- Email: [email protected]
Characterizing Nanopore-Polymer Interactions and Cys-loop Protein Receptor Gating
630a
Wednesday, March 2, 2016
‘‘super-inhibitory’’ ternary complex (SERCA-SLN-PLB), whereby SERCA activity shows 5-fold decreased calcium affinity and 2-fold decreased maximal velocity. Fo¨rster resonance energy transfer (FRET) was used to quantify the complex equilibria of homo- and hetero-oligomeric interactions between SLN, PLB, and SERCA1a: five binary interactions were assayed and three assembly parameters were determined (binding affinity, oligomer number, interprobe distance). FRET results indicate that SLN and PLB show high-affinity self-association into homo-oligomers, and that SERCA forms heterodimers with SLN or PLB when co-expressed with either subunit individually, with SERCA having 3-fold higher affinity for SLN over PLB. To determine inhibition mechanisms of subunit-bound complexes, we used microsecond MD simulation of SERCA1a 5 SLN or PLB in a POPC bilayer, with calcium, proton, or magnesium ions bound to transport sites of the SERCA transmembrane domain. MD results indicate that two potassium ions initially bind and preorganize transport sites for calcium binding (SERCA activation), and that SLN and PLB individually inhibit SERCA by populating a calcium/ potassium-free intermediate state with one bound proton at Glu771 (transport site I). We propose that super-inhibition of SERCA in the ternary complex is mediated by SLN/PLB-synergistic inhibition of proton/potassium exchange. Acknowledgments: this work was funded by grants to DDT (NIH-GM27906) and MEF (AHA-12SDG12060656). 3108-Pos Board B485 In Vitro Demonstration of Light-Driven NaD/HD Pumping by a Microbial Rhodopsin Hai Li, Oleg A. Sineshchekov, Giordano F.Z. da Silva, John L. Spudich. University of Texas Medical School at Houston, Houston, TX, USA. A new subfamily of rhodopsins was discovered in bacteria and proposed to function as dual function light-driven Hþ/Naþ pumps, ejecting Naþ from cells in the presence of Naþ and Hþ in its absence (Inoue et al 2013. Nat. Commun. 4:1678). This proposal was based primarily on light-induced proton flux measurements in suspensions of E. coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the Naþ and Hþ transport definitively. We expressed IAR from Indibacter alkaliphilus in E. coli cells and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta reported earlier. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Naþ pumping activity in vitro, namely Naþ-dependent light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Naþ-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore, showing Hþ pumping activity. The direction of proton fluxes indicated that IAR was inserted inside-out into the sealed LUV system, which we confirmed by site-directed spin-label EPR spectroscopy. The in vitro LUV system proves that the dual light-driven Hþ/Naþ pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo. We are currently investigating ion fluxes through natural anion channelrhodopsins with this system. 3109-Pos Board B486 The Diversity of Light-Driven Ion Pumps and their Conversion into Ion Channels Arend Vogt, Christiane Grimm, Peter Hegemann. Experimental Biophysics, Humboldt Universita¨t zu Berlin, Berlin, Germany. Microbial rhodopsins are integral seven-transmembrane helix proteins, which covalently bind all-trans-retinal as light-sensitive chromophore. They play an extremely important role in neuroscience as optogenetic tools for the modulation of neuronal activity. These rhodopsins are subdivided into sensory rhodopsins, ion channels and ion pumps. Light-driven ion pumps transport protons, sodium or chloride across the plasma membrane against an electrochemical gradient. We analyzed a variety of ion pumps using two-microelectrode voltage-clamp measurements (TEVC) in Xenopus leavis oocytes. We found that all pumps show different electrophysiological behaviors. Especially light-driven proton pumps can be subdivided according to their characteristics at high electrochemical load, i.e. low extracellular pH and negative voltage. Photocurrents of Bacteriorhodopsin and the rhodopsin from the eukaryotic microalga Coccomyxa subellipsoidea (CsR) are always outward directed or inactivate at high load. In contrast, the rhodopsins from Exiguobacterium sibiricum (ESR) and from
the cyanobacterium Gloeobacter violaceus (GR) show inward directed photocurrents at high load. The well-established rhodopsin Arch3 from the archaeon Halorubrum sodomense shows weak inward directed photocurrents at high load. Most recently, the class of sodium pumps was discovered, but so far their underlying mechanism and ion selectivity is poorly understood. Here, we show the first more comprehensive electrophysiological characterization of the light-driven sodium-pumps in native membranes. They are characterized by an unexpected strong pH-dependency. In earlier work we have shown that proton pumps can be converted into operational proton channels via the introduction of selected mutations. Now, we transferred this approach to the sodium pumps and attained also inward directed photocurrents. This observation opens the opportunity to engineer new optogenetic tools with high ion-selectivity. 3110-Pos Board B487 Localization of a Sodium Binding Site in the Sodium Translocating NADH: Ubiquinone Oxidoreductase Katherine G. Mezic, Blanca Barquera. Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY, USA. Many marine, soil and pathogenic bacteria, including Vibrio cholerae, Pseudomonas aeruginosa, Yersinia pestis and Chlamydia trachomatis, possess a unique respiratory enzyme called Naþ-translocating NADH:quinone oxidoreductase (NQR). This enzyme is capable of pumping Naþ across the cell membrane, thus creating an electrochemical gradient that the cell can use for metabolic functions. NQR pumps Naþ by using the energy released by the oxidation-reduction (redox) reaction between NADH and ubiquinone. While the electron transfer reactions have been well characterized in this enzyme, the Naþ translocation mechanism is not yet well known. Several acid groups oriented towards the cytoplasmic surface participate in Naþ uptake, while another group of acidic residues are required for Naþ release; however, there is no clear pathway for Naþ to bind through NQR. A further investigation into one of the residues required for Naþ uptake, NqrE-E95, has led to the discovery of one of the two predicted Naþ binding sites. After determining that this residue is essential for enzyme function, a closer look into the crystal structure revealed that NqrE-E95 is in the proper orientation to form a binding site with NqrE-D99 and NqrE-Y106. It was then found that these residues are also important for the utilization of Naþ by NQR. Further studies are being conducted to elucidate the exact role of NqrE-D99 and NqrE-Y106 in binding and transport of Naþ. Locating binding sites in NQR is the first step in understanding how Naþ is translocated, and ultimately how the translocation is coupled to the redox reactions. 3111-Pos Board B488 Characterizing Nanopore-Polymer Interactions and Cys-loop Protein Receptor Gating Nicholas B. Guros, Jeffery B. Klauda. Chemical and Biomolecular Engineering, University of Maryland, College Park, MD, USA. The first objective is to better understand how the size and charge of an analyte affect the flow of ions through a transmembrane nanopore to support the development of a bio-nanosensor that can measure the mass and identify of complex macromolecules such as DNA. To this end, molecular dynamics (MD) simulations were conducted on a transmembrane nanopore system composed of the b-barrel region of the transmembrane protein a-hemolysin (a-HL), a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer membrane, and an aqueous solution of potassium chloride (KCl), in which the b-barrel forms a channel through the membrane. After the system was allowed to equilibrate, a single molecule of polyethylene glycol (PEG) was added into the channel and a transmembrane potential was applied to one side of the membrane. The ionic current through the channel was measured, and compared to the measured current without a PEG molecule present. This work concluded that a system composed of only the bbarrel region of a-HL (not including the head region) can accurately depict the flow of ions through the nanopore in response to an applied transmembrane potential with greater computational ease than with the entire protein. The second objective is to better understand the response of the 5HT3 receptor to the ligand 5HT (serotonin) in an environment that mimics the surface of a proposed microelectromechanical system (MEMS) device designed to measure the presence of ligands such as neurotransmitters, neuropeptides and drug molecules. To this end, MD simulations were conducted on a system composed of the Cys-loop ligand-gated ion channel protein receptor 5HT3, a 7:7:6 ratio POPC/1-stearoyl-2-docosahexaenoyl-sn-glyerco-3-phosphocholine
Wednesday, March 2, 2016 (SDPC)/cholesterol membrane, and 5HT to determine if binding of the ligand causes protein reassembly and channel opening. 3112-Pos Board B489 Key Differences in Molecular Transport Mechanisms of Uncoupling Proteins Gabriel Macher1, Melanie Ko¨hler2, Anne Rupprecht1, Peter Hinterdorfer2, Elena Pohl1. 1 Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine, Vienna, Austria, 2Institute of Biophysics, Johannes Kepler University, Linz, Austria. Mitochondrial membrane uncoupling protein 1 (UCP1) facilitates proton leak to support non-shivering thermogenesis in brown adipose tissue. The transport function of other UCPs is controversially discussed. It was proposed that in addition to protons, other substrates can be transported. Moreover, the molecular mechanism of UCP regulation is insufficiently understood, although it is generally accepted that its transport is regulated by free fatty acids (FFA) and purine nucleotides (PN). Here we tested the hypothesis that regulation differs between members of UCP family leading to differences in their functions [1]. We evaluated binding forces and the degree of inhibition by PNs, comparing the data obtained using recognition force microscopy and electrophysiological measurements of recombinant proteins reconstituted in planar bilayer membranes [2]. We reveal that, in contrast to UCP1, other UCPs can be fully inhibited by all PNs, as KD increases with a decrease in phosphorylation. Furthermore, three arginines (R84, R183, R277) in the PN-binding pocket are involved in UCP1 inhibition to different extents. This result disagrees with previously proposed mechanisms, suggesting that only R277 is responsible for 100% inhibition. Moreover, FFAs can compete with all PNs bound to UCP1, but only with triphosphate-PNs bound to UCP3. Our results demonstrate the different regulation across a family of highly homologous uncoupling proteins, which, in the case of UCP1 and UCP3, are even expressed in the same tissue. We anticipate that the differences in the molecular mechanism of UCPs can be useful in understanding their physiological functions. [1] Rupprecht A, Sokolenko EA, Beck V, Ninnemann O, Jaburek M, et al. (2010) Biophys J; 98: 1503-1511. [2] Zhu R, Rupprecht A, Ebner A, Haselgrubler T, Gruber HJ, Hinterdorfer P, Pohl EE. (2013) J Am Chem Soc; 135:3640-3646. 3113-Pos Board B490 Water Pathway Analysis of Multi-Drug Efflux Transporter AcrB Tsutomu Yamane1, Ryotaro Koike2, Motonori Ota2, Satoshi Murakami3, Akinori Kidera1, Mitsunori Ikeguchi1. 1 Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan, 2Graduate School of Information Science, Nagoya University, Nagoya, Japan, 3Graduate School of Bioscience & Bioengineering, Tokyo Institute of Technology, Yokohama, Japan. The multi-drug efflux transporter AcrB exists in the inner membrane region of E. coli., and exports wide variety of noxious compounds using proton motive force as an energy source in Gram-negative bacteria. From the results of x-ray crystallography, the followings were found: (1) AcrB adopts asymmetric structure comprising three protomers with different structures, which correspond to access (A), binding (B) and extrusion (E) state of drugs, (2) three titratable residues (Asp407, Asp408 and Lys940), which locate in the middle of the transmembrane domain, form the protonation site (PS), and the Lys940 in E state adopts the different conformation from those of other states. These results suggest that AcrB exports drugs through the cyclic structural change among three states by using proton motive force generated by the change of protonation state in PS, which are called ‘‘functional rotation’’. We have studied the functional rotation mechanism of AcrB by using the Motion-tree method, which is a new procedure to describe the structural change as rigid body motions with a hierarchal manner. Our results have elucidated how convert the proton motive force generated from PS to the large structural change of AcrB. In the present study, to clarify the proton transfer process in the transmembrane domain, we searched the water pathways from the PS by using CAVER and 3D-RISM methods. As the results from CAVER method, A and B state protomers observed pathways from the PS to the periplasm side, and E state protomer observed a pathway from PS to the cytoplasm side. In addition, the results of 3DRISM method suggest that B and E state pathways have an ability to permeate water molecules. These results were consistent with the protonation states of each protomers estimated from structural change process from Motion-tree method.
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Computational Neuroscience 3114-Pos Board B491 Modeling the Oscillating Dipole Properties of Electric Organ Discharge in the Weakly Electric Fish, Eigenmannia Bela Joos1, Michael R. Markham2, Yanna Steimle1, John E. Lewis3, Morris E. Catherine4. 1 Physics, University of Ottawa, Ottawa, ON, Canada, 2Biology, University of Oklahoma, Norman, OK, USA, 3Biology, University of Ottawa, Ottawa, ON, Canada, 4Neuroscience, Ottawa Hospital Research Institute, Ottawa, ON, Canada. One of the most studied weakly electric fish, Eigenmannia produces a continuous high frequency electric organ discharge (EOD) used to sense nearby objects and communicate with conspecifics. The EOD of an individual fish has a characteristic frequency (within the species range, 250-600 Hz) which it shifts when necessary to avoid jamming. The nearly dipolar oscillating electric field yields zero net current and is generated by parallel columns of identical, synchronously discharging electrocyte cells. Recent findings from whole fish respirometry (during high-frequency signaling over a range of frequencies) have renewed interest in the frequency-dependent energetics of the EODs (Lewis et al 2014 J Neurosci 34:197) but the modeling based on past analyses is missing some key features of the in-vivo electrolyte operation. We have constructed a model for the neurally-driven electrocyte action potentials (APs) that underlie the EOD. APs are initiated by brief postsynaptic cation influxes through AChR channels at each electrocyte’s innervated posterior end. This yields a head positive current while the anterior end serves mainly as a capacitor whose discharge yields the head negative current of the oscillating dipole cycle. To maintain the appropriate [Na]in and [K]out levels, Na/K-ATPase pumps run continuously. Modeling the activity of this ATP consuming protein gives us access to electrocyte energetics and frequency-dependent EOD efficiency. Modeling the posterior-anterior current flows gives us access to the electric field patterns produced by the electrocytes. 3115-Pos Board B492 Resonances and Spectral Characteristics of a Neural Network for the Song Motor Pathway in Birds Cristiano Giordani1, Hector Fabio Rivera-Gutierrez1, Ruggero Micheletto2. 1 Universidad de Antioquia, Medellin, Colombia, 2Yokohama City University, Yokohama, Japan. Birdsong is a complex learned behavior regulated in an intricate way. Neuromuscular coordination of different muscular sets is necessary for producing high quality and consistent songs. We developed a realistic neural network that emulates neurons from the High Vocal centre (HVc) and the robust nucleus of the archistriatum (RA) neurons that drive the muscles to generate birdsong sounds. We used modern computational tools and neural architecture to simulate the entire motor pathway up to the physical oscillator muscle system and its spectral characteristics. Several network parameter dependences were analysed and elucidated. An optimal network size within 10 to 25 neurons within which minimal and smooth frequency variations occur, was found. Beyond that range we observe, instead, strong frequency dependence. Moreover, response frequency is influenced by the pathway input current; also in this case frequency response keeps smooth within a certain range of current, but shows interesting resonant values where negative peaks are observed. Resonant values are found in respect to the non-linear dissipation constant of the equation of motion for the bird’s labial oscillation and also in respect of the network background noise level (stochastic resonance). This work demonstrate that is possible to achieve a realistic computational model for the motor pathway leading to generation of sounds in birds, which contributes to the understanding of its spectral and dynamical fundamental properties and characteristics. 3116-Pos Board B493 Noise-Driven Synchronization of Coupled Neural Networks Anis Yuniati, Te-Lun Mai, Chi-Ming Chen. Department of Physics, National Taiwan Normal University, Taipei, Taiwan. The brain is complex network of dynamical system. For this reason, the interaction between different brain areas can be modeled as a large-scale network which is important in functional brain dynamics, and it closely related to the brain disorder. In recent years, the significant progress about the understanding of the relation between the structure and dynamical properties of the networks has developed. It describes that the complex dynamic behavior such as synchronization of coupled dynamical system plays a crucial role in a brain function and dysfunction. In our simulations, we construct a coupled neural
Wednesday, March 2, 2016
‘‘super-inhibitory’’ ternary complex (SERCA-SLN-PLB), whereby SERCA activity shows 5-fold decreased calcium affinity and 2-fold decreased maximal velocity. Fo¨rster resonance energy transfer (FRET) was used to quantify the complex equilibria of homo- and hetero-oligomeric interactions between SLN, PLB, and SERCA1a: five binary interactions were assayed and three assembly parameters were determined (binding affinity, oligomer number, interprobe distance). FRET results indicate that SLN and PLB show high-affinity self-association into homo-oligomers, and that SERCA forms heterodimers with SLN or PLB when co-expressed with either subunit individually, with SERCA having 3-fold higher affinity for SLN over PLB. To determine inhibition mechanisms of subunit-bound complexes, we used microsecond MD simulation of SERCA1a 5 SLN or PLB in a POPC bilayer, with calcium, proton, or magnesium ions bound to transport sites of the SERCA transmembrane domain. MD results indicate that two potassium ions initially bind and preorganize transport sites for calcium binding (SERCA activation), and that SLN and PLB individually inhibit SERCA by populating a calcium/ potassium-free intermediate state with one bound proton at Glu771 (transport site I). We propose that super-inhibition of SERCA in the ternary complex is mediated by SLN/PLB-synergistic inhibition of proton/potassium exchange. Acknowledgments: this work was funded by grants to DDT (NIH-GM27906) and MEF (AHA-12SDG12060656). 3108-Pos Board B485 In Vitro Demonstration of Light-Driven NaD/HD Pumping by a Microbial Rhodopsin Hai Li, Oleg A. Sineshchekov, Giordano F.Z. da Silva, John L. Spudich. University of Texas Medical School at Houston, Houston, TX, USA. A new subfamily of rhodopsins was discovered in bacteria and proposed to function as dual function light-driven Hþ/Naþ pumps, ejecting Naþ from cells in the presence of Naþ and Hþ in its absence (Inoue et al 2013. Nat. Commun. 4:1678). This proposal was based primarily on light-induced proton flux measurements in suspensions of E. coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the Naþ and Hþ transport definitively. We expressed IAR from Indibacter alkaliphilus in E. coli cells and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta reported earlier. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Naþ pumping activity in vitro, namely Naþ-dependent light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Naþ-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore, showing Hþ pumping activity. The direction of proton fluxes indicated that IAR was inserted inside-out into the sealed LUV system, which we confirmed by site-directed spin-label EPR spectroscopy. The in vitro LUV system proves that the dual light-driven Hþ/Naþ pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo. We are currently investigating ion fluxes through natural anion channelrhodopsins with this system. 3109-Pos Board B486 The Diversity of Light-Driven Ion Pumps and their Conversion into Ion Channels Arend Vogt, Christiane Grimm, Peter Hegemann. Experimental Biophysics, Humboldt Universita¨t zu Berlin, Berlin, Germany. Microbial rhodopsins are integral seven-transmembrane helix proteins, which covalently bind all-trans-retinal as light-sensitive chromophore. They play an extremely important role in neuroscience as optogenetic tools for the modulation of neuronal activity. These rhodopsins are subdivided into sensory rhodopsins, ion channels and ion pumps. Light-driven ion pumps transport protons, sodium or chloride across the plasma membrane against an electrochemical gradient. We analyzed a variety of ion pumps using two-microelectrode voltage-clamp measurements (TEVC) in Xenopus leavis oocytes. We found that all pumps show different electrophysiological behaviors. Especially light-driven proton pumps can be subdivided according to their characteristics at high electrochemical load, i.e. low extracellular pH and negative voltage. Photocurrents of Bacteriorhodopsin and the rhodopsin from the eukaryotic microalga Coccomyxa subellipsoidea (CsR) are always outward directed or inactivate at high load. In contrast, the rhodopsins from Exiguobacterium sibiricum (ESR) and from
the cyanobacterium Gloeobacter violaceus (GR) show inward directed photocurrents at high load. The well-established rhodopsin Arch3 from the archaeon Halorubrum sodomense shows weak inward directed photocurrents at high load. Most recently, the class of sodium pumps was discovered, but so far their underlying mechanism and ion selectivity is poorly understood. Here, we show the first more comprehensive electrophysiological characterization of the light-driven sodium-pumps in native membranes. They are characterized by an unexpected strong pH-dependency. In earlier work we have shown that proton pumps can be converted into operational proton channels via the introduction of selected mutations. Now, we transferred this approach to the sodium pumps and attained also inward directed photocurrents. This observation opens the opportunity to engineer new optogenetic tools with high ion-selectivity. 3110-Pos Board B487 Localization of a Sodium Binding Site in the Sodium Translocating NADH: Ubiquinone Oxidoreductase Katherine G. Mezic, Blanca Barquera. Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY, USA. Many marine, soil and pathogenic bacteria, including Vibrio cholerae, Pseudomonas aeruginosa, Yersinia pestis and Chlamydia trachomatis, possess a unique respiratory enzyme called Naþ-translocating NADH:quinone oxidoreductase (NQR). This enzyme is capable of pumping Naþ across the cell membrane, thus creating an electrochemical gradient that the cell can use for metabolic functions. NQR pumps Naþ by using the energy released by the oxidation-reduction (redox) reaction between NADH and ubiquinone. While the electron transfer reactions have been well characterized in this enzyme, the Naþ translocation mechanism is not yet well known. Several acid groups oriented towards the cytoplasmic surface participate in Naþ uptake, while another group of acidic residues are required for Naþ release; however, there is no clear pathway for Naþ to bind through NQR. A further investigation into one of the residues required for Naþ uptake, NqrE-E95, has led to the discovery of one of the two predicted Naþ binding sites. After determining that this residue is essential for enzyme function, a closer look into the crystal structure revealed that NqrE-E95 is in the proper orientation to form a binding site with NqrE-D99 and NqrE-Y106. It was then found that these residues are also important for the utilization of Naþ by NQR. Further studies are being conducted to elucidate the exact role of NqrE-D99 and NqrE-Y106 in binding and transport of Naþ. Locating binding sites in NQR is the first step in understanding how Naþ is translocated, and ultimately how the translocation is coupled to the redox reactions. 3111-Pos Board B488 Characterizing Nanopore-Polymer Interactions and Cys-loop Protein Receptor Gating Nicholas B. Guros, Jeffery B. Klauda. Chemical and Biomolecular Engineering, University of Maryland, College Park, MD, USA. The first objective is to better understand how the size and charge of an analyte affect the flow of ions through a transmembrane nanopore to support the development of a bio-nanosensor that can measure the mass and identify of complex macromolecules such as DNA. To this end, molecular dynamics (MD) simulations were conducted on a transmembrane nanopore system composed of the b-barrel region of the transmembrane protein a-hemolysin (a-HL), a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer membrane, and an aqueous solution of potassium chloride (KCl), in which the b-barrel forms a channel through the membrane. After the system was allowed to equilibrate, a single molecule of polyethylene glycol (PEG) was added into the channel and a transmembrane potential was applied to one side of the membrane. The ionic current through the channel was measured, and compared to the measured current without a PEG molecule present. This work concluded that a system composed of only the bbarrel region of a-HL (not including the head region) can accurately depict the flow of ions through the nanopore in response to an applied transmembrane potential with greater computational ease than with the entire protein. The second objective is to better understand the response of the 5HT3 receptor to the ligand 5HT (serotonin) in an environment that mimics the surface of a proposed microelectromechanical system (MEMS) device designed to measure the presence of ligands such as neurotransmitters, neuropeptides and drug molecules. To this end, MD simulations were conducted on a system composed of the Cys-loop ligand-gated ion channel protein receptor 5HT3, a 7:7:6 ratio POPC/1-stearoyl-2-docosahexaenoyl-sn-glyerco-3-phosphocholine
Wednesday, March 2, 2016 (SDPC)/cholesterol membrane, and 5HT to determine if binding of the ligand causes protein reassembly and channel opening. 3112-Pos Board B489 Key Differences in Molecular Transport Mechanisms of Uncoupling Proteins Gabriel Macher1, Melanie Ko¨hler2, Anne Rupprecht1, Peter Hinterdorfer2, Elena Pohl1. 1 Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine, Vienna, Austria, 2Institute of Biophysics, Johannes Kepler University, Linz, Austria. Mitochondrial membrane uncoupling protein 1 (UCP1) facilitates proton leak to support non-shivering thermogenesis in brown adipose tissue. The transport function of other UCPs is controversially discussed. It was proposed that in addition to protons, other substrates can be transported. Moreover, the molecular mechanism of UCP regulation is insufficiently understood, although it is generally accepted that its transport is regulated by free fatty acids (FFA) and purine nucleotides (PN). Here we tested the hypothesis that regulation differs between members of UCP family leading to differences in their functions [1]. We evaluated binding forces and the degree of inhibition by PNs, comparing the data obtained using recognition force microscopy and electrophysiological measurements of recombinant proteins reconstituted in planar bilayer membranes [2]. We reveal that, in contrast to UCP1, other UCPs can be fully inhibited by all PNs, as KD increases with a decrease in phosphorylation. Furthermore, three arginines (R84, R183, R277) in the PN-binding pocket are involved in UCP1 inhibition to different extents. This result disagrees with previously proposed mechanisms, suggesting that only R277 is responsible for 100% inhibition. Moreover, FFAs can compete with all PNs bound to UCP1, but only with triphosphate-PNs bound to UCP3. Our results demonstrate the different regulation across a family of highly homologous uncoupling proteins, which, in the case of UCP1 and UCP3, are even expressed in the same tissue. We anticipate that the differences in the molecular mechanism of UCPs can be useful in understanding their physiological functions. [1] Rupprecht A, Sokolenko EA, Beck V, Ninnemann O, Jaburek M, et al. (2010) Biophys J; 98: 1503-1511. [2] Zhu R, Rupprecht A, Ebner A, Haselgrubler T, Gruber HJ, Hinterdorfer P, Pohl EE. (2013) J Am Chem Soc; 135:3640-3646. 3113-Pos Board B490 Water Pathway Analysis of Multi-Drug Efflux Transporter AcrB Tsutomu Yamane1, Ryotaro Koike2, Motonori Ota2, Satoshi Murakami3, Akinori Kidera1, Mitsunori Ikeguchi1. 1 Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan, 2Graduate School of Information Science, Nagoya University, Nagoya, Japan, 3Graduate School of Bioscience & Bioengineering, Tokyo Institute of Technology, Yokohama, Japan. The multi-drug efflux transporter AcrB exists in the inner membrane region of E. coli., and exports wide variety of noxious compounds using proton motive force as an energy source in Gram-negative bacteria. From the results of x-ray crystallography, the followings were found: (1) AcrB adopts asymmetric structure comprising three protomers with different structures, which correspond to access (A), binding (B) and extrusion (E) state of drugs, (2) three titratable residues (Asp407, Asp408 and Lys940), which locate in the middle of the transmembrane domain, form the protonation site (PS), and the Lys940 in E state adopts the different conformation from those of other states. These results suggest that AcrB exports drugs through the cyclic structural change among three states by using proton motive force generated by the change of protonation state in PS, which are called ‘‘functional rotation’’. We have studied the functional rotation mechanism of AcrB by using the Motion-tree method, which is a new procedure to describe the structural change as rigid body motions with a hierarchal manner. Our results have elucidated how convert the proton motive force generated from PS to the large structural change of AcrB. In the present study, to clarify the proton transfer process in the transmembrane domain, we searched the water pathways from the PS by using CAVER and 3D-RISM methods. As the results from CAVER method, A and B state protomers observed pathways from the PS to the periplasm side, and E state protomer observed a pathway from PS to the cytoplasm side. In addition, the results of 3DRISM method suggest that B and E state pathways have an ability to permeate water molecules. These results were consistent with the protonation states of each protomers estimated from structural change process from Motion-tree method.
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Computational Neuroscience 3114-Pos Board B491 Modeling the Oscillating Dipole Properties of Electric Organ Discharge in the Weakly Electric Fish, Eigenmannia Bela Joos1, Michael R. Markham2, Yanna Steimle1, John E. Lewis3, Morris E. Catherine4. 1 Physics, University of Ottawa, Ottawa, ON, Canada, 2Biology, University of Oklahoma, Norman, OK, USA, 3Biology, University of Ottawa, Ottawa, ON, Canada, 4Neuroscience, Ottawa Hospital Research Institute, Ottawa, ON, Canada. One of the most studied weakly electric fish, Eigenmannia produces a continuous high frequency electric organ discharge (EOD) used to sense nearby objects and communicate with conspecifics. The EOD of an individual fish has a characteristic frequency (within the species range, 250-600 Hz) which it shifts when necessary to avoid jamming. The nearly dipolar oscillating electric field yields zero net current and is generated by parallel columns of identical, synchronously discharging electrocyte cells. Recent findings from whole fish respirometry (during high-frequency signaling over a range of frequencies) have renewed interest in the frequency-dependent energetics of the EODs (Lewis et al 2014 J Neurosci 34:197) but the modeling based on past analyses is missing some key features of the in-vivo electrolyte operation. We have constructed a model for the neurally-driven electrocyte action potentials (APs) that underlie the EOD. APs are initiated by brief postsynaptic cation influxes through AChR channels at each electrocyte’s innervated posterior end. This yields a head positive current while the anterior end serves mainly as a capacitor whose discharge yields the head negative current of the oscillating dipole cycle. To maintain the appropriate [Na]in and [K]out levels, Na/K-ATPase pumps run continuously. Modeling the activity of this ATP consuming protein gives us access to electrocyte energetics and frequency-dependent EOD efficiency. Modeling the posterior-anterior current flows gives us access to the electric field patterns produced by the electrocytes. 3115-Pos Board B492 Resonances and Spectral Characteristics of a Neural Network for the Song Motor Pathway in Birds Cristiano Giordani1, Hector Fabio Rivera-Gutierrez1, Ruggero Micheletto2. 1 Universidad de Antioquia, Medellin, Colombia, 2Yokohama City University, Yokohama, Japan. Birdsong is a complex learned behavior regulated in an intricate way. Neuromuscular coordination of different muscular sets is necessary for producing high quality and consistent songs. We developed a realistic neural network that emulates neurons from the High Vocal centre (HVc) and the robust nucleus of the archistriatum (RA) neurons that drive the muscles to generate birdsong sounds. We used modern computational tools and neural architecture to simulate the entire motor pathway up to the physical oscillator muscle system and its spectral characteristics. Several network parameter dependences were analysed and elucidated. An optimal network size within 10 to 25 neurons within which minimal and smooth frequency variations occur, was found. Beyond that range we observe, instead, strong frequency dependence. Moreover, response frequency is influenced by the pathway input current; also in this case frequency response keeps smooth within a certain range of current, but shows interesting resonant values where negative peaks are observed. Resonant values are found in respect to the non-linear dissipation constant of the equation of motion for the bird’s labial oscillation and also in respect of the network background noise level (stochastic resonance). This work demonstrate that is possible to achieve a realistic computational model for the motor pathway leading to generation of sounds in birds, which contributes to the understanding of its spectral and dynamical fundamental properties and characteristics. 3116-Pos Board B493 Noise-Driven Synchronization of Coupled Neural Networks Anis Yuniati, Te-Lun Mai, Chi-Ming Chen. Department of Physics, National Taiwan Normal University, Taipei, Taiwan. The brain is complex network of dynamical system. For this reason, the interaction between different brain areas can be modeled as a large-scale network which is important in functional brain dynamics, and it closely related to the brain disorder. In recent years, the significant progress about the understanding of the relation between the structure and dynamical properties of the networks has developed. It describes that the complex dynamic behavior such as synchronization of coupled dynamical system plays a crucial role in a brain function and dysfunction. In our simulations, we construct a coupled neural