well as the formation of transient inter- lective control of protein structure and function, however the fundamental as- actions between protomers that break during unbinding. The unbinding pects of RTIL effects on peptides and proteins remain unclear. Here we pathways observed in simulations may help further elucidate the molecular ba- describe some recent results for the influence of the RTIL 1-butyl-1-methylpyr- sis of protocadherin binding specificity. rolidinium bis(trifluoromethylsulfonyl) imide ([C4mpy][Tf2N]) on the structure of the miniprotein Trp-cage and on the behavior of Xaa-Pro dipeptides, where 2208-Pos Board B528 Xaa is any of the common amino acids. Starting from an unfolded configura- Exploring Reaction Pathways for Peptidylprolyl-Isomerase tion, we find that Trp-cage folds in water at 298 K in less than 500 ns of mo- Hiroshi Fujisaki1, Yasushige Yonezawa2, Motoyuki Shiga3, lecular dynamics (MD) simulation, but exhibits very little mobility in the Luca Maragliano4, Shin-ichi Tate5. RTIL at the same temperature, which is related to the RTIL viscosity. However, 1Nippon Medical School, Musashino, Tokyo, Japan, 2Kindai Univ., Kainan- in simulations carried out at 365 K, the mobility of the RTIL is increased and shi, Wakayama, Japan, 3JAEA, Kashiwa, Chiba, Japan, 4Istituto Italiano di initial stages of Trp-cage folding are observed. We employed scaled MD to Tecnologia, Genova, Italy, 5Hiroshima Univ., Higashi-Hiroshima, expedite sampling, and demonstrate that Trp-cage in the RTIL can closely Hiroshima, Japan. approach the aqueous folded state. While the RTIL is found to restrict Trp- Pin1 enzyme is one of the peptidyl-prolyl cis/trans isomerases (PPIase), which cage motion, cis/trans isomerization of peptide bonds involving proline occur isomerizes an omega bond of specific phospho-Serine/Threonine-Proline mo- that are not observed in aqueous simulations. Therefore, we studied Xaa-Pro tifs, leading to many biological consequences. However, the isomerization dipeptides in several environments, including the same RTIL, water, octanol, mechanism has not been fully clarified yet though there are several experi- and vacuum, in order to further explore this effect. The RTIL is found to restrict mental and numerical studies. For example, Tate and coworkers investigated Ramachandran space sampling of the dipeptides, and for Trp-Pro, isomeriza- a mutated (C113D) Pin1, and found that the isomerization rate is lower than tion of the dipeptide bond to the cis state is observed. This suggests that RTILs that in the wild type [1], but it is difficult to understand the mechanism only can be used to stabilize otherwise infrequently observed dipeptide conforma- from the experiment. Previously, Hamelberg and coworkers carried out molec- tions. Our simulations imply that stacking of the Trp ring and Pro ring in the ular dynamics (MD) simulations of Pin1 with a model substrate, and because cis state versus the trans state might contribute to this effect for the Trp-Pro the isomerization process is a rare event, they used accelerated MD method dipeptide.

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2211-Pos Board B531 simulations to understand what confers the specificity for nucleoside substrates. Exploring the Interaction between MLP1 and NAB2 for mRNA Quality We calculate the relative free energy of binding for these sites and the free en- Control Purposes ergy barriers within the Tsx channel from umbrella sampling simulations. Mohammad Soheilypour1, Mohaddeseh Peyro2, Hengameh Shams1, Additionally, we parametrize the nucleoside analogue drug Floxuridine and Mohammad Mofrad3. compare its translocation to natural substrates of the Tsx channel to explore 1Applied Science and Technology, University of California, Berkeley, the potential usefulness of Tsx proteins as anti-microbial drug delivery path- Berkeley, CA, USA, 2Mechanical Engineering, University of California, ways. Using applied electric field simulations, we calculate the ionic conduc- Berkeley, Berkeley, CA, USA, 3Bioengineering and Mechanical tivity of the channel and explore the gating behavior that modulates the ionic Engineering, University of California, Berkeley, Berkeley, CA, USA. current using the functional mode analysis method. Transcription of mRNA from DNA is followed by several processing and pack- aging steps, which are quality controlled at different stages of RNA biogenesis. 2214-Pos Board B534 One of the specific steps of mRNA surveillance occurs at the entry of the nu- Glycosaminoglycan Sequence Influence on Iduronate Ring Puckering clear pore complex (NPC), involving various types of proteins ranging from Bruno DeMaria, Courtney Burkham, Samantha Mansberger, Owen Ganter, RNA-binding proteins (RBPs) to NPC-associated proteins. Among all the Olgun Guvench. involved proteins, Mlp1, a nuclear basket-associated protein, has been sug- Department of Pharmaceutical Sciences, University of New England, gested to play a substantial role. The interaction between Nab2 (a yeast Portland, ME, USA. RBP) and Mlp1 has been previously explored through in vitro and in vivo ex- Iduronate (IdoA) is a major monosaccharide constituent of the linear polysac- periments. These studies identified a hydrophobic patch in Nab2, centered on charides, called glycosaminoglycans (GAGs), that form covalent conjugates Phe73, which was suggested as the critical residue for the interaction between with core proteins to make proteoglycans (PGs). PGs are known as important the two proteins. However, the exact underlying molecular mechanism is still structural components of extracellular matrix, and are increasingly recognized elusive, limiting the current understanding of mRNA quality control process. for their roles in cell signaling. Toward advancing understanding of the atomic- Here, we employ an integrated computational protocol (including structure pre- resolution structural biology of PGs, we have performed all-atom explicit-sol- diction, docking, and molecular dynamics) to explore the pivotal interaction be- vent molecular dynamics (MD) simulations on IdoA as a monosaccharide and tween Mlp1 and Nab2. Previous studies have shown that mutation of Phe73 to in the context of oligosaccharides corresponding to fragments of the GAG der- Alanine will disrupt this interaction. Our results suggest a key, yet indirect, role matan sulfate (DS). The stereochemistry of the carbon atoms in the pyranose for Phe73 in this interaction. Specifically, Phe73 primes a slight conformational ring of IdoA and the resulting spatial distribution of functional groups attached change in Nab2, enabling the disordered domain of Mlp1 to encircle Nab2 he- to the ring result in a delicate thermodynamic balance between preferred ring lices. This leads to a decreased distance between the involved residues and for- puckering conformation for IdoA. Therefore, Cremer-Pople ring pucker anal- mation of stronger interaction between the two proteins. The relatively large ysis was applied to understand the sequence influence on IdoA in the context disordered region in Mlp1’s C-terminal domain enables it to interact with of DS oligosaccharide vs. IdoA monosaccharide. These results are compared different RBPs. Our proposed binding mechanism, where Mlp1 encircles with analogous simulations for glucuronate (GlcA), which is a single-site Nab2 using its disordered domain, may be relevant for the binding of Mlp1 epimer (at C6) of IdoA, and makes up the related GAG chondroitin sulfate to other RBPs as well. The flexibility of the disordered region allows for bind- (CS). ing of Mlp1 to different proteins of various 3D structures, facilitating mRNA 2215-Pos Board B535 quality control mechanism. Molecular Dynamics Reveals New Insights into Activation of the Insulin- Family Proteins and their Binding Specificity to the Insulin Receptor 2212-Pos Board B532 Anastasios Papaioannou1, Serdar Kuyucak2, Zdenka Kuncic1. Allosteric Coupling of Nucleotide Turnover to Bimolecular Recognition in 1Charles Perkins Centre, The University of Sydney, Sydney, Australia, Kinesin Motors and G Protein Switches 2School of Physics, The University of Sydney, Sydney, Australia. Barry J. Grant. The insulin-family proteins, which include insulin, as well as insulin-like University of Michigan, Ann Arbor, MI, USA. growth factors I (IGF-I) and II (IGF-II), share high homology in their structure Kinesin motors and homologous G proteins switches coordinate a wide array of and sequence. Insulin and IGFs are implicated in chronic diseases such as dia- important subcellular processes, from division and growth to intracellular betes and cancer, respectively. Interestingly, while the insulin-family proteins communication and self-organization. These functionally diverse families bind to their own receptors, IGF-II can also bind to the A-isoform of the insulin contain a similar core structure supporting a common mode of nucleotide receptor (IR-A), activating unique and alternative signalling pathways, from dependent allostery. However, a complete atomistic scale picture of key struc- those of insulin, to stimulate cancer cell growth. This provides a mechanism tural dynamic mechanisms and their adaptation to control distinct functional in- by which cancer cells can appear to be resistant to treatments targeting the teractions remains elusive. Here we describe results from a comparative IGF-1 receptor. Consequently, the way in which IGFs interact with and bind approach that couples bioinformatics, molecular simulation and experimental to the IR-A is of particular interest. Although extensive studies of insulin protein engineering across kinesin and G protein families. This includes the have revealed that its activation is associated with the opening of the prediction and experimental engineering of allosterically decoupled constitu- B-chain-C-terminal (BC-CT), the activation mechanisms of the IGFs still tively active heterotrimeric G proteins. Prediction and experimental verifica- remain unknown, yet are essential for the prediction and design of therapeutic tion of allosterically enhanced and mechanochemically distinct kinesin-5. antibodies. To this end, we performed Molecular Dynamics (MD) simulations Computational and experimental dissection of kinesin-microtubule interactions to elucidate the activation dynamics and energetics of IGFs, to identify the dif- along with the rational modulation of kinesin processive motility - the ability of ferences and similarities in their activation behaviour, and to contrast them an individual motor to take multiple steps along its microtubule filament. against those of insulin. Our MD simulations revealed, in particular, that insulin Collectively, this work furthers our understanding of the evolutionary adapta- and IGFs exhibit stochastic dynamics during their activation, which involves tion of a ubiquitous allosteric motif and informs mechanistic studies on many two opening locations in the IGFs compared to the only one in insulin. More- related nucleotide triphosphatases that constitute nearly 5% of human protein over, the occurrence of these two openings is simultaneous in IGF-I, but not in coding genes. IGF-II, which exhibits two independently occurring openings. This suggests Software and other material related to this work can be found at:http:// that the IGFs undergo different activation mechanisms for receptor binding. thegrantlab.org/. The probabilities of the active/inactive states of the proteins are further sugges- tive of IGF-II having a hyperactive nature compared to IGF-I and insulin. Sum- 2213-Pos Board B533 marising, our MD simulations have elucidated the crucial differences and Tsx as a Nucleoside Channel similarities in the activation mechanisms of the insulin-family proteins, Shreyas S. Kaptan1, Bert van den Berg2, Ulrich Kleinekatho¨fer1. 1 2 providing new insights into their binding specificity to the insulin receptor Physics and Earth Sciences, Jacobs University, Bremen, Germany, Institute that could not readily be gleaned experimentally. for Cell and Molecular Biosciences, Newcastle University, Newcastle, United Kingdom. 2216-Pos Board B536 Tsx proteins form a family of widespread bacterial outer membrane channels Exploiting Tumor Acidity in Designing a Novel Cancer Targeting Agent that show a strong substrate specificity for nucleosides. X-ray crystallographic Chitrak Gupta, Blake Mertz. structures for E. coli Tsx were previously determined with the channel sub- Chemistry, West Virginia University, Morgantown, WV, USA. strates uracil and thymidine. The substrates bind at two binding sites inside Developing more effective targeting of chemotherapeutics is currently one of the channel. We use these structures as the starting points to study the nature the most pressing needs in oncology. One promising approach is to utilize of interactions of the channel with the nucleosides using molecular dynamics the acidic microenvironment of cancer tumors, which is a universal property

BPJ 7869_7874 Tuesday, February 14, 2017 451a of all cancers. pH (Low) Insertion Peptide (pHLIP) is a soluble peptide that 2219-Pos Board B539 binds and inserts into cell membranes under acidic conditions. However, devel- Acceleration of Cardiac Simulations for Cloud Computing Resources oping pHLIP into a novel cancer targeting agent will require informed design of Delvin Huynh, Yuanfang Xie, Daisuke Sato. a variant that can distinguish between the pH range of 7.2 (healthy tissue) and University of California Davis, Davis, CA, USA. 6.8 (most cancers). One of the key steps in pHLIP function is the process of Computer simulation has greatly facilitated our understanding of the cardiovas- folding into an a-helix before insertion. Partial helical formation of pHLIP un- cular system and is boosting the development of personalized medicine for car- der alkaline conditions has been reported in literature, and recent results from diac diseases. The simulation of the heart is computationally intensive due to our group [1] support this claim. These observations, coupled with biophysical the complexity and multi-scale essence of the cardiovascular system. Parallel theory of peptide folding [2], leads to our hypothesis that the apparent pKa of computing has been one of the commonly used solutions for such large-scale insertion of pHLIP is tied to its helix-forming propensity in solution. We have simulations. More recently, rapidly growing cloud computing resources, such tested this hypothesis through long timescale (~ms) molecular dynamics simu- as Amazon Elastic Compute Cloud and Google Compute Engine, provide a lations of pHLIP in implicit solvent. Comparison with the insertion behavior new infrastructure for parallel computing. However, the high latencies of the observed in two variants of pHLIP that use non-natural amino acids [3] allows network limit the application of cloud computing. In this study, we developed us to tie the unique properties of pHLIP to its primary amino acid sequence. a novel algorithm to facilitate the utilization of cloud based computing re- Application of the Lifson-Roig model of helix-coil transition to our results sources. By exploiting the fact that the time scale of diffusion of ions is yielded a complete thermodynamic description of state I of pHLIP. Collec- much slower than the time scale of reaction in ion channels/pumps, this algo- tively, our results are the first steps towards informed design of pHLIP variants rithm improves the simulation speed by more than twice as much; the larger the with properties ideally suited to target cancer cells. latencies in networks, the faster our algorithm performs the simulations. Tar- [1] Renet. al. Proteins., (Submitted); geted computer models at many system scales will be drastically optimized [2] Almeida et al., BBA, 1818:178 (2012); in terms of efficiency including cardiac tissue models, models of subcellular [3] Onyango et al., Angew. Chem. Intl. Ed., 54:3658 (2015). calcium cycling, and a combination of them. Techniques developed here can be also applied for any number of other complex reaction-diffusion systems Computational Methods and Bioinformatics II in other fields.

2217-Pos Board B537 2220-Pos Board B540 Extending Rule-Based Modeling to the Spatial Domain with Virtual Cell Re-Docking by Analyzing the Profile of Protein-Protein Interaction (VCELL) Yosuke Amano1, Wataru Nemoto1, Nobuyuki Uchikoga2. 1 2 James C. Schaff, Dan Vasilescu, Ion I. Moraru, Leslie M. Loew, Dept. Sci. & Eng., Tokyo Denki University, Saitama, Japan, Dept. Phys., Michael L. Blinov. Chuo University, Tokyo, Japan. Center for Cell Analysis and Modeling, University of Connecticut School of Various proteins exert molecular functions through forming a protein complex. Medicine, Farmington, CT, USA. Elucidation of a complex structure is essential to understand its molecular func- When a kinetic model has to account for all the potential molecular complexes tions. At present, the number of complex structures in PDB is not enough for us and interactions between multivalent or multistate molecules, the reaction to understand the information coded on the protein-protein interaction network. network may be too large and complex to be manually specified. Rule-based In silico analyses, docking simulation has been applied to augment the lack of modeling solves this problem by expressing molecular interactions in the complex structure information. In general, software for docking simulation ro- form of reaction rules that serve as generators of reactions. Thus, the individual tates a protein (ligand) around its interaction partner protein (receptor) to species and the reactions connecting them are generated automatically. generate many complex structure candidates. Hereafter, these candidates are Recently, we introduced rule-based modeling into the popular Virtual Cell referred to as decoys. Similar complex structures to the native structure (VCell) modeling framework (Schaff et al., 2016), even permitting manually (near-native structures) are expected to exist among the decoys. However, in specified reaction networks to be merged with rule-based model constituents. some protein pair cases, there is no near-native structure in a set of decoys The same set of rules can be simulated using both network generation (Bio- because of the shortage of docking space, that is one of the important problems NetGen, Faeder et al., 2009) and network-free simulation (NFSim, Emonet et to be solved. This problem was addressed by expanding the docking space al., 2011). Here we present a compartmental extension of VCell rule-based ca- based on re-docking strategy [Uchikoga et al. 2013 PLOS ONE 8:e69365]. pabilities. The VCell paradigm in which every species and location has an as- Re-docking is a second round docking step after the initial docking. Then, an- signed compartment, was extended to the rule-based description. Both alyses of Interaction FingerPrints (IFPs) [Uchikoga and Hirokawa 2010 BMC BioNetGen and NFSim engines were modified to account for compartments. Bioinformatics 11:236-245] have been proved to obtain near-native structures Every reactant and product pattern and every rule have their assigned locations. efficiently by focusing on the docking space corresponding to a decoy. Hence, Moreover, to prevent involuntary translocation of species between compart- we developed a software named Pftkool that is a tool for re-docking by calcu- ments, a new feature of rule-based modeling was introduced, allowing anchoring lating and classifying IFPs. Pftkool uses k-means algorithm to search receptor a molecule to a given compartment. This way, any species that has this molecule surface for target residues to perform re-docking. In general, the number of k of as one of its molecular components, will remain in the anchored compartment. In k-means algorithm should be specified by a user. However, our analyses show addition to ODE, stochastic kinetic and NFSim simulators within VCell, this that k is automatically determined in the case of re-docking. now permits VCell users to apply rule-based models to reaction-diffusion in 2221-Pos Board B541 complex geometries using either the VCell PDE deterministic solvers or the A Docking Based Approach to Analyze Interaction Surfaces of Virus-Host Smoldyn stochastic simulator. The expanded VCell framework was used to Protein-Protein Interactions simulate several biological systems, such as membrane-bound clustering of Yuri Matsuzaki1, Jaak Simm2, Nobuyuki Uchikoga3, Yutaka Akiyama1. Nephrin-Nick-NWasp proteins. (Supported by NIH grant P41 GM103313). 1Tokyo Institute of Technology, Tokyo, Japan, 2KU Leuven, Leuven, 3 2218-Pos Board B538 Belgium, Chuo University, Tokyo, Japan. Cloud Computing for All-To-All Protein-Protein Docking on Azure HPC Core elements of cell regulation are made up of protein-protein interaction Masahito Ohue1, Yuki Yamamoto1, Takanori Hayashi1, Yuri Matsuzaki2, (PPI) networks. Predicting relevant interacting partners from their tertiary Yutaka Akiyama1. structure is a challenging topic where computer science methods have potential 1Computer Science, Tokyo Institute of Technology, Tokyo, Japan, 2ACLS, to contribute to biology. Protein-protein rigid docking has been applied for this Tokyo Institute of Technology, Tokyo, Japan. purpose by several projects. Docking-based approaches have advantages such Cloud computing environments, such as Amazon AWS, Microsoft Azure, Goo- that (i) they can suggest binding poses of predicted binding partners that would gle Cloud Platform, etc., achieve computational performance improvement help understanding the interaction mechanisms, (ii) comparing docking results remarkably in recent years, and is also useful in parallel computing (high-per- of both non-binders and binders can lead to understanding the specificity of PPI formance computing, HPC) fields. Cloud enables users to get thousands of CPU from structural viewpoints. However, the prediction power is limited mainly cores and GPU accelerators casually, and several software are used very easy because of poor correlation between docking score and actual protein-protein by cloud images. We have transplanted original protein-protein interaction pre- binding affinity. To improve state of the art we propose a machine learning diction (protein-protein docking) software, MEGADOCK, to the Microsoft approach to improve docking-based PPI predictions by using residue profiles Azure HPC environment. We have obtained strong scaling value of 84% obtained by rigid docking. A possible application of our method is a problem with virtual machines of Azure A9 instance up to 1,200 CPU cores. In future, in which we have one ‘receptor’ protein and pickup proteins that have potential MEGADOCK will link to web interface and GUI local client on Azure cloud to to interact with it from a pool of candidate ‘ligand’ proteins. We define an inter- be more easy-to-use for non-computer scientists. face fingerprint of a pair of proteins as a collection of residues of a ‘receptor’

BPJ 7869_7874 452a Tuesday, February 14, 2017 protein, with information of how often each residue are included in binding [4] Zhan D., Claudio C., and Juswinder S., Structural interaction fingerprint sites of high scoring docking models. We applied multiple methods of machine (SIFt): a novel method for analyzing three-dimensional protein-ligand binding learning to discriminate binders and non-binders using those profiles as input. interactions. Journal of Medicinal Chemistry, 47(2):337–344, 2004. In this poster we present an evaluation of our method by applying it to proteins [5] Da C., and Kireev D., Structural protein–ligand interaction fingerprints of Protein Docking Benchmark ver. 5.0, bacterial chemotaxis, and host-virus (SPLIF) for structure- based virtual screening: Method and benchmark study. protein interactions.We further applied our method to analyze proposed host- Journal of Chemical Information and Modeling, 54(9):2555–2561, 2014. virus PPI interaction surfaces by post-docking analysis to compare surfaces [6] Richard A. F., et al., Glide: a new approach for rapid, accurate docking and of PPIs between the host proteins. scoring. 1. method and assessment of docking accuracy. Journal of Medicinal Chemistry, 47(7):1739–1749, 2004. 2222-Pos Board B542 In Silico Screening for Chemical Scaffolds as Suitable Natural Inhibitors of Optical Spectroscopy: CD, UV-VIS, Vibrational, Kinesin EG5 Divulges Morelloflavone, a Biflavonoid, as Potential Anti- Fluorescence I cancer Compound Tomisin Happy Ogunwa, Takayuki Miyanishi. 2224-Pos Board B544 School of Fisheries and Environmental Sciences, Nagasaki university, New Thiol-Reactive Eu-Complex for Distance Measurements by LRET Nagasaki, Japan. Felix Faschinger1, Mirjam Zimmermann1, Guenther Knoer2, Natural products remain a source of chemical scaffold pool for drug design and Hermann Gruber1. development. Kinesin Eg5 has emerged as a clinical target for anticancer 1Institute of Biophysics, Johannes Kepler University, Linz, Austria, 2Institute agents. In our search for potent natural inhibitors of kinesin Eg5, we employed of Inorganic Chemistry, Johannes Kepler University, Linz, Austria. in silico tools to screen selected diverse chemical scaffolds (compounds) that Crystallography and NMR spectroscopy are ideally suited to resolve the 3D were obtained from medicinal plants. Surprisingly, the molecular interaction structures of biomolecules but the material and time demand for each structure analysis for the selected compounds adjudged morelloflavone (a biflavonoid) is high. Fluorescence resonance energy transfer (FRET) provides less structural as a potential ATP-noncompetitive inhibitor of kinesin Eg5 protein which information but is better suited to study conformational changes and structure- occupied the putative L5/a2/a3 allosteric pocket on the protein. Compared to function relationships by screening a large number of mutants or experimental STLC with binding energy value 10.0 Kcal/mol, morelloflavone displayed conditions. Moreover, FRET allows for real time monitoring of conformational binding energy value of 10.2 Kcal/mol and a 90 percent binding site similar- changes induced by specific ligands. Usually, FRET yields only a crude esti- ity. It is also worth noting that morelloflavone was embedded within the cavity mate of the donor-acceptor distance, due to the fact that the relative orientation formed by amino acid residues Ile-136, Glu-116, Glu-118, Trp-127, Gly-117, of donor and acceptor are rarely known. Luminescence resonance energy trans- Ala-133, Glu-215, Leu-214, Tyr-211 and hence, displayed a reliable tendency fer (LRET) is much better suited for distance measurements because the orien- to block the enzymatic catalysis of kinesin Eg5 allosterically. The compound tation factor (and thus the Forster distance) are known, and because energy established hydrogen bonds with Glu-118 and Tyr-211 having minimum length transfer is measured by a change of lifetime, rather than of signal intensity. ˚ of 2.97A and hydrophobic interactions occurred with alkyl side chain of resi- In LRET-experiments the ideal donors are highly stable Eu/Tb-complexes, dues Gly-117, Glu-116, Ala-218, Ile-136, Arg-119 and Asp-130 while p-stack- with a single lifetime that is not influenced by attaching the complex to a ing interaction is observed between the aromatic ring of morelloflavone and biomolecule. Several terpyridine-based Eu-complexes described in literature Arg-119. These interactions anchored morelloflavone into the binding site. have promising properties concerning uniform lifetimes after protein labeling The results obtained in this work indicate the strong affinity and inhibitory po- but all described complexes have rather long linkers which prevent accurate tential of this compound on kinesin Eg5, hence lending credence to the yet un- distance measurements. In this study, a new terpyridine-based Eu-complex tapped anticancer capacities of morelloflavone. We therefore suggest in vitro with maleimide very close to the metal ion center was synthesized and found and ex vivo evaluation of this compound as anticancer agent targeting kinesin to have ideal properties for distance measurement by LRET: After linking to Eg5 protein. the single cysteine of BSA, the complex showed a quantum yield of 30%, a sin- gle lifetime of 1.2 ms, comparable to the best known Eu-complexes, and the 2223-Pos Board B543 lifetime was unaffected by phosphate or EDTA. In conclusion, this new Eu Development of Postprocessing Method of Protein-Ligand Docking using complex appears ideally suited for reliable measurement of intra- or intermo- Interaction Fingerprint lecular distances. Nobuaki Yasuo1, Masakazu Sekijima1,2. The work was supported by grant W0125 of the Austrian science fund (FWF) 1Department of Computer Science, Tokyo Institute of Technology, Tokyo, Japan, 2Advanced Computational Drug Discovery Unit, Tokyo Institute of 2225-Pos Board B545 Technology, Tokyo, Japan. A Novel FRET Technique to Characterize the Oligomerization State of Protein-ligand docking is an important method in Structure-based Drug Dis- Protein-Protein Interactions covery [1]. Although many programs have been developed for docking [2], Philipp J. Heckmeier, Mark G. Teese, Dieter Langosch. the accuracy is still insufficient due to the difficulty in the scoring function Lehrstuhl fur€ Chemie der Biopolymere, Technische Universit€at Munchen,€ [3]. Interaction fingerprint is one of the solutions, which generate fingerprints Freising, Germany. of ligands using the interactions between the ligand and the protein. Interac- Protein-protein interactions are the fundamental driving force of numerous tion fingerprints use the information of known compounds so that compounds cellular processes and cell signaling pathways. Characterizing whether proteins that have similar interaction to the known active ligands are expected to find interact as dimers, trimers, or higher oligomers is essential to understanding through the virtual screening. However, existing interaction fingerprints such these interactions. Several microscopy and advanced imaging techniques as SIFt [4] and SPLIF [5] only assess the existence or the distance of the in- relying on Fo¨rster resonance energy transfer (FRET) between identical fluoro- teractions and do not consider the strength correctly. In this study, we made a phores (homo-FRET) have been developed to estimate protein stoichiometry. new scoring function of protein-ligand docking called SIEVE-Score (Simi- The increased FRET in oligomers is detected by measuring depolarization or larity of Interaction Energy VEctor-Score), which can consider the strength emission time. Homo-FRET methods have a strong advantage in requiring of each interaction explicitly. SIEVE-Score is calculated based on the only a single fluorophore, greatly simplifying sample preparation in compari- similarity of the interaction energy vector, which is the list of interaction son to conventional hetero-FRET methods. However, most homo-FRET energy between the ligand and each residue of the protein. We also evaluate methods require sophisticated imaging equipment, and both theoretical models the accuracy of virtual screening using SIEVE-Score after the docking by and applications have been restricted to the study of membrane-bound proteins. Glide [6]. Using a simple bulk homo-FRET and laser photobleaching approach, we [1] Chiba, S., et al. Identification of potential inhibitors based on compound demonstrate the feasibility of characterizing the oligomerization state of an in- proposal contest: Tyrosine-protein kinase Yes as a target. Scientific reports teracting protein in-vitro. To simulate oligomers in a proof of concept, we con- 5:17209, 2015. structed an extensive repertoire of fusion proteins with 1-6 consecutive green [2] Elizabeth Y., Jessica H., and Paul A. R., Improvements, trends, and new fluorescent protein (GFP) domains. We show how the resulting homo-FRET ideas in molecular docking: 2012–2013 in review. Journal of Molecular Recog- (measurable via steady-state anisotropy or fluorescence polarization) is propor- nition, 28(10):581–604, 2015. tional to the oligomerization state of proximal GFP domains. For the first time, [3] Yan L., Li H., Zhihai L., and Renxiao W., Comparative assessment of this is demonstrated with soluble proteins. In both membrane and soluble pro- scoring functions on an updated benchmark: 2. evaluation methods and general teins, oligomerization increases FRET and therefore anisotropy. However for results. Journal of Chemical Information and Modeling, 54(6):1717–1736, soluble proteins oligomerization also slows fluorophore rotation, leading to a 2014. size-dependent decrease in anisotropy. Through gradual photobleaching of

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fluorophores these two effects can be distinguished, and the oligomerization 16PRE27130004, USA SURF Program, and the Abraham Mitchell Cancer state of a labeled protein of interest can be estimated. We therefore show Research Fund. how the theoretical framework developed for membrane proteins needs to be adjusted to account for this additional degree of freedom in soluble proteins. 2228-Pos Board B548 Overall, bulk homo-FRET and laser photobleaching is a promising method Fluorescent Proteins for Super-Resolution Microscopy 1 1,2 to determine the oligomerization state of a protein of interest, which can Karin Nienhaus , Gerd U. Nienhaus . 1Institute of Applied Physics, Karlsruhe Institute of Technology, Karlsruhe, have a low concentration (0.1-0.5 mM) and needs only a single fluorescent la- 2 bel. The method requires only a photometer or microplate reader capable of Germany, University of Illinois at Urbana-Champaign, Urbana, IL, USA. measuring steady-state anisotropy. Super-resolution fluorescence microscopy is the method of choice to monitor cellular and subcellular biological processes in live cells. Among the different 2226-Pos Board B546 fluorescent labels presently available, fluorescent proteins (FPs) of the GFP Optimizing a Time-Resolved Spectrometer for All Time Scales family have the key advantage of being genetically encodable. Localization- Christian Litwinski, Sebastian Tannert, Manoel Veiga, Felix Koberling, based super-resolution microscopy approaches require photoactivatable FPs Marcus Sackrow, Michael Wahl, Olaf Schulz, Marcelle Koenig, (PA-FPs) that will change their spectral properties upon irradiation with light Rainer Erdmann. of a particular wavelength. To be able to distinguish individual, activated fluo- PicoQuant GmbH, Berlin, Germany. rophores from the background and to localize them with high precision, a high Time-resolved fluorescence spectroscopy is a spectroscopist’s most valuable photon yield in the activated state and a high dynamic range, i.e., the contrast tool for the investigation of excited state dynamics in molecules, complexes, ratio between the fluorescence of the activated (on) and deactivated (off) states, or semi-conductors. In recent years, the study of luminescence properties has are essential. In stimulated emission depletion (STED) super-resolution micro- gained in popularity in many scientific fields, including Chemistry, Biology, scopy, the sample is raster-scanned by a tightly focused excitation beam fol- Physics, as well as in Life, Material or Environmental Sciences. The investiga- lowed by a red-shifted, donut-shaped depletion beam. Any FP used for tions to be carried out in each of these fields impose different requirements. On STED must be exquisitely photostable because it has to go through multiple one side, monitoring dynamic processes in the excited state necessitates high excitation-depletion cycles while the sample is scanned near its location. More- time resolution that can be achieved by fast pulsed lasers and detectors along over, it must be excitable and de-excitable by the lasers that are typically with appropriate time-correlated single photon counting (TCSPC) units and installed in commercial STED microscopes. Therefore, far-red emitting FPs small monochromators. On the other hand, high spectral resolution is desirable are preferred. for fluorophore characterization, requiring detectors with high quantum effi- We have selected the green-to-red photoconvertible mEosFPthermo and the ciencies, flash lamps for phosphorescence measurements and large monochro- far-red emitting mGarnet as templates for targeted protein engineering. Consid- mators. Up to now, spectrometers have been usually targeted towards either one ering that FPs are all very similar and share the same scaffold, an obvious strat- of these two specifications. Spectrometers equipped with hybrid detectors, ver- egy was to identify specific amino acid residues that elicit certain properties to satile TCSPC cards with optional longer time ranges, and pulsed lasers capable one FP and introduce the corresponding amino acid in the other FP variant by of working in a burst mode can offer an combined solution, covering most of using site-directed mutagenesis. As we will show, such simple rational engi- the demands of either high time or spectral resolution. We will demonstrate neering approaches often do not meet with success, which clearly shows that the performance of such a spectrometer in terms of its time resolution, the abil- our current understanding of the physics of proteins is far from being complete. ity to measure long decays and record time-gated spectra using laser drivers with burst capabilities. This type of instrument is of great value for analytical 2229-Pos Board B549 facilities in research centers, as it offers a wide range of possible spectroscopic Wide Scale Investigation of Protein Interactions by Automation of Fluo- applications in a single, easy to use instrument. rescent Polarization and Fluctuation Analysis Tuan A. Nguyen, Grace H. Taumoefolau, Youngchan Kim, Henry L. Puhl, 2227-Pos Board B547 Steven S. Vogel. Hyperspectral Measurements Allow Separation of FRET Signals from NIAAA, NIH, Rockville, MD, USA. Non-Uniform Background Fluorescence Monitoring changes in molecular conformation is essential for studying protein Savannah J. West1, Chase Hoffman2, Naga S. Annamdevula2, interactions within and between complexes. Methods such as FRET or FCS Kenny T. Trinh3, Thomas C. Rich2, Silas J. Leavesley3. reveal limited aspects of these changes, which in many cases is insufficient 1 Biomedical Sciences, University of South Alabama, Mobile, AL, USA, for interpretation. Fluorescent Polarization and Fluctuation Analysis (FPFA), 2 3 Pharmacology, University of South Alabama, Mobile, AL, USA, Chemical a time-correlated single-photon counting technique that combines homo- and Biomolecular Engineering, University of South Alabama, Mobile, AL, FRET and FCS was developed to address this problem. In FPFA changes in USA. protein complex mass and/or shape, the number of fluorescent subunits per In recent years Fo¨rster resonance energy transfer (FRET) has become a stan- complex, as well as subunit proximity (1 – 10 nm), is simultaneously detected. dard imaging approach to gain insight into localized biochemical processes This multimodal approach was validated with series of 6 fluorescent oligomers within cells. These processes include changes in protein colocalization, second composed of between 1 and 6 concatenated Venus molecules, and successfully messenger concentration, and activation of effector proteins such as protein employed to investigate structural dynamics of calcium/calmodulin-dependent kinase A (PKA). However, there are several limitations in FRET measurements protein kinase II (CaMKII) – a multimeric protein kinase that is enriched in that are not often considered. In recent years our group has used hyperspectral synaptic spines and dendrites that is involved in memory and synaptic modula- imaging approaches to address a subset of these limitations, including the tion, as well as microtubule-associated protein 1A/1B-light chain 3 (LC3) – a inherently low signal-to-noise ratio of standard two and three filter set FRET soluble protein that is a key component of autophagy, a homeostasis process measurements. Here we present data demonstrating that hyperspectral imaging responsible for the turnover of cellular components. Here we have extended approaches can be used to correct for non-uniform background fluorescence in FPFA method by developing a robotic microscope that can measure up to 96 low intensity FRET measurements. We utilized the FRET-based cAMP probe samples automatically. This FPFA automation enables wide-scale biophysical H188. The H188 probe contains a cAMP binding domain between donor (Tur- analysis of protein-protein interactions, and hence facilitates the identification quoise) and acceptor (Venus) fluorescent proteins. The probe was transfected of drugs that target protein complexes as potential therapeutic sites for diseases into pulmonary microvascular endothelial cells plated on glass coverslips. Hy- of aggregation and abnormal protein-protein interactions. perspectral image stacks were acquired using a Nikon A1R inverted confocal microscope. Changes in cAMP were triggered by addition of 0.1 mM isoproter- 2230-Pos Board B550 enol (a beta adrenergic receptor agonist) 0.1 mM PGE1 (a prostanoid receptor Comparison Study on Fluorescence Quenching Ability of DNA Wrapped agonist) or 50 mM forskolin (an adenylyl cyclase activator). Data were analyzed Single- and Multi-Walled Carbon Nanotubes using Nikon Elements software and custom MATLAB scripts. Results demon- Shusuke Oura, Katsuki Izumi, Kazuo Umemura. strate that non-uniform background fluorescence associated with glass cover- Tokyo University of Science, Shinjuku-ku, Japan. slips can contaminate FRET measurements from weak fluorescence signals. In terms of superior physical properties of carbon nanotubes (CNTs), CNT has Linear unmixing approaches were able to separate the abundances of back- been intensively studied for various applications including biosensors and drug ground, donor and acceptor fluorescence signals. Thus, results from this study delivery. For medical applications, hybridization of single-stranded DNA demonstrate that hyperspectral unmixing approaches can readily separate non- (ssDNA) and CNT (ssDNA-CNTs) is key to utilize them multiply because uniform background fluorescence from other fluorescence signatures, allowing DNA can bind to various biomolecules. For example, Li et al. used ssDNA- for more accurate quantification of FRET efficiency. This work was supported CNTs for nucleic acid detection by applying the reaction of fluorescent mole- by NIH P01HL066299, NIH S10RR027535, NIH T32HL076125, AHA cules and CNTs. It is known that, due to electron transfer, fluorescence

BPJ 7869_7874 454a Tuesday, February 14, 2017 emission of fluorescent dyes are quenched by CNTs when fluorescent mole- interrogated without the use of expensive specialized equipment. Additionally, cules directly adsorbed on CNT surfaces. the implementation of TIRF imaging and spatially-restricted analysis allows for In this study, we investigated the fluorescence quenching ability of single- and improved levels of detail within individual firing events. Furthermore, the rela- multi-walled carbon nanotubes (SWNTs and MWNTs, respectively) by tive ease of accessibility of the software and hardware comprising this system measuring the fluorescence quenching ratio of fluorescence emission when would allow for the integration of our platform into an existing experimental fluorescein-labeled single-stranded DNA (Fluor-ssDNA) is reacted with the hy- setup. brids of 30-mers of thymine (T30) and SWNTs/MWNTs. As the base- sequences of Fluor-ssDNA, we adopted dT30 and dA30 (Fluor-T30 and 2233-Pos Board B553 Fluor-A30, respectively). Edible Luminescent Probes as Sensors of Food Quality:Identification and Fluorescence measurements revealed that the fluorescence quenching ratios of Selection 1 1,2 1 3 4 the mixture of Fluor-T30 and T30-SWNT/MWNT were 28 5 3.1% and 36 5 Rahul Chib , Bogumil Zelent , Yan Wang ,AnLe, Maria G. Corradini , 1 2.0% relative to free fluorescein at the same concentration, respectively. On the Richard D. Ludescher . 1Department of Food Science, Rutgers The University of New Jersey, New other hand, the those of Fluor-A30 with T30-SWNT/MWNT hybrids were 11 2 5 1.9% and 32 5 1.9%, respectively. One of the explanations of fluorescence Brunswick, NJ, USA, Department of Biochemistry and Biophysics, quenching might be exchange reaction of Fluor-ssDNA and T30 molecules on Perelman School of Medicine, University of Pennsylvania,, New Brunswick, NJ, USA, 3School of Engineering and Applied Science, Yale University, New SWNT surfaces. We conducted fluorescence measurements at various temper- 4 atures in order to estimate the thermodynamic parameters of the exchange re- Haven, CT, USA, Food Science, University of Massachusetts, Amherst, action. The change of the enthalpy was positive value, indicating that the MA, USA. exchange reaction is endothermic process. These results suggest that SWNT Foods contain a plethora of aromatic molecules—natural colors, synthetic dyes, hybrids have good resolution for the base-sequences of Fluor-ssDNA, while flavors, vitamins, antioxidants—that are luminescent, exhibiting prompt fluo- MWNT hybrids evenly and strongry could quench Fluor-ssDNA regardless rescence or delayed phosphorescence. Although food autofluorescence has of its base-sequences. been used to assess food composition (e.g., quantification of vitamins), to detect specific contaminants (e.g., aflatoxins) or, more recently, to authenticate spe- 2231-Pos Board B551 cific foods (e.g., luminescence fingerprinting of olive oil), much of the potential Using Real-Time Quantification of Autofluorescence Spectrum Shape of using the optical luminescence of intrinsic molecules for sensing properties to Distinguish between Metabolic Responses Involving Nadh-Utilizing of foods is unrealized. Relatively low quantum yields, short lifetimes under Pathways conditions of interest in food testing and/or lack of comprehensive knowledge Paul Urayama, Symeon Stefan, Dylan Palo, Madhu Gaire, Nazar Al Aayedi. on the sensitivity of luminescence signals from native or commonly added Physics, Miami University, Oxford, OH, USA. chromophores to food quality attributes are some of the reasons that hinder In recent studies, autofluorescence measurement of excited-state lifetime or their utilization. anisotropy decay have revealed multiple forms (e.g., conformations) of intra- We summarize here work characterizing the photophysical properties of some cellular NADH. Because proportions of these forms respond differently to edible, generally recognized as safe (GRAS) chromophores and their sensitivity metabolic conditions, the often-used ‘‘free’’ and ‘‘protein bound’’ assignments to, and thus potential for sensing, various physical and chemical properties of for NADH may not be adequately descriptive, possibly leading to ambiguities food known to reflect or be indicative of food quality, stability, and safety. when identifying metabolic state. Here, we describe an accessible method for Edible luminescent molecular rotors offer a non-disruptive and highly sensitive sensing changes in cellular NADH forms, based on the real-time tracking of alternative to conventional mechanical methodologies to evaluate the physical UV-excited autofluorescence spectrum shape quantified using spectral phasor properties of foods. Flavonols that exhibit dual fluorescence and a ratiometric analysis. The analytic power of a phasor approach is utilized, testing for and concentration independent response to their solvent environment are partic- whether changes in spectrum shape are consistent with two-state behavior. ularly promising probes of water activity (aw). Food grade compounds with We show that spectral phasor quantification of spectrum shape can distinguish known active triplet states provide a feasible way to assess the stability of between metabolic responses induced by the additions of cyanide, glucose, and food matrices by monitoring molecular mobility and oxygen diffusion in amor- various alcohols to Saccharomyces cerevisiae suspensions. Controls are pre- phous solids. sented, evidencing the physiological origins of these spectral responses. The A thorough characterization and interpretation of the luminescent signals from potential for measuring dynamical aspects of metabolic responses, e.g., in rela- edible chromophores can expand the repertoire of analytical techniques avail- tion to Crabtree-positive overflow metabolism and to cellular respiratory activ- able to monitor quality, and even safety, of the food supply at various stages of ity, is discussed. the production and distribution chain or at point of sale. The potential of edible fluorophores as food quality sensors, their properties and limitations are dis- 2232-Pos Board B552 cussed herein. Spatially-Resolved Fluorescence Lifetime Measurement for Optical Inter- rogation of Electrically Dynamic Biological Systems 2234-Pos Board B554 Vaughn Spurrier1, Wenli Dai1, Eric Gauchat1, Devin Harrison1, Evan Kiefl1, Confocal Raman Microscopy Tracks Cutaneous Delivery of Flufenamic Andre´s Moya-Rodrı´guez1, Amar Risbud1, Alan Selewa1, Hallie Sussman1, Acid using Lipophilic Versus Hydrophilic Penetration Enhancers Yifan Zhou1, Justin Jureller2, Adam Hammond1. Yelena Pyatski, Qihong Zhang, Richard Mendelsohn, Carol R. Flach. 1Biophysical Sciences, University of Chicago, Chicago, IL, USA, 2IBD Chemistry, Rutgers University, Newark, NJ, USA. NanoBiology Facility, University of Chicago, Chicago, IL, USA. Recent technological advances have led to an increase in the use of confocal The excited-state lifetimes of fluorophores in biological samples are valuable Raman microscopy to tackle biophysical and pharmacological issues in topical measurements, reporting a concentration-independent measurement of the delivery and dermatology. Advantages of the technique include the ability to physical environment of the fluorophore. We describe the development of an optically section intact, full thickness skin without the need to microtome or instrument based on a total internal reflection microscope capable of measuring otherwise physically segment the skin. In addition, both exogenous agents fluorescence lifetimes within user-defined regions while simultaneously and perturbations to endogenous structure can be monitored without the intro- acquiring full field-of-view fluorescent images. Collecting photons only from duction of probe molecules. In the current work, flufenamic acid (FluA), a non- regions defined via spatial light modulation is critical for measuring localized steroidal anti-inflammatory drug, is a model agent for investigating the influ- fluorescence signals, such as fluorescence from a small region on a cell mem- ence of lipophilic versus hydrophilic penetration enhancers on the outer barrier brane. Our platform, which utilizes time-correlated single photon counting layer of skin. For topical delivery to be effective, a drug must cross the stratum from a selected space, would be especially suitable for complex biological con- corneum (SC) barrier into viable tissue. In separate experiments, FluA in octa- texts where fluorescence is used as an indicator for expeditious, transient phys- nol or propylene glycol/ethanol (75/25) is applied to skin for varying times fol- ical changes. Recent development of a Genetically Encoded Voltage Indicator lowed by confocal Raman microscopic mapping of drug and enhancer spatial (GEVI), ASAP1, has provided a new method for optically measuring neuronal distribution. The FluA pathway is tracked by the C=C stretching mode at membrane potential. To demonstrate the utility of the instrument, we hope to 1618 cm1. The exogenous enhancers are spectroscopically differentiated follow the changes in membrane potential in two ASAP1-expressing cultured from the endogenous lipids by utilizing deuterated versions of the enhancers cell models, PC-12 neurites and H9c2 myotubes, by measuring the voltage- which do not affect penetration parameters. Discrete pockets (5-20 mm) of dependent lifetime of the ASAP1 fluorescence signal. Using a standard tabletop both enhancers are observed throughout the SC. High concentrations of FluA microscope and spatial light modulation, alongside easy-to-access software and are co-localized with octanol inclusions which appear to provide a pathway hardware solutions to implement the necessary components, broadly-acting to the viable epidermis for the drug. In contrast, FluA concentrates in the upper electrically dynamic biological environments can be easily and effectively SC when using the hydrophilic agent and endogenous lipids are unperturbed in

BPJ 7869_7874 Tuesday, February 14, 2017 455a regions outside the enhancer pockets. The ability to examine perturbations to their biomechanical properties have not been identified. The mechanical endogenous ultrastructure and molecular structure in skin while tracking pene- strength of the VWF-collagen anchorage relative to VWF-platelet adhesion re- tration pathways provides insight into delivery mechanisms. mains unclear. This work examines the mechanical unbinding between A3, the collagen-binding domain of VWF, and human type I collagen, one of the major 2235-Pos Board B555 constituents of the subendothelium. Single-molecule force measurements of A3- Investigation into the Physico-Chemical and Phytochemical Properties of collagen interactions were conducted via atomic force microscopy. Depending Cola Lepidota (K. SCHUM) Seed on the loading rates, the unbinding forces between recombinant A3 and collagen Sarah O. Oni1, O.A. Oladimeji2, R.O. Owoade1, R.E. Obon1, A.K. Akinlabi3. 1 or between VWF multimers and collagen ranged from 50 to 105 pN. When using Department of Biochemistry, Lead City University, Ibadan, Nigeria, multimeric VWF constructs bearing the loss-of-function, type 2M mutations in 2Department of Chemical Sciences, Ondo State University of Science and 3 the A3 domain, either no or decreased unbinding forces were observed. The data Technology, Okitipupa, Nigeria, Department of Chemistry, Federal is consistent with the bleeding phenotypes of these mutations observed in pa- University of Agriculture, Abeokuta, Nigeria. tients with von Willebrand disease (VWD). Moreover, A3-collagen interactions Medicinal plants are rich sources of active ingredients used in drugs synthesis are only stronger than A1-platelet interactions when the pulling force is greater and development. Monkey kola is a common name given to some edible wild than 15 pN. Together, the data suggest that the A3-collagen interaction is suit- relatives of West African kola nut. In this study, the physico-chemical and able for mediating platelet adhesion under high shear flow and that VWD A3 phytochemical profile of Cola Lepidota (monkey kola) were assessed using mutations impair the mechanical properties of the interaction. standard chemical methods. The proximate analysis showed that the seed con- tained moisture (9.4050.02 %), ash (4.5750.03 %), fibre (4.5850.01 %), fat 2238-Pos Board B558 (16.7050.02 %), protein (23.7850.04 %), and carbohydrate (40.9750.05 %). A Computationally Designed Protein-Ligand Interaction is Mechanically The Fourier transform infra- red (FTIR) spectrometer analysis revealed the Robust presence of functional groups like Hydroxyl (-OH), Nitrile (C-N) and carbox- William J. Van Pattten1, Robert Walder1, Ayush Adhikari1, ylic acid (COOH) in the seed. Micro PIXE/RBS analysis carried on the seed Rashmi Ravichandran2, Christine E. Tinberg2, David Baker2, revealed the presence of elements like sodium, magnesium, aluminum, sili- Thomas T. Perkins1. cone, phosphorus, sulphur, chlorine, potassium, calcium, manganese, iron, 1JILA, NIST & Univ. of Colorado, Boulder, CO, USA, 2Dept. of zinc, bromine. The percentage compositions of carbon was 57.53%, oxygen Biochemistry, University of Washington, Seattle, WA, USA. 39.96% and nitrogen 0.84%, signifying that the cola is a good source of carbon. Quantifying the energetics and dynamics protein-ligand interactions is critical The phytochemical contents (mg/100g) obtained in the sample were as follows; to understanding molecular recognition. Computational design provides an Alkaloids 4239.3750.29, Flavonoids 10.7250.14, Saponins 22.5050.02, exciting means by which to expand the repertoire of naturally existing Tannins 288.44527.00 and Terpenoids 1032.00541.70. This result shows protein-ligand interactions for diverse biomedical applications. In both cases, that Cola Lepidota (monkey kola) has some medicinal values and contains rapid and precise determination of the fundamental physical parameters that essential elements needed for human body growth and development. govern the strength of a protein-ligand bond is needed. A particularly powerful Force Spectroscopy and Scanning Probe technique is single molecule force spectroscopy (SMFS), which generally reports the off-rate at zero applied force (koff) and the distance to the transition Microscopy I state (Dxz). Here, we used constant-force SMFS to characterize the computa- tionally designed protein DIG10.3 binding to its target ligand, the steroid 2236-Pos Board B556 digoxigenin. To improve the precision and throughput of molecular recognition Apobec3G Transitions from a Mobile to Static Binding State through studies using atomic force microscopy, we integrated a number of technical Dimerization Mediated by the N-Terminal Cytidine Deaminase Domain improvements, including low-drift cantilevers, site-specific coupling, and Michael Morse1, Ran Huo1, Linda Chelico2, Ioulia Rouzina3, corrected pulling geometries. Our enhanced precision allowed us to not only 4 1 z ˚ 1 determine koff (= 4 5 0.110 s ) and Dx (= 8.3 5 0.1 A,), but also the Mark Williams . z 1Northeastern University, Boston, MA, USA, 2University of Saskatchewan, height of the transition state (DG = 6.3 5 0.2 kCal/mol) and the shape of Saskatoon, SK, Canada, 3Ohio State University, Columbus, OH, USA. the energy barrier at the transition sate (linear-cubic potential). Importantly, APOBEC3G (A3G) is a human enzyme with anti-viral properties and is a by doing so in an automated and relatively rapid manner (<2 days of instrument particularly potent inhibitor of HIV-1 infectivity in the absence of viral infec- time), we anticipate that these measured energy landscape parameters can pro- tivity factor. A3G is a deoxycytidine deaminase which creates C to U base mu- vide valuable experimentally feedback to further optimize computational tations on HIV minus strand DNA during replication, reducing viral viability. design of protein-ligand interactions. However, A3G can still inhibit HIV infectivity even in the absence of deami- nase activity. It has been proposed that A3G bound to minus stand viral DNA 2239-Pos Board B559 physically blocks reverse transcription of the complementary plus strand and Heterobifunctional Polyprotein for Efficient Characterization of Mechan- that A3G’s ability to form this roadblock depends on its ability to form oligo- ically Labile Proteins 1 1 1 mers while bound to DNA. Using single molecule methods, we measure the ki- Marc-Andre LeBlanc , Devin T. Edwards , Robert Walder , David Rabuka2, Thomas T. Perkins1, Marcelo C. Sousa1. netics of A3G binding to single-stranded DNA and the subsequent formation of 1 2 bound A3G oligomers. We repeat these experiments using A3G variants with University of Colorado Boulder, Boulder, CO, USA, Catalent Biologics, mutated cytidine deaminase domains in order to determine the role of each of Emeryville, CA, USA. the two domains in oligomer formation. We determine that A3G first tran- Atomic force microscopy (AFM) is a powerful tool for single molecule force siently binds DNA as a monomer then forms dimers through interactions be- spectroscopy, allowing researchers to directly probe the energy landscape of tween the N-terminal domains. While monomers freely slide along DNA and protein folding and unfolding. Despite the power of this technique, it has dissociates entirely on the timescale of a minute, dimerization prevents sliding been hindered by non-specific attachment of protein to the AFM surface and and increases binding time by more than an order of magnitude. Our results tip. Non-specific attachment allows for simple experimental set up, but results suggests viral DNA deamination is performed by monomeric A3G while in low attachment efficiency, multiple attachments, incomplete unfolding, and reverse transcription is inhibited by the formation of A3G dimers. This new adhesion to the surface which can mask low force unfolding events. Not only model helps explain how a small number of A3G subunits packaged in the does this make data collection incredibly time consuming, it also makes it diffi- virion can both rapidly deaminate viral DNA and disrupt reverse transcription cult to test mechanically labile proteins. Despite recent advances in tip and sur- on two greatly different timescales. face functionalization, protein labeling continues to be a major hurdle for many labs. Furthermore, most current labeling protocols require cysteine tag modifi- 2237-Pos Board B557 cation which is unsuitable for cysteine containing proteins. To address this Biophysical Mechanisms of Von Willebrand Factor-Collagen Interactions issue, we developed a simple and versatile protein cassette utilizing aldehyde X. Frank Zhang1, Yan Xu1, Thomas A.J. McKinnon2, Wei Zhang1. tags to facilitate the specific attachment of proteins to both the AFM surface 1Mechanical Engineering and Bioengineering, Lehigh University, and tip. Combining our labeled protein cassette with newly developed tech- Bethlehem, PA, USA, 2Haematology, Hammersmith Hospital, Imperial niques to functionalize the surface and tip resulted in a 75-fold increase in College, London, United Kingdom. attachment efficiency while greatly reducing the number of incomplete unfold- Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that medi- ing events and surface adhesion. We have shown the ability to quickly insert ates platelet adhesion onto bleeding blood vessels by binding to both sub- new proteins into our cassette and rapidly characterize their response to force. endothelial collagen and platelets. VWF-collagen interactions are capable of Moreover, we were able to rapidly collect a dynamic force spectrum for a3D, resisting the substantial force imposed by blood flow or captured platelets, yet an a-helical protein which unfolds at very low force. Our protein cassette is a

BPJ 7869_7874 456a Tuesday, February 14, 2017 powerful tool that not only increases the efficiency of AFM based single mole- based on atomic force microscopy (AFM) promises a powerful tool to measure cule force spectroscopy, but also enhances the ability to test mechanically labile the force and distance in great precision and accuracy simultaneously while proteins. SMD simulations are able to provide valuable detailed information about the mechanical unfolding details that cannot be reached by SMFS. In addition, 2240-Pos Board B560 SMD simulations are also used to predict proteins’ mechanical properties ahead Investigating the Scaling Behavior of Multidomain Proteins under Force of experiments. Many b-grasp proteins with terminal b-stands of sheer topology using Single Molecule and Ensemble Force-Clamp Spectroscopy have been proven mechanical stable. It was predicted that this shear topology is Kirill Shmilovich, Narayan P. Dahal, Luai R. Khoury, Ionel Popa. a common feather for mechanical stable proteins and even nonmechanical pro- Physics, University of Wisconsin Milwaukee, Milwaukee, WI, USA. teins can have significant mechanical stability. Streptokinase b domain (SKb) A large number of proteins operating under force in vivo are segregated into and staphylokinase (SAK) are b-grasp proteins with similar terminal b-stands multidomains, and integrate the unfolding of some of these domains into their of sheer topology. Molecular dynamics simulations suggest that they both mechanical response. Due to the vectorial nature of the force, these proteins are have high mechanical stabilities as the hydrogen bonds between terminal best studied with force spectroscopy techniques. Here we combine single mole- b-stands provide high mechanical resistance. In this work, we use SMFS exper- cule magnetic tweezers with a new technique capable of measuring protein hy- iments to investigate the mechanical properties of SKb and SAK. Surprisingly, drogels using constant force protocols. For our studies we engineer constructs we found that SAK is mechanically much weaker than SKb and many other me- with the B1 domain of protein L, a model alpha-beta protein from Peptostrep- chanical stable proteins with terminal b-stands of sheer topology. In addition, tococcus magnus. Using magnetic tweezers and HaloTag covalent attachment, through chemical denaturation experiments, we draw the free energy land- we measure the unfolding and refolding of protein L in various force and solu- scapes of the unfolding of these two kinase proteins together with SMFS exper- tions regimes. At single molecule level, domain unfolding is measured as step- iments. Longer distance between native state and transition state is responsible increments in end-to-end length. The measured extensions and unfolding rates for the unexpected low unfolding force of SAK. However, the reason of the dif- as a function of force allow us to estimate the energy barrier between the folded ference of the mechanical properties of these two kinases remains unclear. and unfolded states to ~12 kT and validate the of the use worm-like chain model to describe the step size as a function of force, as previously reported 2243-Pos Board B563 by J. Valle-Orero et al, BBRC 460 (2015). Crosslinking multidomain protein Structural and Mechanistic Insights into the Copper-Modulated Unfolding L constructs engineered with eight repeats results in protein hydrogels, which Pathways of Azurin are a few mm long and experience mN forces while extending several microns. Anju Yadav, Sanjoy Paul, Ravindra Venkatramani, We find that hydrogels based on protein L show at constant force a non- Sri Rama Koti Ainavarapu. exponential smooth response to force and the presence of chemical denaturants Department of Chemical Sciences, Tata Institute of Fundamental Research, or osmolytes have a significant effect on their elasticity. We propose a 3D Mumbai, India. model to explain the measured behavior of these protein hydrogels which is Characterization of energy landscape of metalloproteins is challenging because based on the free energy of a single protein domain under force and which as- of the subtle changes imparted by metal binding on the stability and conforma- sumes a random orientation and placement of the molecules inside the gel. This tional flexibility of proteins. Here we present copper-binding induced changes model reproduces qualitatively the measured force-clamp response of protein in the mechanical unfolding pathways of azurin, a b-barrel protein with type-I hydrogels and is the first step toward understanding the transition from the copper center, through a joint experimental-computational study. Single- probabilistic response of single molecules to the deterministic response that molecule force spectroscopy experiments reveal an intermediate along the un- characterizes the functioning of tissues and organs. folding pathway of apo-azurin in half of population. Steered molecular dynamics simulations attribute the native and intermediate unfolding transition 2241-Pos Board B561 states (TSs) to the rupture of interactions between the pairs of b-strands, 2B-8 Disulfide Bonding in the Contractile Work of Titin and 4-7, respectively. We show that the copper-binding does not change the Jaime Andres Rivas Pardo, Edward Eckels, Jessica Valle-Orero, first TS of azurin and its mechanical properties, but influences the second Julio M. Fernadez. TS. The rupture of 4-7 b-strand pair is delayed in holo-azurin because of con- Department of Biological Sciences, Columbia University, New York, NY, straints imposed by the copper coordination sphere and occurs after the second USA. TS. Our experimental and computational approach extracts unprecedented de- Titin, composed of more than 36,000 amino acids, is the largest protein in the tails along the unfolding landscape for apo- and holo- forms of azurin. human body, and is responsible for the passive elasticity of muscles. Recently, we demonstrated that titin also contributes to muscle shortening through the 2244-Pos Board B564 folding contraction of its immunoglobulin-like (Ig) domains. Cysteine residues Mechanical Modulation of Protease Activity Captured at the Single- are abundant in titin, particularly within the elastic I-band region, where they Molecule Level can oxidize to form intra-Ig disulfide bonds. How these disulfide bonded-Ig do- David Giganti1, Ainhoa Lezamiz Herrero2, Guillaume Stirnemann3. mains contribute to muscle contraction is unknown, and may be fundamental to 1Physics Department, King’s College London, London, United Kingdom, muscle physiology. To understand the effect of titin’s disulfide bonds, we have 2Randall Division, King’s College London, King’s College London, United studied a disulfide bonded-Ig (I9132C/75C) using Magnetic Tweezers-based sin- Kingdom, 3Institut de Biologie Physico-Chimique, CNRS, Paris, France. gle molecule force spectroscopy. Our results show that the probability of Ig- Recent discoveries conducted at the single molecule level highlight the close folding changes when the disulfide bond is present. The oxidized I9132C/75C relation between mechanical forces and proteolysis in vivo. While much has domain can fold against a pulling force of 13.4 pN, double the force observed been discovered about protein enzymology in the recent decades, the question for the wild type I91 domain (6.6 pN). While reduced and oxidized Ig domains of how a mechanical perturbation affects enzymatic catalysis remains vastly operate in different force ranges, the expected value of the mechanical work elusive. We propose a new single-molecule approach, based on force-clamp delivered by protein folding is comparable between both molecules (~50 zJ). spectroscopy, molecular biology engineering and bioinformatics structural Our results suggest that titin is able to generate the same contractile work at analysis to elucidate the mechanism that underlies protease catalysis under me- different loading regimes by recruiting a mixture of reduced and oxidized Ig chanical force.In our single-molecule assay, the enzymatic activity relies on the domains. From this perspective, formation of disulfide bonds in titin cause enzyme:substrate assembly that can be modulated when the substrate is the folding contraction to begin at much longer sarcomere lengths, and may stretched by force. Two different mechanisms govern this mechano- effectively double the amount of weight that the muscle is able to lift. regulation. First, upon mechanical unfolding, proteins unveil their buried sub- strate sites thus favoring the formation of the enzyme:substrate complex and 2242-Pos Board B562 ultimately permitting the subsequent chemical reaction. Second, adjustment Proteins with High Structural Similarity can have Different Mechanical of the applied force largely affects the conformation of the unfolded substrate Unfolding Behaviors and the association kinetics. Chengzhi He1,2, Chunguang Hu3, Xiaodong Hu3, Xiaotang Hu3, Hongbin Li2,3. Biosensors 1Precision Instrument, Tianjin University, Tianjin, BC, Canada, 2Chemistry, University of British Columbia, Vancouver, BC, Canada, 3Precision 2245-Pos Board B565 Instrument, Tianjin University, Tianjin, China. High Resolution Ketone Measuring Method using Enzyme Reactions and Single molecule force spectroscopy (SMFS) and steered molecular dynamics Electrical Chemical Analysis (SMD) simulations play crucial roles in studying mechanical properties of sin- Naoyuki Yokoyama, Takeshi Sugimoto, Satoshi Hashizume, Emi Kagami. gle molecule protein. Many mechanical and nonmechanical proteins have been Department of Artificial Organs, National Institute of Technology, Numazu studied by SMFS and SMD simulations during the last two decades. SMFS College, Numazu-City, Shizuoka, Japan.

BPJ 7869_7874 Tuesday, February 14, 2017 457a

Diabetes Mellitus (DM) is caused by the pancreas insufficient produce of insu- binding of PEG inside the pore gives rise to blocks of ionic current, with the lin or the body cells not responding properly to the blood insulin, which leads to degree of block being detectably different for PEG molecules differing in disorder of carbohydrate metabolism. When the body has deficient insulin, tis- size by only one monomer1,2. sues cannot take glucose in from blood stream. As an alternative metabolic In order to extend the range of application of Np-SMMS, and to obtain infor- pathway, burning fatty acids produces ketones (b-hydroxybutyric acid: mation on the mechanism underying mass sensitivity of polymer-induced pore 3-HB) into bloodstream as well as create energy. Too much concentration of block, we have begun to test the interaction of other neutral polymers with bio- blood ketone body is called ketoacidosis, and it can cause serious disturbance logical nanoores. Here, we report on poly(dimethylacrylamide) (PDMA), a of consciousness, or result in death at the worst case. Therefore it is important water-soluble neutral polymer. Under conditions used for Np-SMMS of PEG to measure blood ketone body concentration and control it. Standard method of with alpha-hemolysine (4M KCl, þ40 mV), we obtained current blocks with measuring blood ketone is venous blood test. Therefore, there is many prob- polydisperse PDMA (Mn 1500 g/mol by MALDI) that were low in frequency lems such as 1) difficulty of periodic measurement, 2) risks of infection, 3) (<0.1 Hz/ mM) short (tau <100 ms) and noisy, resulting in little mass resolution pain of injection, 4) waiting time for a measurement result. In this study, we compared to PEG. We reasoned that we might take advantage of a specific salt 3 developed a novel enzymatic reactor and H2O2 electrode aiming at the applica- effect of fluoride anion reported for the aHL pore in order to increase blocking tion to measure urine/sweat ketone body non-invasively. To evaluate properties frequency and dwell time. Using electrolyte solutions consiting of 4M Kþ as of trial product sensors, we prepared test flow circuit which consisted of contin- cation and various proportions of Cl- and F- as anions (1:1, 2:1, 3:1, 4:1) we uous flow pump, manual injector, and incubator (30/37C). Within phosphate were able to obtain longer events (tau up to 400 ms) and higher frequencies buffer solution, different concentration (0.5-113.83 [mM]) of ketone body (up to 0.15 Hz/mM), allowing significantly better mass resolution for PDMA which mixed in 1.0mL/min of continuous flow path could be detected with than in 4 M KCl. However, the large noise component in the blocked current high accuracy. levels for PDMA as opposed to PEG remained, still compromising the peak- to-valley ratio of histograms. These findings suggest that the specific salt effect 2246-Pos Board B566 of F- on polymer-protein interaction is independent of the polymer and may be Microvolume Dielectric Spectroscopy and Molecular Dynamics of Amino useful in tuning polymer pore interaction for a range of analytes. Acids (1) Robertson et al. Single-Molecule Mass Spectrometry in Solution Using a Daniel Havelka, Ondrej Krivosudsky, Jiri Prusa, Michal Cifra. Solitary Nanopore. Proc. Natl. Acad. Sci. U S A 2007, 104, 8207-8211. Institute of Photonics and Electronics, Czech Academy of Sciences, Prague, (2) Baaken et al. High-Resolution Size-Discrimination of Single Nonionic Syn- Czech Republic. thetic Polymers with a Highly Charged Biological Nanopore. ACS Nano 2015, Accurate knowledge of electromagnetic properties of biosystems is essential 9, 6443-6449. for the novel physical methods for therapy and diagnostics in medicine and (3) Rodrigues et al. Hofmeister Effect in Confined Spaces: Halogen Ions and biotechnology. Single Molecule Detection. Biophys. J. 2011, 100, 2929-2935. To study the dynamics and electrical properties of biomolecules, we have de- signed, fabricated, and tested novel grounded coplanar waveguide-based chip 2248-Pos Board B568 for dielectric spectroscopy of liquid samples in 0.5 - 40 GHz band made on A Novel Capacitive Biosensor for the Detection of Small Molecule S-Nitro- RO4350B substrate and we implemented a method for the permittivity extrac- sothiols tion based purely on the extraction from measured scattering parameters. Nikki M. Meyer1, Spencer Burton1, James N. Bates2, Benjamin Gaston1, To our knowledge, the proposed bio sensing method represents an advantage Stephen J. Lewis1, James M. Seckler1. over the current known sensing chips because the dielectric function of sample 1Pediatrics, Case Western Reserve University, Cleveland, OH, USA, is obtained purely by using precision calibration techniques (NIST multi-line 2Anesthesiology, Univsersity of Iowa, Iowa City, IA, USA. TRL) and computational optimization (CST Microwave Studio) without any Small molecule S-nitrosothiols are a class of endogenous chemicals which are a priori assumption about the dielectric model of measured sample. This produced by various forms of nitric oxide synthase. The regulation of these chip is designed with well-defined active area to achieve the high sensitivity molecules has been shown to play a role in control of a variety of bodily pro- to dielectric change, enables perfect repeatability of sample position without cesses and disease models including breathing, blood pressure, pulmonary hy- the need of any microfluidics. Furthermore, due to its sensing size, the chip per- pertension, and asthma. However, these molecules are extremely labile, making mits to work with only 250 microliter sample volume. in vivo detection extremely challenging as most small molecule S-nitrosothiols In the pioneering experiments, we focused on dielectric (i.e. polarization) prop- exists at very low concentrations in the body. To overcome this challenge, we erties of solutions of amino acids, as building blocks of proteins. Alanine has have developed a capacitive biosensor which employs an organic semicon- been selected. The polar nature of amino acids determines their behavior in ductor that readily covalently crosslinks to all free amines, free thiols, and aqueous solutions and due to fact that at least one relaxation process is antici- S-nitrosylated thiols in solution. Samples were treated with formaldehyde to pated in microwave band, it allows us to study their dynamics and structure block all free amines and free thiols, leaving only S-nitrosylated thiols. S- nitro- which employ using broadband dielectric spectroscopy. Data extracted from sothiol bonding to the semiconducting surface of the sensing electrode changes experiment are in an exact agreement in comparison with widely used commer- its capacitance, allowing for extremely sensitive detection of S-nitrosothiols cial reflection method by coaxial probe (85070E Dielectric Probe Kit). Data in biological samples. We will present evidence of attomolar detection of show the trend of rising static permittivity at low frequencies with rising con- S-nitrosocysteine which can be abolished by the addition of Mercury to the centration of amino acids and also show the shift of relaxation times. We also fixing buffer, or by exposing the sample to UV light during fixing, both performed molecular dynamics simulations to predict the complex permittivity methods of degrading S-nitrosothiols. We will also present evidence of the of amino acid solution and obtained good agreement with experimental data. presence of small molecule S-nitrosothiols in blood, saliva, and other biological The presented procedure enables simple experimental verification of molecular samples. dynamics of biomolecules by the dielectric spectroscopy using our microvo- lume chip. 2249-Pos Board B569 We acknowledge support by the Czech Science Foundation, project no. P102/ Multiparametric Characterization of Single, Unlabeled Proteins in 15-17102S. Authors participate in COST Action BM1309 and bilateral ex- Solution Jared Houghtaling1,2, Michael Mayer2. change project between Czech and Slovak Academies of Sciences, no. SAV- 1 15-22. Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA, 2Adolphe Merkle Institute, Universite´ de Fribourg, 2247-Pos Board B567 Fribourg, Switzerland. Tuning Polymer-Protein Interaction with Salt With large diversity in structure and function, as well as clinical relevance, pro- Monasadat Talarimoghari1, Aleksandra Dylewska1, Marcel Hoffmann1, teins represent an important target for biophysical characterization. Resistive Gerhard Baaken2, Dalila Chouikhi3, Jean-Francois Lutz3, Jan C. Behrends1. pulse-based nanopore sensing is a compelling platform for this task, as it pro- 1Physiology, University of Freiburg, Freiburg, Germany, 2Physiology, Ionera vides information about individual proteins during their translocation through Technologies GmbH, Freiburg, Germany, 3Institut Charles Sadron, the zeptoliter sensing volume inside of a nanopore. Previous work in our group University of Strasbourg, Strasbourg, France. used lipid-coated synthetic nanopores to extract five distinct protein descriptors Biological nanopores are known to interact with synthetic and biological poly- by analyzing modulations in ionic current, DI, resulting from the translocation mers, enabling their use in label-free single-molecule analytical tasks such as and rotation of individual lipid-tethered proteins [1]. However, while coating of sequencing and/or mass discrimination. The latter, called nanopore-based sin- nanopore walls with fluid lipid bilayers and tethering proteins with lipid an- gle molecule mass spectrometry (Np-SMMS) has, to date, only been shown for chors is useful for preventing non-specific protein adsorption, extending trans- one synthetic polymer, poly(ethyleneglycol) (PEG). It is based on the fact that location times, and increasing specificity, it is technically demanding and its

BPJ 7869_7874 458a Tuesday, February 14, 2017 success depends on nanopore diameter, geometry, and surface chemistry. This solid-state nanopores is of great interest as it offers the opportunity to control work explores the possibility of using the detergent Tween-20 for nanopore sur- the macroscopic response of nanofluidic devices. In this work, we present a face coating [2] combined with high bandwidth recording electronics to char- simple and straightforward method to modify SiN nanopores based on the acterize freely translocating, untethered proteins on a single molecule level. self-polymerization of dopamine. Previous researches have proven that polydo- Here, we utilize the dependence of DI on the orientation of non-spherical pro- pamine(PDOPA) performs well as binding agents for coating inorganic sur- teins transiting a nanopore to determine their intrinsic shapes, volumes, and faces, forming strong covalent and non-covalent interaction with substrates. dipole moments in solution. The ability to thoroughly examine unlabeled, By soaking the SiN nanopore in dopamine hydrochloride solution, a thin natively-folded proteins in an aqueous sample on a single molecule level sig- PDOPA layer can be formed both on the surface and in the inner wall of the nifies an important step toward the use of nanopores for proteomic and diag- nanopore. We used Atomic force microscopy (AFM), X-ray photoelectron nostic applications. spectroscopy (XPS), contact angle measurements and Transmission micro- [1] Yusko, E.C., et al., Real-time shape approximation and 5-D fingerprinting scopy (TEM) to characterize the PDOPA overlayer. We also employed I-V of single proteins. arXiv preprint arXiv:1510.01935, 2015. characteristics to measure the conductivity of the SiN nanopore before and after [2] Hu, Rui, et al. Intrinsic and membrane-facilitated a-synuclein oligomeriza- the polymerization modification. The responsive behavior of the PDOPA- tion revealed by label-free detection through solid-state nanopores. Scientific modified SiN nanopore was also tested, hence we can evaluate the stability reports 6, 2016. of the PDOPA-modified nanopores. Our experiment data indicate that the self-polymerization of dopamine provides an effective way to adjust the dimen- 2250-Pos Board B570 sion of the pore and also can improve the rectification properties and stability of Mechanotransduction at the Nuclear Envelope the SiN nanopore. This innovation to modify solid-state nanopores based on 1 2 3 1 The´ophile De´jardin , Patricia Davidson , Cynthia Seiler , Philippe Girard , PDOPA polymerization offers a promising framework to explore new design 4 2 5 6 Damien Cuvelier ,Ce´cile Sykes , Edgar Gomes , Cadot Bruno , concepts in nanofluidic devices. Nicolas Borghi1. 1IJM CNRS UMR 7592, Paris, France, 2Intitut Curie CNRS UMR 168, Paris, 3 4 2253-Pos Board B573 France, Institut Gustave Roussy, Paris, France, Institut Pierre Gilles de Organelle-Targeting of Apollo-NadpD Requires Careful Selection of Gennes, Paris, France, 5Instituto de Medicina Molecular, Lisbon, Portugal, 6 Fluorescent Proteins to Maintain pH Independence Institut de Myologie, INSERM U974, Paris, France. William D. Cameron1, Jonathan Rocheleau1,2. LINC complexes are transmembrane protein assemblies that physically con- 1Institute of Biomaterials and Biomedical Engineering, University of nect the nucleo- and cytoskeletons through the nuclear envelope. Dysfunctions Toronto, Toronto, ON, Canada, 2Toronto General Research Institute, of LINC complexes are associated with pathologies such as cancer and University Health Network, Toronto, ON, Canada. muscular dystrophies. The mechanical roles of LINC complex proteins in these We recently developed a spectral family of genetically encoded homoFRET contexts are poorly understood. To address this issue, we have developed sensors to measure NADPH/NADPþ redox state based on changes in anisot- genetically encoded FRET biosensors that measure molecular tension in ropy/polarization due to oligomerization of glucose-6-phosphate dehydroge- LINC complex proteins. nase (Apollo-NADPþ). In cells such as insulin-secreting beta-cells, the We have evidenced that LINC complex proteins are under tension generated by NADPH/NADPþ redox state supports the scavenging of the reactive oxygen the cytoskeleton and sensitive to various external and internal mechanical con- species H2O2 by the glutathione/thioredoxin antioxidant pathway. A loss of straints in fibroblastic and epithelial cells. In addition, we have shown that some beta-cell mass due to oxidative stress leads to type 2 diabetes. A major source of the same constraints result in the localization at the nuclear envelope of of H2O2 production is the mitochondrial electron transport chain (ETC), with alpha-catenin, an actin- binding protein required for intercellular adhesion overproduction a characteristic of many metabolic disorders including type 2 that can also act as a transcription regulator. These results are consistent with diabetes. Although H2O2 can cross cell membranes through facilitated diffu- a mechanotransduction activity of LINC complex proteins, the targets of which sion, the NADPH/NADPþ redox is compartmentalized within organelles. We we currently investigate. therefore explored targeting Apollo-NADPþ to various organelles including: 2251-Pos Board B571 the mitochondria, nucleus, plasma membrane, peroxisomes, Golgi apparatus, A Fluorescent Protein Voltage Sensor in the Endoplasmic Reticulum and endoplasmic reticulum. Unlike the cytoplasm, however, the pH of organ- Masoud Sepehri Rad1, Lawrence B. Cohen1,2, Bradley J. Baker1. elles such as the mitochondria are dynamic and therefore require pH- 1Center for Functional Connectomics, Korea Institute of Science & independent sensors. Here we found that pH significantly affects the anisotropy Technology, Seoul, Korea, Seoul, Korea, Republic of, 2Department of of fluorescent proteins with high pKa (ex. Venus), while fluorescent proteins Molecular and Cellular Physiology, Yale University School of Medicine, with low pKa values (ex. Cerulean3, Turquoise2) maintained stable anisotropy values across a wider range of pH values (4.0-8.0). We also found that dimeric New Haven, CT, USA. þ In eukaryotic cells, the endoplasmic reticulum (ER) is the largest continuous Turquoise2-tagged Apollo-NADP was stable from pH = 5.0-8.0, making it suitable for use in most cellular compartments. We are now investigating the membrane-enclosed network which surrounds a single lumen. Using a newly þ designed genetically encoded voltage indicator (GEVI), we applied the patch ability to simultaneously measure the NADPH/NADP redox state in various clamp technique to HEK293 cells and found that there is a direct electrical organelles of beta-cells as well as using these sensors in high throughput interaction between plasma membrane and ER membrane. We have optically screening. Overall, this project demonstrates how homoFRET-based sensors monitored the voltage changes in both of these membranes simultaneously. may be adapted for specific organelles while revealing a novel use of the The optical signal of the GEVI in the plasma membrane is consistent from trial intensity-independence property of homoFRET towards simultaneous single- to trial. However, the ER signal decreases in size with repeated trials. This dy- colour measurements and high-throughput assays. namic behavior of the internal signal suggests that voltage may stress the ER causing it to remodel and change its resistance. Our findings further suggest 2254-Pos Board B574 that the ER may transfer electrical signals from the plasma membrane to the Energy Landscape Modeling of Escape Time Distributions Reveals the nuclear envelope. Molecular Mechanism of a-Synuclein Translocation through a VDAC This work was supported by the World Class Institute (WCI) Program of the Nanopore 1 2 2 National Research Foundation of Korea (NRF) funded by the Ministry of Ed- David P. Hoogerheide , Philip A. Gurnev , Tatiana K. Rostovtseva , 2 ucation, Science and Technology of Korea (MEST) (NRF Grant Number: WCI Sergey M. Bezrukov . 1Center for Neutron Research, National Institute of Standards and 2009-003), KIST Institutional Program (Project No. 2E24310) and US NIH 2 Grant U01 NS099691. Technology, Gaithersburg, MD, USA, Section on Molecular Transport, Eunice Kennedy Shriver National Institute of Child Health and Human, 2252-Pos Board B572 National Institutes of Health, Bethesda, MD, USA. A Bioinspired Surface Chemistry for Solid-State Nanopores Modification Elucidating the motion of uniformly charged polymers in nanoscale channels Qimeng Huang1, Yunsheng Deng1, Yue Zhao1, Lei Zhao2, Weihua Hu2, has been a scientifically and technologically fruitful enterprise. It has long Deqiang Wang1. been recognized that underlying this motion is a free energy landscape to which 1Research Centre of Precision Medicine and Single-Molecule Detection, both entropy and electrostatics contribute, but this landscape has proven diffi- Chongqing Institute of Green and Intelligent Technology, Chinese Academy cult to measure experimentally. Here we use a non-uniformly charged ‘‘diblock of Sciences, Chongqing, China, 2Institute of Clean Energy and Advanced copolymer’’-like neuronal protein, a-synuclein, to probe the energy landscape Materials, Southwest University, Chongqing, China. governing passage through a nanoscale pore. a-Synuclein is a naturally occur- Solid-state nanopores have been wildly studied in the past decade due to their ring, intrinsically disordered polypeptide associated with Parkinson disease pa- capability of single molecule detection. To modify the surface property of thology and mitochondrial bioenergetics. The motion of this electrically

BPJ 7869_7874 Tuesday, February 14, 2017 459a heterogeneous polymer through an outer mitochondrial membrane passive expanding the OmpG nanopore analyte library and investigating the effects of a transport channel, the voltage-dependent anion channel (VDAC), depends on double-ligand on kinetics. the electrical and membrane association properties of both the charged and un- charged regions of a-synuclein. We introduce complementary models that 2257-Pos Board B577 describe this motion in two limits: first, a simple Markov model accounts for Characterization of Protein Aggregation by Solid-State Nanopore 1 2 the simultaneous interaction of multiple a-synuclein molecules with VDAC Mitu C. Acharjee , Jiali Li . 1Microelectronics-Photonics, University of Arkansas, Fayetteville, AR, USA, for high membrane surface a-synuclein coverage. Second, the detailed energy 2 landscape of this motion in the dilute limit can be reconstructed from the Physics, University of Arkansas, Fayetteville, AR, USA. entropic, electrostatic, and membrane binding components by optimizing a In this report, we use solid-state nanopore device to characterize native state drift-diffusion framework to the experimental data. The models predict the aggregation of ß-lactoglobulin variant A (bLGa) protein as a function of salt probability of a-synuclein translocation across VDAC pore, with immediate concentration, temperature, pH, and electric field strength. Silicon nitride implications for the (patho-)physiological role of a-synuclein in mitochondrial nanopores used in this work, 5-20 nm in diameter, are fabricated by low en- functioning. Finally, we show that the time-dependent effect of a-synuclein on ergy ion beam sculpting method. The main component of a nanopore device the electrical properties of VDAC reports on the motion of the junction between is a nanopore supported by a silicon substrate which separates two PDMA the charged and uncharged regions of the polymer through the pore. chambers containing salt solution. When a charged protein molecule or a protein aggregate passes through a nanopore driven by an applied voltage 2255-Pos Board B575 across the nanopore chip, a protein aggregate that has larger volume than Structural Investigations of Quiet Outer Membrane Protein G Nanopores a single protein molecule,will generate larger current blockage, therefore a Raghavendar Reddy Sanganna Gari, Patrick Seelheim, Binyong Liang, nanopore device can be used to characterize protein aggregation. We present Lukas Tamm. data of bLGa self-association and aggregation at pH 4.6 as a function of University of Virgina, Charlottesville, VA, USA. temperature measured in 2M and 0.1M salt. The nanopore geometry is cali- Interest in nanopore technology has been growing due to their unique capabil- brated by a standard that has known geometry such as a long rod shaped ities in small molecule sensing, measurement of protein folding, and low cost dsDNA molecule. The volume of a translocating proteins or protein aggre- DNA and RNA sequencing. The E. coli outer membrane protein OmpG is an gates are estimated using the calibrated nanopore. We show that by using excellent alternative to the heptameric a-hemolysin (aHL) because its pore a reference dsDNA molecule, solid-state nanopore method is capable of is formed by a single polypeptide chain. OmpG is a 14-stranded b-barrel pro- measuring protein aggregation number and the aggregation number distribu- tein with seven flexible extracellular loops. It has been shown that loop 6 con- tion in the conditions that is close to their native aqueous solution trols pore access in a pH-dependent manner and its dynamics result in environment. spontaneous gating of the open pore current. This phenomenon ultimately 2258-Pos Board B578 limits the applications of OmpG in traditional biosensing. In this work, we Hybrid Nanopore for Molecular Sensing Applications modified the OmpG nanopore by selectively deleting several residues in and Uppiliappan Rengarajan1,2, Hiofan Hoi1,2, Manisha Gupta2,3, near loop 6. Electrophysiological measurements show that these modifications Carlo Montemagno1,2. eliminate the flickering between open and closed states across wide pH range. 1Chemical and Materials Engineering, University of Alberta, Edmonton, AB, We used nuclear magnetic resonance (NMR) spectroscopy to assess the struc- Canada, 2Ingenuity Lab, Edmonton, AB, Canada, 3Electrical and Computer tural role of these modifications on nanopore function by solving the structure Engineering, University of Alberta, Edmonton, AB, Canada. of our quiet OmpG. Our data show that chemical shift perturbations are more Molecular sensing is of great interest from fundamental understanding of a prominent near the modification site indicating possible structural rearrange- molecule and from biosensing application perspective also. Nanopores are ments. We further modified the inside of the quiet OmpG derivative with a cop- now widely used for molecular sensing and utilize the ionic current for detec- per chelating moiety to facilitate the detection of small molecules such as tion. Molecules passing through a nanopore cause a step change in the ionic catecholamines, amino acids, and nucleotides. For instance, we can concur- current; which depends on the length and width of the molecule. Nanopore sen- rently detect glutamic acid and ATP using this approach. sors can be classified as biological and solid-state nanopores. Integration of the solid-state nanopore with a biological one helps overcome many of the limita- 2256-Pos Board B576 tions like size reproducibility, translocation velocity and analyte specificity. Selective Detection of Protein Homologues by Outer Membrane Protein G Here we present the development of a hybrid nanopore by integrating a silicon Bib Yang1, Monifa AV Fahie2, Hui Wang3, Patanachai Limpikirati1, nitride (SiNx) based solid-state nanopore with a beta-barrel porin OmpG. Richard Vachet1,2, Sankaran Thayumanavan1,2, Min Chen1,2. 1 Solid-state nanopore were fabricated on a free standing membrane. 50 nm thick Department of Chemistry, University of Massachusetts Amherst, Amherst, SiN membranes with a dimension of 50 x 50 mm2 were fabricated using chem- MA, USA, 2Molecular and Cellular Biology, University of Massachusetts x 3 ical vapor deposition, photo-lithography, reactive ion and chemical etch. Hour- Amherst, Amherst, MA, USA, US Army Natick Soldier RD&E Center, glass shaped nanopores, with a half cone angle of 20, ranging from 2 nm to Natick, MA, USA. 8nm in size were sputtered out on the SiNx membrane using JOEL 2200FS Conventional nanopore platforms rely on rigid nanopore structures that pro- TEM. The pore current was then measured in a specialized cell filled with ionic duce a steady open pore current. Decrease of the ionic current indicates the solution. Bias was applied using Ag/AgCl electrodes interfaced to Axon-patch detection of an analyte, which may be translocation-based or non- 200B amplifier. translocation based. Since these detection schemes are based on the decrease The characterized solid-state nanopores were tested for their sensing ability. of the ionic current, analytes with a similar size and properties may generate The desired molecule was added to cis side of the device; the charged molecule similar current changes, limiting specificity in addition to size limitations. was then electrophoretically pulled through the pore onto the electrode on trans Outer membrane protein G (OmpG) is a 32kDa monomeric beta-barrel porin side. Hybrid nanopore has been achieved by inserting OmpG protein onto a with 7 flexible extracellular loops, the most flexible being loop 6. Contrary 4.1 nm or a 4.7 nm sized solid-state nanopores. The intrinsic gating character- to rigid nanopores, these flexible loops generate an indigenous pattern of cur- istic of the protein remained observed, demonstrating the proper-functioning of rent fluctuations, which we call gating. We exploit OmpG’s loops by intro- the protein. Molecular translocation through this hybrid nanopore would be ducing a cysteine into loop 6, allowing an analyte-specific ligand to be observed and the results from this study will be presented. tethered to OmpG, generating a nontranslocation-based OmpG nanopore sensor. 2259-Pos Board B579 Using this approach, we found that binding of an analyte changes the dynamic Translocation and Binding in the Recognition of Short Oligonucleotides by behavior of the loops, thus changing the gating pattern of the OmpG nanopore. a Biological Nanopore Using a biotin ligand tethered to OmpG, we successfully detected and discrim- Mordjane A. Boukhet1,2, Ibrahim Halimeh2, Gerhard Baaken1, inated between biotin-binding homologues due to each analyte’s ability to Jan C. Behrends2. generate a different and distinguishable gating pattern when bound to the ligand 1Ionera Technologies GmbH, Freiburg, Germany, 2Physiology, University of on OmpG. We’ve also detected two human carbonic anhydrase isoforms using Freiburg, Freiburg, Germany. a benezenesulfonamide inhibitor ligand. These analytes range from 30kDa to Polymer interactions with pore-forming membrane proteins, as evidenced by over 150kDa in size, illustrating the OmpG nanopore’s ability to detect a the resulting block of ionic current, may be classified into two types: (1) the wide range of analytes independent of size. Furthermore, OmpG retains its threading of an extended polymer chain of which only a small length contrib- discriminatory ability in a sample mixture of fetal bovine serum. In addition utes to the block and (2) a binding reaction, where the polymer enters the pore to OmpG’s sensing capabilities, we are able to extract single molecule kinetic entirely and interacts as a particle. The former phenomenon typically occurs in data, revealing differences in affinity among homologues. Future works include dilute aqueous electrolyte solutions (e.g. KCl <1 M) and requires long and

BPJ 7869_7874 460a Tuesday, February 14, 2017 charged molecules and can allow sequencing (e.g. of DNA). In contrast, the 2. J. J. Kasianowicz, E. Brandin, D. Branton, D. W. Deamer, Proc. Natl. Acad. binding reaction typically occurs with short, neutral polymer molecules, re- Sci. U. S. A. 1996, 93, 13770. quires high salinity (e.g. KCl 3-4 M) and enables the high-resolution discrim- 3. D. Branton, D. W. Deamer, A. Marziali, H. Bayley et al. The potential and ination of polymer masses1. The aerolysin nanopore has recently been shown to challenges of nanopore sequencing. Nat Biotechnol. 2008, 26, 1146. strongly interact with short adenine oligonucleotides (A3-A10) and this inter- 4. C. Cao, Y.-L. Ying, Z.-L. Hu, D.-F. Liao, H. Tian, Y.-T. Long, Discrimina- action allows mass discrimination on the basis of the depth of block of ionic tion of oligonucleotides of different lengths with a wild-type aerolysin nano- current induced by the binding of the analyte,2 suggesting a binding-type inter- pore. Nat. Nanotechnol. 2016, 11, 713. action. Using low-noise current recording, we have indentified strong dynamics of this interaction between DC and 50 kHz and indentified short visits to deeper 2262-Pos Board B582 blocked states preceding and following the principal, mass-dependent state Controlling the Capture Process of 50BP DNA by Solid-State Nanopores (pre- and post-blocks). Statistical analysis indicates that the probability of Martin Charron, Aidan Baker-Murray, Vincent Tabard-Cossa. post- but not pre-blocks decreases with (1) oligomer length and (2) transmem- Physics, University of Ottawa, Ottawa, ON, Canada. brane voltage. We interpret this finding in terms of a combined translocation We quantify the frequency of short DNA fragments interacting with solid-state and binding interaction, probably involving several binding sites for DNA in nanopores under different experimental conditions. By measuring the time be- the pore. In line with this hypothesis, longer polynucleotides (A30) showed sta- tween adjacent single-molecule translocation events, we can extract the capture ble and long-lasting interactions with near-complete block of current. rate from the ionic current signal. For this study, we use >50 individual solid- (1) Robertson et al. Proc. Natl. Acad. Sci. U S A 2007, 104, 8207-8211. state nanopores ranging in sizes between 2.5 and 4nm and fabricated in ultra- (2) Cao et al. Nature Nanotechnology 2016, 1-7. thin 10nm SiN membranes using controlled breakdown (CBD). We investigate the factors that affect the reliability and variability of the capture process of 2260-Pos Board B580 50bp DNA molecules, such as applied voltage and electrolyte type, and identify Comparison of Multi-Dyes Quenching by Single-Walled Carbon Nano- the optimal conditions for maximizing the capture rate of such short fragments. tubes Dispersion with Single-Stranded DNA and Double-Stranded DNA These results help establish solid-state nanopores fabricated by CBD as reliable Ying Tan1, Katsuki Izumi2, Kazuo Umemura2. single-molecule Coulter counters for accurately determining molecular concen- 1Physics, Tokyo University of Science, Tokyo, Japan, 2Physics, tokyo trations of short DNA polymers in solution. university of science, Tokyo, Japan. 2263-Pos Board B583 A fluorescence quenching phenomenon of a wide range of fluorophores could be Observation of Adsorption Process of Single Stranded DNA to Single- observed in the present of single-walled carbon nanotubes (SWCNTs) suspen- Walled Carbon Nanotubes Surfaces by Fluorescence Quenching sion. In this study, as a consequence of attaching to SWCNT surface, quenching Shizuma Sato1, Gilbert Bustamante2, Jing Yong Ye2, Kazuo Umemura1. effect of three kinds of dyes, uranine (Ur), acridine orange (AO), and Rhodamin 1Tokyo University of Science, Shinjuku-ku, Japan, 2The University of Texas B (RB), were compared and investigated. Then employed SWCNT suspension at San Antonio, San Antonio, TX, USA. with single-stranded DNA and double-stranded DNA, three groups of compar- Single walled carbon nanotubes (SWNTs) are one of the promising nanomate- ison experiment of quenching properties of Ur vs. AO, Ur vs. RB, and AO vs. rials for various applications in nanotechnology. In biomedical fields, hybrids RB, were carried out respectively. For the process of adding two dyes into of DNA and SWNT (DNA-SWNT hybrids) are expected to be attractive nano- SWCNT suspension, ‘‘simultaneous adding’’ was emphasized because the devices for drug delivery and DNA sensors. For preparing DNA-SWNT hy- competitive adsorption of two dyes onto the SWCNT surface was admired in brids, mixture of SWNT and DNA solutions are prepared via sonication. order to investigate the quenching performance. Based on fluorescence results Although DNA-SWNT hybridization techniques have been established, the and calculated quenching efficiency, it was found that AO adsorbed onto diverse sonication conditions in every paper may affect structures of DNA- SWCNT-ssDNA surface prior to Ur and RB, while Ur showed stronger SWNT hybrids. Here we report a method of monitoring adsorption process quenching-restrain performance over RB. Overall, SWCNT-dsDNA’s quench- of DNA to SWNT surfaces using fluorescent labeled single-stranded DNA ing capability was weaker than SWCNT-ssDNA. The strengthening fluores- (Fluor-ssDNA). It is known that fluorescent emission of fluorescent dyes is cence stem from the AO-dsDNA moiety, and the suppressed emission due to quenched when the dye is adsorbed on SWNT surfaces. Using this unique AO-SWCNT moiety, existed at the same time. The emission shift of RB in quenching phenomenon, we evaluated adsorption process of Fluor-ssDNA on AO/RB and Ur/RB assay maybe generated by the superimposing of light. The SWNT surfaces. In the experiments, 0.25 mg of SWNT powder, 50 uL of competitive adsorption model was established based on Langmuir adsorption Fluor-ssDNA solution (1.0 mg/mL in tris (hydroxymethyl aminomethane equation. The dispersion strategy of fluorophore/dye into SWCNT suspension buffer solution, 200mM, pH 7.5)), and 450 uL of the buffer were mixed, and without sonicate treatment, and the fundamental quenching effect found in then sonicated under three different sonication conditions as follows: probe this work were necessary for multi-nanosensor applications that could be useful type with amplitude 60% of 130 W (P60), probe type with amplitude 20% of for fluorophores/dyes detection and quantification in aqueous solution. 130 W (P20), and bath type with 80 W (B80). In order to monitor the adsorption process, samples were characterized by fluorescent spectroscopy and atomic 2261-Pos Board B581 force microscopy. In the case of P60, fluorescent intensity of Fluor-ssDNA Direct Identification of Adenine, Thymine, Cytosine and Guanine using decreased 98% within 2 min. This suggests that most of Fluor-ssDNA rapidly Aerolysin Nanopore adsorbed on SWNT surfaces. However, AFM images demonstrated that it took Chan Cao1, Matteo Dal Peraro2, Yitao Long1. 1 more time to form mono-dispersed DNA-SWNT hybrids. In the case of P20 and Key Laboratory for Advanced Materials, School of Chemistry & Molecular B80, 30 min was needed for 98% quenching. In addition, reproducibility of the Engineering, East China University of Science and Technology, Shanghai, 2 B80 data was much higher than that of P20. This is an advantage of bath type China, Institute of Bioengineering, School of Life Sciences, Ecole sonicator. Polytechnique Fe´de´rale de Lausanne, Lausanne, Switzerland. Direct identification of four nucleobases (adenine, thymine, cytosine and gua- 2264-Pos Board B584 nine) in single-stranded DNA is the cornerstone for clinical diagnosis, person- Afm-Based Nanosensor and Super-Resolution Immunofluorescence alized medicine and genomic studies1. Nanopore technology is one of the most Studies: A Correlative Approach to Assess the Role of HER2 Cancer promising next-generation sequencing methods2, 3. Our recent results have Biomarker demonstrated that aerolysin pore-forming toxins exhibit an unexceptional Elena Ambrosetti1,2, Alessandro Bosco3, Loredana Casalis4. sensitivity for the detection of single oligonucleotides both in current separa- 1University of Trieste, Trieste, Italy, 2INSTM-ST Unit, Trieste, Italy, tion and duration, which already falls within the optimal reading rate for 3Department of Biochemistry and Biophysics, Karolinska Institutet, DNA sensing4. Here, we reported the ability of aerolysin nanopore for direct Stockholm, Sweden, 4Nanoinnovation lab, Elettra Sincrotrone Trieste identification of adenine, thymine, cytosine and guanine in DNA oligomers. S.C.p.A, Trieste, Italy. Combining the distinct current levels with the characteristic durations of the Human Epidermal Growth Factor Receptor 2 (Her2) is a transmembrane pro- four DNA oligomers, we could distinguish the differences of single nucleo- tein overexpressed in many tumours and so considered an important indicator bases in a mixed sample. This highly sensitive, accurate and simple signal read- in cancer diagnosis, prognosis and therapeutic drug monitoring. Her2 tumour outs indicated that aerolysin nanopore is a powerful protein pore for single potential is strictly related to its dimerization process, that determines the acti- nucleobase identification. vation of the signal cascade pathways and the cleavage (shedding) of its extra- Reference: cellular domain (ECD). The dimerization seems to take place in a specific 1. H. Y. Xiong, B. Alipanahi, L. J. Lee, H. Bretschneider, D. Merico et al. The subfamily of membrane rafts, (caveolae), also involved in the biogenesis of human splicing code reveals new insights into the genetic determinants of dis- exosomes and hence in exosome-driven tumor cell communication and metas- ease. Science 2015, 347, 1254806. tasis occurrence.

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We present here an integrative approach based on advanced microscopies enabling biocolloid in this technique as they carry a diverse variety of geneti- and nanotechnology-based diagnostic tools in order to clarify the clinical sig- cally rich cargo, including proteins, RNA, and DNA. Hence, studying exo- nificance of circulating ECD-Her2 and to elucidate the correlation of Her2 somes could potentially provide vital information about undesirable genetic biology with the biogenesis of exosomes. We performed immunofluores- deviations occurring in their cell of origin. Rapid isolation of exosomes from cence studies in order to visualized overexpression/oligomerization status blood, urine or other body fluids remains a key challenge in this growing field. of Her2 and/or truncated Her2 on living cell membranes. Then we co- Deterministic lateral displacement (DLD) pillar arrays have proven an effective localized Her2 with membrane lipid rafts, and in particular with the caveolae means to sort, segregate, and enrich micron-size particles, such as parasites and raft subfamily; we characterized Her2 presence in exosomes isolated from blood cells. Here, we have developed a nanoscale DLD devices, containing gap cell medium. For the detection and quantitative analysis of ECD-Her2 sizes as small as ~25nm, with nanoscale sorting resolution of biological parti- released in the cell medium and for the characterization of Her2-enriched cles. This development in nanofluidics and engineering has enabled us to sort exosomes we developed and optimized a highly sensitive Atomic Force colloidal particles at the tens of nanometers scale. Additionally, we have devel- Microscopy-based Nanoarray capable to perform label-free analysis in small oped predictive computational models to provide key insights into the behavior sample volumes. Nanoarrays are generated from the loading of DNA- of particles in these systems. Furthermore, we have successfully demonstrated antibody conjugates, specific for antigen of interest, on DNA nanospots on-chip, size-based separation of exosomes, indicating the potential of this created by nanografting. For biorecognition we tested monoclonal mouse technology for sorting plasma, urine, serum or circulating tumor-derived Antibodies and innovative camelid Nanobodies. By measuring spot height exosomes. variation we were able to detect biomarkers with high sensitivity (up to pico- molar range). 2268-Pos Board B588 Novel Biosensor Design Reveals the Role and Regulation of GEF-H1 in 2265-Pos Board B585 Cell Migration Electronic Detection of Single Cancer Cells with Graphene Field Effect Mihai L. Azoitei1, Jungsik Noh2, Maria J. Sandi3, Philippe Roudot4, Transistors Robert Rottapel3, Gaudenz Danuser2, Klaus M. Hahn1. James Froberg1, Prajakta Kulkarni2, Sanku Mallik2, Yongki Choi1. 1University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 1 2 Physics, North Dakota State University, Fargo, ND, USA, Pharmaceutical 2University of Texas Southwestern Medical Center, Dallas, TX, USA, Sciences, North Dakota State University, Fargo, ND, USA. 3University of Toronto, Toronto, ON, Canada, 4University of Texas Here, we present electronic-type biosensors for ultra-sensitive detection of Southwestern Medical Center, Dalals, TX, USA. pancreatic cancer cells, based on graphene field effect transistors. The devices Rho family GTPases are molecular switches that control cellular movement. was decorated with bi-functional pyrene-iRGD peptides in which the pyrene Their activity is regulated with precise spatio-temporal dynamics by guanine moiety binds to the graphene though pi-pi interaction and the iRGD peptides exchange factors (GEFs). While biosensors have revealed the dynamics of binds to neuropilin receptors in the pancreatic cancer cells. Real-time electronic Rho GTPases in polarized motility, little is known about the subcellular distri- detection enabled detecting few cancer cells in the presence of the human bution and timing of GEF activation. Guanine exchange factor H1 (GEF-H1) serum, which is a critical for the early detection of cancer diagnosis and treat- activates RhoA at the leading edge of moving cells and plays a critical role ments. We acknowledge the support of ND EPSCoR New Faculty Award and in focal adhesion turnover and mechanosensing. GEF-H1 is unique among ND NASA EPSCoR RID Grant. GEFs because it is sequestered on microtubules (MTs) in its inactive state and its activation requires microtubule dissociation. GEF-H1 activity is thought 2266-Pos Board B586 to be regulated by MT remodeling and to be essential for crosstalk between the Regulation of Glucokinase in Pancreatic Beta Cells MT and actin networks. We identified a new mode of GEF-H1 regulation and Kendra M. Seckinger. used it to design a fluorescent biosensor that reports GEF-H1 activation in Physiology, University of Maryland School of Medicine, Baltimore, MD, living cells. The biosensor was designed using general structure-based princi- USA. ples that can be extended to the other 68 members of the Dbl family of Glucokinase (GCK) is the main glucose sensor in pancreatic b cells and con- GEFs. Live cell imaging experiments of GEF-H1 biosensor and MT þTIP trols insulin secretion. Because of this, GCK is an important target for type 2 labelling in the same cell showed that GEF-H1 is localized at the MTs in its diabetes mellitus therapies. The mechanism of how GCK, a monomeric inactive state, and that GEF-H1 was active away from the MTs. We developed enzyme with a single binding site for glucose, exhibits cooperativity is still a new image analysis method to detect MTs disassembly through detection and unknown although there are many proposed models. Our lab has shown that tracking of MT þTIPs. We then correlated MT disassembly frequency with nitric oxide (NO) enhances GCK activity, but the underlying mechanism of GEF-H1 activity in space and time using local windows adaptive to cell mem- activation and the relationship between GCK S-nitrosylation and cooperativ- brane fluctuation. Using this techniques, we measured that MT instability reg- ity are unclear. To further investigate GCK regulation, we created a novel ulates GEF-H1 activity at the cell edge with a time delay of 10 seconds. Current single-color GCK reporter. This Fo¨rster resonance energy transfer (FRET) studies that investigate the role of kinases in mediating this GEF-H1 activation biosensor allows for more precise and quantitative measurements than tradi- will be described. tional two-color FRET reporters. Here we show that our single-color GCK sensor accurately measures changes in GCK conformation in response to increasing concentrations of glucose. 7.5mM glucose is representative of 2269-Pos Board B589 post-prandial blood glucose levels and also corresponds to half-maximal Multifunctional High-Throughput Single-Cell Analysis using Reconfigura- GCK activation. We show that 7.5mM also has the highest degree of vari- ble Amplifier Array ability further highlighting the need to elucidate the mechanism of GCK Kevin A. White, Geoffrey Mulberry, Brian N. Kim. activation. Introducing known activating and inactivating GCK point muta- Department of Electrical and Computer Engineering, University of Central tions into our GCK FRET sensor provide evidence for two conformations of Florida, Orlando, FL, USA. GCK. We measured distinct FRET efficiencies for the both the active and In recent years, an introduction of CMOS bioelectronic systems to biotech- inactive states that converged on precise anisotropy values. Addition of nology gave unprecedented opportunities in extremely high-throughput the NO donor, SNAP, stimulates formation of the more active GCK (1k - 10M parallel recordings) and improved noise performance. However, conformation. Together, these findings provide support for a two state recent developments of CMOS bioelectronic devices mostly focused on GCK model with NO facilitating formation of the more active GCK state. Application Specific Integrated Circuits (ASIC) in which the mode of oper- Learning how GCK is regulated is important for understanding the pathol- ation is fixed from the design phase. Thus, ASIC devices offer little flexi- ogy of diabetes. bility and a new design is mandatory to support alternative modes. We developed a reconfigurable design that can switch into different operational 2267-Pos Board B587 modes to suit the type of demanded analysis. The presented CMOS device On-Chip Liquid Biopsy: Progress in Isolation of Exosomes for Early contains a reconfigurable array of 32 32 electrodes and amplifiers. Mem- Diagnosis of Cancer ory cells are embedded in each unit of the array to store the amplifier config- Sung Cheol Kim, Navneet Dogra, Benjamin H. Wunsch, Joshua T. Smith, uration and allow for rapid re-programming. The benefit of this approach is Stacey M. Gifford, Gustavo Stolovitzky, Huan Hu, Pablo Meyer. the full customizability, conceptually similar to a field-programmable gate IBM, Yorktown Heights, NY, USA. array (FPGA). With the demonstrated multifunctionality of the CMOS- In contrast to a standard biopsy, the so-called ‘liquid biopsy’offers a rapid, non- based biosensor design, various types of single-cell analysis, including am- invasive, and cost effective alternative for cancer diagnosis. Exosomes, which perometry, cyclic voltammetry, and patch-clamp, can be conducted from a are secreted by most eukaryotic cells and range in size from 30-150nm, are the low cost, high-throughput device.

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2270-Pos Board B590 accessible and straightforward pipetting step leading to stable DIBs between 3D Printed Real-Time PCR Machine for Infectious Disease Diagnostics two aqueous reservoirs with a proton gradient across the bilayer interface. Geoffrey Mulberry, Kevin A. White, Brian N. Kim. When a pore-forming peptide is present, the pH of the reservoirs change, which Electrical and Computer Engineering, University of Central Florida, Orlando, can be detected by the unaided eye based on the color change of a pH sensitive FL, USA. dye. This approach takes advantage of bio-inspired amplification in the sense Diagnosing infectious diseases is a complex task with many different tech- that one ion channel, after analyte-triggered activation, facilitates the flux of niques to obtain the results. According to the World Health Organization thousands of ions across the DIB. We describe our attempts to impede Hþ/ some diseases such as Malaria and HIV, Polymerase Chain Reaction (PCR) OH- flux using known pore blockers in order to detect alterations in the rate must be used to obtain results and to determine the proper treatment. Ideally, of color change in the presence of these molecules. We also discuss the possi- PCR would be used as often as possible because it gives increased reliability bility of using pore-forming peptides engineered to alter their function in the and accuracy, but the cost and the difficulty to obtain equipment used for presence of a specific analyte in order to detect this analyte within a sample. PCR diagnostics outside of a lab environment effectively forces the use of Our ultimate goal is to develop an assay to screen samples of biofluids in a an inferior method of a more accessible nature or treatment without proper broadly-accessible and robust manner that can be adapted to various analytes diagnosis. The main goal of this research is to develop a system to perform and biomarkers. PCR diagnostics that can be manufactured using simple tools, off-the-shelf parts, and a 3D printer. Using a 3D printer allowed for the manufacture of 2273-Pos Board B593 complex parts that would otherwise have to be machined or injection molded, Optical Probes for Imaging Signal Mediating Phospholipids both processes that are out of reach for many. In keeping with this theme, the Samsuzzoha Mondal, Ananya Rakshit, Shafali Gupta, printed circuit boards were also routed and drilled using the milling tool head Ravindra Venkatramani, Ankona Datta. of the 3D printer. This was to avoid the complexity of a photochemical pro- Department of Chemical Sciences, Tata Institute of Fundamental Research, cess or the poor reliability of a heat transfer method. Few challenges, Mumbai, India. including difficulty decreasing cycle times, which required designing a heat- Phospholipids mediate essential cell signaling pathways. Dynamic changes in ing block that satisfied light-path and thermal requirements, and reading of phospholipid concentrations and locations on biological membranes act as real-time fluorescence data, were addressed. The resulting device is initiating flags for signaling events. Tracking spatiotemporal dynamics of battery-operated and can perform a complete 40-cycle PCR with real-time the phospholipids on the cell membrane is important for obtaining mechanistic fluorescence reading of intercalating dyes. Ultimately, the files for the 3D insights into signaling processes. Optical imaging is apt for visualizing printed parts can be published and printed out on any 3D printer. This will phospholipid-dynamics at high spatiotemporal resolution within live cells. enable the construction and use of this device in all corners of the globe The major requirement is fluorescent probes for quantitatively and selectively and without the need for a complex manufacturing process and only standard monitoring the signal mediating phospholipids. Toward this goal, we have tools. developed fluorescent sensors for signaling phospholipids like phosphatidyl- serine and phosphoinositides. We have used both protein and peptide-based 2271-Pos Board B591 scaffolds for selective phospholipid binding. By using lanthanide-bound pro- Development of a Surface Plasmon Resonance and Plasmon-Waveguide teins we have developed a ‘turn-on’ sensor for phosphatidylserine.[1] Ratio- Resonance Combined Chip for Studying the Transport Behaviors of Cell metric fluorescent sensors for phosphoinositides have been developed by Membrane Transporters conjugating polarity-sensitive dyes to peptides.[2] The phosphoinositide Yu-Ting Lin, Ling Chao. probes are cell-permeable and have been tested in both live cells and in a Chemical Engineering, National Taiwan University, Taipei, Taiwan. C. elegans model. I will present the rationale behind our phospholipid sensor Surface plasmon resonance (SPR) is a powerful label-free and contact-free designs, results on in vitro and in vivo sensing of phospholipids, and our recent technique for chemical and biological sensing experiments. However, the attempts toward applying molecular dynamics simulation to fine-tune the traditional use of SPR instruments is for molecular interactions on the surface sensor designs. of metallic film. In order to extend the application of SPR, we integrated the [1] Gupta, S.; Mondal, S.; Mhamane, A.; Datta, A. Inorg. Chem. 2013, 52, concept of plasmon-waveguide resonance (PWR) and proposed the idea of 12314. PWR/SPR combined chip in order to measure the transport behaviors of [2] Mondal, S.; Rakshit, A.; Pal, S.; Datta, A. ACS Chem. Biol. 2016, 11, 1834. cell membrane transport proteins. The PWR/SPR combined chip is composed of a silica layer with sub-micron sized pores on a thin gold film. The geom- Biophysics Education etry allows us to use SPR to detect the refractive index change in the pore region, which is correlated to the target species concentration inside the 2274-Pos Board B594 pore, and PWR to simultaneously monitor the change of refractive index at Responsive Web-Based Molecule Viewer for 3D Communication, Collabo- the top silica surface. We deposited the giant plasma membrane vesicles ration, and Virtual Reality 1 1 2 2 (GPMV) derived from cells onto the PWR/SPR combined chip to construct Merry Wang , Aditya Dhoot , Andrew Kimoto , David Parker , 2 2 2 2 the lipid membrane with native membrane proteins suspending over the Michael Zyracki , Malte Tinnus , Peter Jones , Drew Hylbert , Dion Amago2, Florencio Mazzoldi2. sub-micron sized pores. Consequently, the detection area can be divided 1 2 into two regions including the space inside the pores and the region above Autodesk Research, Toronto, ON, Canada, Autodesk Research, San the grating structure. By using COMSOL simulation, we confirmed that Francisco, CA, USA. this system allows us to simultaneously measure the change of refractive Communication for molecular visualization is a fundamental component of any indices in the two regions across the lipid membrane. We experimentally research, collaboration, and education pipeline between scientists across disci- demonstrated how the platform can be used to study the glucose transport plines and knowledge base. However, existing industry standard tools provide through the corresponding membrane transport proteins (Glut 1, Glut2). In limitations whereby complex 3D spatial data is reduced to 2D images. Novel the future, we plan to use these platforms to monitor how various inhibitors approaches which utilize recent innovative technological advances are needed or ligands could influence the transport dynamics of interested membrane to adequately facilitate communication workflows. Autodesk Molecule Viewer transport proteins. leverages web-based, cloud-enabled 3D viewing technology to overcome lim- itations in accessibility and 3D sharing of curated molecular structural content 2272-Pos Board B592 to enable effective communication. Optimized for large-scale data, the Viewer Broadly Accessible Assays with Signal Amplification across Lipid Bilayers demonstrates usage scenarios of author-curated 3D structural content through Anirvan Guha1, Thomas B.H. Schroeder2, Sarah Grunsfeld3, Jerry Yang4, multiple saved camera and model states (such as measurements, annotations, Michael Mayer1. captions, representations, color, etc.) to be shared through a URL. The shared 1University of Fribourg, Fribourg, Switzerland, 2University of Michigan, Ann URL contains presentation views of the authored content in its native 3D Arbor, MI, USA, 3Massachusetts Institute of Technology, Cambridge, MA, environment, which can be viewed through any internet-accessible device USA, 4University of California, San Diego, San Diego, CA, USA. such as phones, tablets, and laptop devices. As such, novel methods of presen- Over the past decade, droplet interface bilayers (DIBs) have emerged as a tation, education, and exploration can be leveraged, such as immersive robust platform for performing studies across lipid membranes with the poten- smartphone-enabled virtual reality using any affordable Cardboard VR device. tial for high-throughput applications. Here we explore the potential of DIBs for Molecule Viewer applies innovative technological trends and breakthroughs to detecting the presence of analytes in a heterogeneous sample without the need molecular visualization to accelerate efforts in communication, collaboration, for an external power source. We discuss the formation of DIBs by a broadly and education.

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2275-Pos Board B595 ical context, and mechanism of protein function. The lab course is an A Ratiometric Method to Measure Viscosity Inside Mesoporous Silica Par- extension of successful undergraduate research efforts at RIT and Dowling ticles using Protein-Bound Fluorescent Probe College. The modules that are developed have been disseminated to the sci- Pegah S. Nabavi Zadeh, Bjo¨rn A˚ kerman. entific community via a web site (promol.org), including both protocols and Chemistry and Chemical Engineering, Chalmers, Go¨teborg, Sweden. captioned video instructions for the techniques involved. Over the course of Mesoporous silica particles are used for immobilization of enzymes in order to the project, we have followed changes in faculty and teaching assistant increase enzyme stability, facilitate product purification and reuse of enzyme. competence in two areas: effective teaching with both in silico and in vitro Many efforts have been done to investigate how the environment inside the tools and the development of skills in the area of measuring learning gains pores changes after enzyme immobilization and how it can affect activity by students. We are also analyzing the flexibility of the curriculum for adop- and stability of immobilized enzymes. One of these environmental changes tion as a whole in a single course or in parts across multiple courses. As we can be viscosity inside the pores and comparing the difference with the bulk conduct the lab on the different campuses, we are focusing on advantages of solution. our approach and barriers to implementation that exist on each campus., from Here, we investigate viscosity inside mesoporous silica particles, SBA-15, 1mm the level of student acceptance and faculty training, to resources that are diameter with 9nm pore diameter, based on two different cyanine fluorophores needed, to changes in the culture at the departmental and institutional levels. which are sensitive to viscosity, Cy3 and Cy5 using a ratiometric method. The We are now optimizing the curriculum for wider dissemination, and we seek probes can bond covalently to lipase as enzyme used in two different condi- input from additional potential adopters about their level of interest and the tions: a. both probes seated on one enzyme, b. they can be attached to the barriers that they anticipate on their campuses. This project is supported in enzyme separately. We work with a ratiometric method to measure only viscos- part by NSF IUSE 1503699. ity and no other solvent effects. In this study, Cy5 can be used to normalize for polarity other than viscosity. in addition, we take advantage of the energy trans- 2278-Pos Board B598 fer (FRET) between probes when they are seated on one enzyme and measure Expanding the Scope of Single Molecule FRET Spectroscopy towards the distance between them. Primarily Undergraduate Institutions Jesse Howe, Gregory Walters, Kambiz Hamadani. 2276-Pos Board B596 CSU San Marcos, Vista, CA, USA. Making a Puzzle Microscope as a Strategy to Recruit High School Girls Single molecule fluorescence methods provide a unique and ultrasensitive set and Minorities in Biophysics of probes for monitoring the conformations and dynamics of protein chains Yuly E. Sanchez. within physiologically relevant contexts. In particular, single molecule fluores- Universidad Nacional de Colombia, Bogota, Colombia. cence resonance energy transfer (smFRET) is capable of monitoring the dis- Low participation of woman in science is an actual fact that has been dis- tances between two fluorescent dyes with sub-nanometer spatial and cussed by different sources, women’s organizations, prestigious universities nanosecond temporal resolution. Unfortunately, smFRET remains inaccessible and different journals. According to UNESCO ‘‘the world needs science to many biophysical researchers and most of all to students at primarily under- and science needs women’’, that’s why last February 11th 2016 was estab- graduate research institutions. This is in part due to the difficulties involved in lished as the first International Day of Women and Girls in Science, UNES- making dual-labelled protein samples but also stems from the expense and/or CO’s message was clear - the new Agenda will not meet its promise complexity of existing single-molecule fluorescence detection platforms. To without investing in women’s and girls’ empowerment through and in science. address the first issue, we have developed a sample generation protocol suitable Unfortunately women in Colombia have been victims of both armed conflict for making large libraries of dual-labelled proteins and ribosome-bound and a society run by men and her role in scientific areas is not increasing. Ac- nascent chains (RNCs). This method consists of using a purified and reconsti- cording to recent research conducted at the National University of Colombia, tuted in-vitro translation system for protein expression, the incorporation of women have little participation in university courses related to the exact sci- azide or alkyne-bearing non-standard amino acids, and finally click- ences, which is easily verified with statistics about of women’s participation chemistry-based dye attachment. Here, we show that this approach is simple in science careers as technology, engineering and mathematics (STEM). As and robust enough to be used by undergraduate students to make large libraries a contribution to address this topic, we proposed an activity aimed at recruit- of dual-labelled protein samples in about a day. To enable smFRET-based ing high school girls in biophysics and science in general. Our proposal was to screening of these libraries we have also built the first-ever confocal smFRET build a puzzle wooden microscope kit by Echo LaboratoriesÒ, this activities microscope at a primarily undergraduate institution. This instrument was built where performed with 4 different groups of 25-30 girls and 3 groups of 25-30 for less than $20K largely using second-hand optical components. It has a students from minorities as afro-Colombian and older students, as a comple- 3-axis piezo-scanning stage which enables single-molecule imaging and trajec- ment we explored different tissues with our microscope and we compared it tory analysis on surface-immobilized molecules. It also has alternating laser with a conventional school microscope. Is well known that puzzles are also excitation capabilities which enable digital sorting of single molecules using an important educational learning tool for young children as they provide multi-dimensional histograms. This work marks the first implementation of many skills and mental learning benefits and opportunities, we used this smFRET as a research tool at a primarily undergraduate research institution. concept too for elderly students. We found that after they build the wooden scopes, they were able to conclude that was much like a science experiment. 2279-Pos Board B599 This proposal incorporating this microscope as educational puzzles into to- Light, Imaging, Vision: An Interdisciplinary Undergraduate Course day’s school curriculum is a great way to encourage their participation in Philip Nelson. science. Physics/Astronomy, Univ Pennsylvania, Philadelphia, PA, USA. Students in physical and life science, and in engineering, need to know about 2277-Pos Board B597 the physics and biology of light. In the twenty-first century, it has become Combining Molecular Visualization with Bench Methods in a Hypothesis- increasingly clear that the quantum nature of light is essential both for the latest Driven Undergraduate Biochemistry Lab Course imaging modalities and even to advance our knowledge of fundamental life Thomas Holt1, Rebecca Roberts1, Julia R. Koeppe2, Paul A. Craig3. processes, such as photosynthesis and human vision. But many optics courses 1Ursinus College, Collegeville, PA, USA, 2Chemistry, SUNY Oswego, remain rooted in classical physics, with photons as an afterthought. Oswego, NY, USA, 3Rochester Institute of Technology, Rochester, NY, I’ll describe a new undergraduate course, for students in several science and en- USA. gineering majors, that takes students from the rudiments of probability theory We are developing an undergraduate biochemistry lab curriculum based on to modern experimental methods like fluorescence imaging and Fo¨rster reso- authentic inquiry. Students on six campuses are combining computational nance energy transfer. After a digression into color vision, students then see (in silico) and wet lab (in vitro) techniques as they characterize enzymes how the Feynman principle explains the apparently wavelike phenomena asso- whose three dimensional structures are known but to which functions have ciated to light, including applications like diffraction limit, subdiffraction im- not been previously ascribed. The in silico modules include protein visualiza- aging, total internal reflection and TIRF microscopy. Then we see how tion with PyMOL, structural alignment using Dali and ProMOL, sequence scientists documented the single-quantum sensitivity of the eye seven decades exploration with BLAST and Pfam, and ligand docking with PyRX and Au- earlier than ‘ought’ to have been possible, and finally close with the remarkable todock Vina. The goal is to predict the function of the enzymes and to identify signaling cascade that delivers such outstanding performance. promising substrates for the active sites. In the wet lab, students express and A new textbook, to be published in April 2017, allows others to replicate this purify the target enzymes and perform kinetic assays with substrates selected course. from their docking studies. Their learning as students and their growth as sci- Partially supported by the United States National Science Foundation under entists is being assessed in terms of research methods, visualization, biolog- Grant PHY-1601894.

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2280-Pos Board B600 the images. All aspects of the device and software design will be made pub- Development of a Non-Radioactive Aminoacylation Assay for Quality lically available in the hopes that others will build and use this setup in their Control of Purified and Reconstituted In Vitro Translation Systems teaching of super resolution techniques at colleges and universities Judith Flores1, Jesse Howe1, Lizbeth Reyes1, Kambiz M. Hamadani2. worldwide. 1Chemistry and Biochemistry, California State University San Marcos, San Marcos, CA, USA, 2Chemistry and Biochemistry, CSU San Marcos, San 2283-Pos Board B603 Marcos, CA, USA. Teaching Some Concepts of Transport Mechanism to Middle School Stu- In order to understand how proteins folding during translation on the ribosome, dents in Valledupar Colombia we require efficient and highly controllable in-vitro translation systems capable Eliana M. Ramos, Claudia A. Blanco, Yuly E. Sa´nchez. of faithfully synthesizing nascent protein chains. Purified and reconstituted in Fı´sica, Universidad Nacional de Colombia, Bogota´, Colombia. vitro translation (e.g. PURE IVT) systems meet this need. However, to ensure Biophysics is an unknown field in Colombian schools; the factor that can influ- high-fidelity translation the numerous activities involved must each be properly ence this is lack of connection between teaching biology with physical princi- characterized prior to reconstitution of full-scale translation. In this study, we ples, which can be an obstacle to his real learning. At skill standards defined by focused on improving a non-radioactive spectrophotometric assay that facili- the Education Council of Colombia, mechanisms of cellular transport teaching tates the determination of aminoacyl-tRNA synthetase activities. This assay, is included as a subject of compulsory education in the early grades of second- monitors pyrophosphate/phosphate generation as a proxy for aminoacylation ary basic education since their understanding is especially important when and can thus be used to monitor the activities of all 20 amino-acyl tRNA syn- dealing with processes such as respiration, circulation, photosynthesis and thetase enzymes required for translation. Phosphate generation is coupled to a excretion in later grades.Based on this standards and in order to divulgate spectrophotometric change using purine nucleoside phosphorylase (PNPase) and start with the biophysics formation from an early age, was implemented which catalyzes phosphorylation of MESG substrates (Amax~330nm) to make a proposal at public school of the village of Aguas Blancas, in the city of Val- AMMP (Amax~360nm) products. The main objective of the present study ledupar, in the Colombian Caribbean, with students from middle school with was to clone, express, and purify highly purified (i.e. phosphate-free and ages between 10-12 years old and from a very low income families. We ATPase-free) PNPase for the determination of PURE IVT system component develop as a part of a science class, teaching concepts of passive cellular trans- activities. This assay will enable the non-radioactive characterization of the port mechanisms: diffusion and osmosis, using problem-based learning activities of natural as well as engineered aminoacyl-tRNA synthetases as method. Activities performed include 11 sessions which was oriented attend required for high-fidelity unnatural amino acid incorporation for co- 34 children to solve a specific problem-based situation, which was a story about translational protein folding studies. a girl from 2085 who traveled to another planet, she should explain to the planet’s people how her pet, a fish, couldn’t survive out of water, and shouldn’t 2281-Pos Board B601 be taken out of seawater. The concepts addressed in the study of the situation A Hands-On Freshman Seminar Course in DNA Origami were: concentration through experience ‘‘Build your dreams’’ semi- Ryan Shriver, Sarah Veatch. permeability, osmosis, and as a part of this experience we implemented the Di- University of Michigan, Ann Arbor, MI, USA. fussion lesson plans available at the Biophysical Society web site released in In the fall semester of 2016, the biophysics program at the University of Mich- 2015 as ‘‘BASICS: Biophysics - A Step-by-Step Introduction to Concepts for igan offered a hands-on seminar course in the theory and practice of DNA Students’’. At the end of the experience we found that the students was moti- origami aimed at first year undergraduate students with varying scientific back- vated to learn more about this topics and they found very interesting the grounds. The course began with lectures on theoretical underpinnings of self- biophysics field as a new way to understand the nature. assembly, and quickly moved into group work where students learned to design DNA origami structures using freely accessible software tools (cadnano: http:// 2284-Pos Board B604 cadnano.org/ and CanDo: http://cando-dna-origami.org/). Students then assem- What’s Wrong with the Hodgkin-Huxley Model? An Exercise in Critical bled an instructor-designed single layer origami and characterized its assembly Thinking using electrophoresis, atomic force microscopy (AFM) and super-resolution H. Richard Leuchtag. fluorescence localization microscopy. The course ended with discussions of Retired, Kerrville, TX, USA. current research aimed at using origami and other self-organizing systems to Critical thinking is fundamental to science: let us examine an assumption of the approach fundamental problems in biology and medicine. The goal of the Hodgkin-Huxley model of excitable membranes. The circuit elements of the course is to introduce young students to several fundamental biophysical con- HH model are a capacitance C and parallel ionic conductances g in series cepts and approaches. This poster will discuss the positive and negative out- with their electromotive forces. HH assumes that C is constant while the gs comes of this class, as well as several obstacles encountered in course design vary with voltage and time. This assumption, adapted to voltage-sensitive and implementation. ion channels, places the entire burden of explaining channel function on the gs. While the assumption of constant C—and its dimensionless coefficient, 2282-Pos Board B602 dielectric permittivity ε—is a good approximation for a membrane patch, its Storm-Lite: An Inexpensive Tool to Demonstrate Stochastic Super Resolu- application at the macromolecular nanoscale is dubious, considering the ubiq- tion Microscopy Techniques in the Classroom uitous presence of branched-chain amino acids—isoleucine, leucine and Anthony Wu1, Lark Moreno1, Maxim Prigozhin2, Sharlene Denos1. valine—in the channels. The sidechains of these amino acids (and no others) 1University Laboratory High School, Urbana, IL, USA, 2Standford strongly affect ε in certain molecules with structures resembling channel seg- University, Palo Alto, CA, USA. ments. These ferroelectric liquid crystals, such as 4’-3M2CPOOB, exhibit ε Studying sub-cellular structures is central to biophysics research. However, values as high as 3000 in their ferroelectric state. Changes in channel ε affect diffraction limits the resolution in light microscopy to a few hundred nanome- all electrostatic interactions, including the mutual repulsions between the posi- ters and the finest structures of interest can no longer be resolved. To address tively charged residues in S4 segments, key components of voltage sensors. this obstacle, a variety of ‘‘super-resolution’’ microscopy techniques have The Channel Activation by Electrostatic Repulsion (CAbER) hypothesis as- been developed. These techniques make use of the photophysics of fluores- sumes the resting channel, at a high electric field, is in a polar, far-from- cent molecules to increase the resolution to several tens of nanometers.We equilibrium state with high ε, and that threshold depolarization converts it to describe here a device that will allow classrooms worldwide to easily simulate a nonpolar state with much lower ε. The consequently increased Coulomb super-resolution microscopy techniques such as photoactivation localization repulsion between arginines and lysines explains the observed outward motion microscopy (PALM) and stochastic reconstruction microscopy (STORM). of the S4 segments, which then drives the opening of the pore domain to sto- These techniques are extremely expensive and cumbersome to demonstrate chastic ion currents. Does critical thinking support constant-C or CAbER? in a classroom setting, limiting students’ exposure to them. By contrast, our Problem: Two proton charges on a helix of radius 0.80 nm are 0.45 nm apart device is inexpensive, lightweight, and portable, using only an LED array axially, with an angular separation of 300. What is their repulsion energy to simulate the fluorophores, frosted glass to simulate the resolution limit, (kJ/mol) when ε is 500? When it is 4? Further reading: HR Leuchtag, and a camera and user interface developed for the Raspberry Pi to process Voltage-Sensitive Ion Channels, Springer, 2008.

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