results suggests that hydrogen bonding of the peptide backbone to water and lipid molecules and the creation of membrane defects are responsible for the exceptionally slow transport rate. 3617-Pos Board B478 Long Distance Lipid: Protein Coupling in the Protein Translocon Ana-Nicoleta Bondar 1 , Douglas J. Tobias 2 , Stephen H. White 3 . 1 Freie Universitaet Berlin, Department of Physics, Berlin, Germany, 2 University of California at Irvine, Department of Chemistry, Irvine, CA, USA, 3 University of California at Irvine, Department of Physiology and Biophysics, Irvine, CA, USA. The SecY/Sec61 protein translocon is the central component of the sophisti- cated translocase machinery essential for the biosynthesis of membrane and se- creted proteins in all branches of life. To incorporate newly synthesized transmembrane (TM) proteins into the lipid membrane, SecY opens a helical gate formed by TM helices 2 and 7 towards the bilayer. Opening of a lateral helical gate to release TM proteins requires close communication between the translocon and the lipid membrane. Here we report prolonged molecular dynamics computations on wild-type and mutant translocons with perturbed lipid interactions. We find a remarkable long-distance coupling between the translocon and the surrounding lipid mem- brane. Mutating amino acids that in the wild type interact with lipids affects the shape of the lipid membrane surrounding the translocon. Importantly, the status of the lipid:translocon interactions is rapidly communicated to the plug - a struc- tural element whose displacement is thought to be part of the translocon open- ing. The rapid propagation of structural perturbations throughout the translocon is likely facilitated by the extensive hydrogen-bonding network of the translo- con. The tight coupling between the translocon and the lipid membrane dem- onstrated here could explain the experimental observations that the lipid membrane composition affects the proper assembly and functioning of the translocase machinery. Research supported in part by the National Institute of General Medical Sci- ences (GM-74637 and GM-86685 to S.H.W), the NSF (CHE-0750175 to D.J.T), and an allocation of computer time from the NSF through the TeraGrid resources at TACC (Ranger). A.-N.B is supported in part by Marie Curie Inter- national Reintegration Award FP7-PEOPLE-2010-RG 276920. 3618-Pos Board B479 Substrate Binding and Transport by a Bacterial Multidrug MFS Trans- porter Ryan Steed, Kristin Trone, Hassane Mchaourab. Vanderbilt University, Nashville, TN, USA. Bacterial multidrug efflux pumps confer resistance to a broad range of antibi- otics, thus reducing the efficacy of clinical treatment of bacterial infection. EmrD is the only structurally-characterized representative of a drug:H þ anti- porter of the major facilitator superfamily (MFS). However, the determinants of substrate binding and transport by EmrD remain unclear. Unlike other MFS transporters, EmrD contains no membrane-embedded acidic residues thought to be necessary for coupling drug:H þ antiport. In an effort to charac- terize the determinants of substrate binding and mechanism of transport by EmrD we have tested the binding and transport of several fluorescent substrates by anisotropy and quenching, respectively. EmrD bound Doxorubicin and Hoechst33342 with micromolar affinity whereas substrates with constitutive positive charges bound with low affinity, suggesting a preference for neutral species. Additionally, EmrD carried out DpH-driven transport of Hoechst in inside-out membrane vesicles. Interestingly, we found that no single acidic res- idue, including the conserved Asp68 in the MFS signature motif, is essential for Hoechst transport. 3619-Pos Board B480 Revealing the Interaction of Binding Protein with ABC Transporter by Fluorescence Correlation Spectroscopy Joanna Ziomkowska, Johanna Heuveling, Erwin Schneider, Andreas Herrmann. Humboldt University Berlin, Berlin, Germany. ATP binding cassette (ABC) transporters can be found in all organism form bacteria to man. Dysfunction of these transporters is often associated with dis- eases, like multi drug-resistance in cancer cells. These transporters translocate substances across the cellular plasma membrane under the expense of ATP, consist of dimers of ATPase subunits known as the ATP binding cassette and of two transmembrane domains (TMDs). The histidine permease (HisQMP 2 ) of S. typhimirium is a well characterised classic canonical ABC importer. For substrate translocation HisJ (binding protein (BP) with specificity for histidine) or LAO (BP with specificity for lysine/arginine/ornithine) are required. The BP interacts with the extracellular loops of the TMD and the binding constants for both proteins were found to be in a micro molar range. To assess the conformational dynamics of BP-TMD complexation during the catalytic cycle of substrate translocation, a Fluores- cense Correlation Spectroscopy based binding assay is designed to investigate the dependency of this interaction on the catalytic state of the transporter. HisQMP 2 is functionally reconstituted into large unilamellar vesicles and a monocysteine variant of the BP is chemically labelled with Alexa Fluor 488. By trapping the transporter in different steps of ATP hydrolysis, the change in diffusion rates of the fluorescent BP is supposed to reveal the dynam- ics of the BP association during substrate translocation. 3620-Pos Board B481 A Unifying Concept of Serotonin Transporter Associated Currents Walter Sandtner 1 , Klaus Schicker 1 , Michael Freissmuth 1 , Gary Rudnick 2 , Harald H. Sitte 1 . 1 Medical University of Vienna, Vienna, Austria, 2 Yale University, New Haven, CT, USA. Serotonin (5-HT) uptake by the human serotonin transporter (hSERT) is driven by ion gradients. The stoichiometry of transported 5-HT and ions is predicted to result in electroneutral charge movement. However, hSERT mediates a cur- rent when challenged with 5-HT. This discrepancy can be accounted for by an uncoupled ion flux. Here we investigated the mechanistic basis of the un- coupled currents and its relation to the conformational cycle of hSERT. Our observations support the conclusion that the conducting state underlying the uncoupled ion flux is in equilibrium with an inward-facing state of the trans- porter with Kþ bound. We identified conditions associated with accumulation of the transporter in inward facing conformations. Manipulations that in- creased the abundance of inward facing states resulted in enhanced steady- state currents. We present a comprehensive kinetic model of the transport cycle, which recapitulates salient features of the recorded currents. This study provides a framework for exploring transporter-associated currents.This work was supported by the FWF (P18706 and SFB35-06 to H.H.S.) and by the NIH (DA007259 to G.R.). 3621-Pos Board B482 Effects of Flavonoids on Dipole Potential of Sterol-Containing Membranes Svetlana S. Efimova, Olga S. Ostroumova. Institute of Cytology of RAS, St-Petersburg, Russian Federation. Flavonoids are hydroxylated polyphenoles from plants which show antioxidant, antiinflammatory, antitumoral, and other therapeutic properties. It is known that flavonoids are membrane-active agents and some of them are able to influ- ence on the membrane structural and dynamic properties. The role of the mem- brane dipole potential (Vd) is a particular interest due to a powerful impact of Vd on the membrane permeability and lipid-protein interactions. It is known that the flavonoids, phloretin and phloridzin, significantly reduce Vd of phos- pholipid bilayers [Andersen et al., 1976], but their effects on sterol- containing bilayers are unknown. We estimated the changes of dipole potential (DVd) of phosphocholine bilayers containing 33 mol % of cholesterol (the ma- jor membrane sterol of mammalian cells) or ergosterol (the major membrane sterol of fungi) after addition into bilayer bathing solution (0.1M KCl pH7.4) of phloretin, phloridzin, genistein, quercetin and biochanin A up to 20 mM. The corresponding calculations were performed assuming that the membrane Kþ-nonactin steady-state conductance is related to Vd by the Boltzmann dis- tribution [Andersen et al., 1976]. We found that in the presence of phloretin DVd of phosphocholine membranes was equal to À130 5 10 mV, of phospho- choline:cholesterol bilayers is À75 5 10 mV, and of phosphocholine:ergos- terol membranes is À150 5 10 mV. In contrast to phloretin introduction of phloridzin, genistein, quercetin and biochanin A changes Vd of bilayers of above composition in a similar manner. The average values of DVd were À55 5 10 mV, À40 5 10 mV, À110 5 10 mV, and À80 5 10 mV, respec- tively. The study was supported in part by RFBR (#09-04-00883), SS- 3796.2010.4 and the Program of Presidium of the RAS «Molecular and Cell Biology». 3622-Pos Board B483 Escherichia Coli Regulates Cell Division by Modulating Membrane Potential Catalin Chimerel, Christopher M. Field, Silvia Pin ˜ero-Fernandez, Silvia M. Hernandez-Ainsa, David K. Summers, Ulrich F. Keyser. University of Cambridge, Cambridge, United Kingdom. 714a Wednesday, February 29, 2012