Cholesterol Influence on the Interaction of Cell Penetrating Peptides (CPPs) with Model Membranes

Monday, February 29, 2016 nonlinear functions of the voltage, area and volume density. As a result, strong similarities exist between the response of biological and artificial membranes. Since biological membranes display phase transitions close to physiological temperature, this has to be included in the electrical models of the cell membrane when interpreting electrophysiological data. Here we show how electric data commonly interpreted as gating currents of proteins and inductance can be explained by the nonlinear dynamics of the lipid matrix itself. 1218-Pos Board B195 Buried Charges and their Effect on Ion Channel Selectivity. Analytical Solutions, Numerical Calculations and MD Simulations Marı´a Queralt-Martı´n, Antonio Alcaraz, Marcel Aguilella-Arzo, Vicente M. Aguilella. Laboratory of Molecular Biophysics, Department of Physics, Universitat Jaume I, Castello´n de la Plana, Spain. The ionic selectivity of large protein ion channels is primarily determined by the electrostatic interactions of mobile ions with charged residues of the protein. Here we discuss the widely spread paradigm that the charges determining the channel selectivity are only those that can be considered solventaccessible because of their location near the permeation pathways of ions and water molecules. Theoretical predictions for the electric potential and average ion densities inside the pore are presented using several approaches of increasing resolution: from analytical and numerical solutions of electrostatic equations in a model channel up to all-atom Molecular Dynamics simulations and continuum electrostatic calculations performed in a particular biological channel, the bacterial porin OmpF. The results highlight the role of protein dieletric properties and the importance of the initial choice of the residue ionization states in the understanding of the molecular basis of large channel selectivity irrespective of the level of resolution of the computational approach used. 1219-Pos Board B196 Structural Determinants of the IF-OF Transition in Human Glucose Transporter GLUT1 Mrinal Shekhar, Javier Baylon, Emad Tajkhorshid. UIUC, Urbana, IL, USA. Glucose transporters (GLUTs) are membrane proteins that facilitate the diffusion of glucose across the plasma membrane. Recently, the crystal structures of human GLUTs (e.g., GLUT1 and GLUT3) have been resolved in the inward-facing (IF) and outward-facing (OF) states, enabling a detailed study of their transport mechanism.In this work, we aim to characterize the IF-OF transition of GLUT1, the major glucose transporter in the human body. Starting from the GLUT1 IF state, we performed an extensive search for an optimal reaction coordinate for the transition. Employing nonequilibrium simulations, we induced the reorientation of multiple combinations of transmembrane (TM) helices of GLUT1 to trigger the transition to the OF state for each TM combination. The relevance of the reaction coordinates was then assessed by the non-equilibrium work required for the transition, as well as the induced structural rearrangement of GLUT1. As a starting point for the transitions, an OF target model of GLUT1 was first generated based on available structures of its close homologue OF-GLUT3, and further refined once the optimal reaction coordinate was obtained. Based on the minimum non-equilibrium work required to drive the IF-OF transition, we identified a specific set of TM helices whose motion is required to reach the OF state. Using this reaction coordinate, we were able to obtain previously uncharacterized stable GLUT1 OF state, and characterize multiple intermediate states connecting the IF and OF states, allowing us to identify important interhelical interactions and the gating helices suggesting a local gating mechanism apart from global large scale transition involving a proline-rich TM segment that mediates the conformational transition in GLUT1. Given the structural similarity among GLUTs, we expect that the our detailed description of the GLUT1 transition will provide a basis to study other human GLUTs. 1220-Pos Board B197 Bilayer Modifying Effects of Antipsychotics of Different Generations R. Lea Sanford, Olaf S. Andersen. Biophysics and Physiology, Weill Cornell Medical College, New York, NY, USA. Antipsychotics are used to manage the symptoms of schizophrenia (delusions, hallucinations, etc.), to treat bipolar mania, and are commonly used as adjective

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agents in the treatment of depression. They are known to alter the function of diverse membrane proteins, and for certain antipsychotics (e.g. quintepine), this polypharmacology is thought to be desirable. Because antipsychotics are amphiphiles that modulate the function of different, structurally unrelated membrane proteins, we investigated whether antipsychotics alter lipid bilayer properties at concentrations where they alter membrane protein function. To this end, we used a gramicidin-based fluorescence assay (GBFA) as well as single-channel electrophysiology and we find that antipsychotics like chlorpromazine increase the lifetime and appearance rate of gramicidin (gA) channels, thus shifting the gA monomer dimer equilibrium toward the conducting dimers. We are currently examining other commonly prescribed antipsychotics with the goal of exploring whether there are systematic differences between antipsychotics from the first and second generations. The expectation is that the newer antipsychotics will be less bilayer active and therefore would be expected to have fewer undesired effects. Our other studies have found that an increased alteration of bilayer properties, above a given threshold, may indicate toxicity. Thus, we were keen to learn whether antipsychotics substantially altered bulk bilayer properties and more specifically, we hope to observe whether or not there is a correlation between how well an antipsychotic is tolerated (and thus more commonly prescribed) and its bilayer modifying propensity. 1221-Pos Board B198 Cholesterol Influence on the Interaction of Cell Penetrating Peptides (CPPs) with Model Membranes Viviana E. Silva1, Fanny Guzma´n2, Patricio Sotomayor1, Luis F. Aguilar1. 1 Instituto de quı´mica, Pontificia Universidad Cato´lica de Valparaı´so, Valparaı´so, Chile, 2Nu´cleo de Biotecnologı´a Curauma, Valparaı´so, Chile. A number of high efficiency peptidic transporters are known, which are called CPPs, and which use three mechanisms of cell incorporation. The models proposed to explain the direct translocation of CPPs across biological membranes include the ‘‘inverted micelle model’’, models involving the formation of membrane pores and the ‘‘carpet model’’1. Studies have demonstrated that cholesterol prevents interaction of the cell-penetrating peptide transportan with model lipid membranes2. Other studies show that higher cholesterol content and tighter packing of the membrane predominantly reduces accumulation of transportan, TP10 and the model amphipathic peptide (MAP) in vesicles, indicating that the internalization of CPPs takes place preferentially via the more dynamic membrane regions3. Our circular dichroism results show a change in the secondary structure of the peptides upon interaction with model membranes. In addition, fluorescence spectroscopy studies have demonstrated changes in the interaction of peptides with LUV ternary mixtures of DMPC/DMPG/CHO as a function of the cholesterol content. Acknowledgements: We thank Project FONDECYT 1140800 for funding and CONICYT for the PhD scholarship for VSS. 1.Trabulo S., Cardoso A.L., Mano M., y Pedroso de Lima M.C., Pharmaceuticals 3, 961-993 (2010). 2.Arsov1 Z., Nemec1 M., Schara1 M., Johansson H., Langel U. y Zorko M., Journal of Peptide Science 14(12), 1303-1308 (2008). ¨.y 3.Paea J., Sa¨a¨likb P., Liivama¨gia L., Lubenetsa D., Arukuuskc P., Langelc U Pooga M. Journal of Controlled Release 192, 103-113 (2014). 1222-Pos Board B199 Unraveling the Outer Membrane Translocation Mechanism of a Protein Antibiotic using Single-Molecule Microbiology and Computational Biophysics Patrice Rassam1,2, Kathleen R. Long2, David J. Williams2, Matthieu Chavent1, Anna Duncan1, Mark Sansom1, Colin Kleanthous1, Christoph G. Baumann2. 1 Biochemistry, University of Oxford, Oxford, United Kingdom, 2Biology, University of York, York, United Kingdom. Colicins are natural protein antibiotics deployed by Escherichia coli to kill closely related bacterial competitors - they act by delivering a toxic protein domain intracellularly. A mechanistic understanding of how colicins manipulate endogenous protein-protein interactions to gain cell entry may lead to the development of novel therapeutics against antibiotic-resistant bacteria. By combining in vivo, in vitro and in silico biophysical techniques, we recently discovered that promiscuous interactions occur between the outer membrane proteins (OMPs) BtuB and OmpF, which are the receptor and translocator proteins for colicin E9 (ColE9), respectively. These promiscuous proteinprotein interactions may contribute to the observed self-association of