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Flip-Flop Promotion by Membrane-Spanning Sequences in the ER Membrane Proteins
Wednesday, March 2, 2016
Membrane Dynamics 2799-Pos Board B176 Flip-Flop Promotion by Membrane-Spanning Sequences in the ER Membrane Proteins Hiroyuki Nakao1, Keisuke Ikeda2, Yasushi Ishihama1, Minoru Nakano2. 1 Kyoto University, Kyoto, Japan, 2University of Toyama, Toyama, Japan. Phospholipid flip-flop in the endoplasmic reticulum (ER) is very rapid and non-specific to phospholipid headgroups, which is necessary to maintain lipid homeostasis in the membrane. Much research suggested that the rapid flip-flop in the ER is mediated by membrane proteins. However, ‘‘flippases’’, which are responsible for the flip-flop, have not been identified yet. We have previously demonstrated that peptide sequences that contain a hydrophilic residue in the transmembrane region are effective to promote the flipflop and that such sequences can be seen in the transmembrane region of ER proteins predicted by SOSUI. Therefore, we hypothesized that ER proteins with hydrophilic residues in the transmembrane region may promote the flip-flop. In this study, we synthesized peptides with membranespanning sequences of several ER proteins and investigated their flip-flop promotion abilities by using fluorescent-labeled phospholipids. We found that the sequences of EDEM1 and SPAST promoted the flip-flop of fluorescent lipids non-selectively with respect to the glycerophospholipid structure. These two peptides contain both positively charged Arg and noncharged His near the center of the sequences. Thus, we next examined how the flip-flop promotion abilities were altered by substitution of hydrophobic Ala for Arg or His. The Arg to Ala mutation inhibited the flip-flop promotion abilities of each peptides completely, while the His to Ala mutation did partly. These results suggested that both Arg and His are responsible for the flip-flop promotion by the peptides and Arg plays a vital role in the activities. Considering the high activity of the EDEM1 peptide observed at significantly low peptide concentrations, EDEM1 protein might act as a ‘‘flippase’’. 2800-Pos Board B177 Molecular Dynamics Simulations of Inter-Leaflet Dependence in Asymmetric Lipid Membranes Michael D. Weiner1, Gerald W. Feigenson2. 1 Field of Physics, Cornell University, Ithaca, NY, USA, 2Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA. Different lipid compositions are found on the cytosolic and exoplasmic leaflets of eukaryotic plasma membranes. We study the effects of this compositional asymmetry through Molecular Dynamics simulations. In particular, we consider the influence between a phosphatidylcholine-rich exoplasmic leaflet’s phase, either liquid ordered or liquid disordered, and a cytosolic leaflet either rich in phosphatidylethanolamine or rich in both phosphatidylserine and cholesterol. Changes to lipid order and lateral pressure are assessed. We examine the flip-flop of cholesterol between leaflets over the course of several microseconds by tracking the net movement of cholesterol due to compositional asymmetry. We consider the time scale for equilibration of cholesterol movement and the relative cholesterol concentrations in each leaflet. 2801-Pos Board B178 Controlling Membrane Dynamics by Tuning the Hydrophobic Mismatch and Lipid Compostion Butler D. Paul1,2, Elizabeth Kelley1, Rana Ashkar3, Robert Bradbury1,4, Andrea Woodka5, Michihiro Nagao1,4. 1 NIST, Gaithersburg, MD, USA, 2Chemical and Biomolecularl Engineering Dept, University of Delaware, Newark, DE, USA, 3Biology and Soft Matter division, Oak Ridge National Laboratory, Oak Ridge, TN, USA, 4Center for Exploration of Energy and Matter, Indiana University, Bloomington, IN, USA, 5West Point Military Academy, West Point, NY, USA. Lipid membranes undergo an array of conformational and dynamic transitions, ranging from individual lipid motions to undulations of micron-sized patches of the membrane. However, the dynamics at intermediate length scales are largely unexplored due to experimental challenges in accessing the appropriate length and time scales. Here we use neutron spin echo spectroscopy (NSE) to provide unique insights into these elusive dynamics and measure both bending and collective thickness fluctuations in model lipid bilayers. We build on our previous direct measurements of thickness fluctuations in single component lipid vesicles and extend the use of NSE to study more complex two component systems. We show that hydrophobic mismatch between lipids with different acyl chain lengths tunes the thickness fluctuation amplitude and relaxation time in a way not achievable in single component
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systems. In the mixed lipid systems, the fluctuation amplitude is enhanced in the fluid phase and reaches approximately 20% of the bilayer thickness, presumably due to an increase in the bilayer compressibility. Meanwhile the fluctuation relaxation time is comparable to the result for single component bilayers in the fluid phase but slows upon gelation of the longer-tailed lipid. Interestingly, our results suggest a decoupling of the fluctuation amplitude with the relaxation time, implying the potential for independent control over the fluctuation space and time domains and providing new insights into the role of lipid diversity in controlling the rich dynamics of biomembranes. 2802-Pos Board B179 Stochastic but Fine-Tuned: Dualism in Cell Membranes’ Organization as Revealed by Computer Simulations Roman G. Efremov. Lab. of Biomolecular Modeling, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation. Model lipid bilayers exhibit atomic-scale structural/dynamic properties, which help to understand complex macroscopic phenomena observed in membranes of living cells. These are: local phase separation, clustering, mosaicity of water-lipid interface, water dynamics, ability to bind external molecules (e.g., proteins). Atomistic simulations are invaluable in exploration of such effects, which still resist easy characterization in experiments. We used molecular dynamics simulations to assess physicochemical parameters of a large set of hydrated bilayers composed of lipids with different polar heads and acyl chains. Special attention was given to molecular mechanisms determining dynamic clustering of lipids and fine-tuned interplay of interactions between membrane components [1-3]. Putative relationships between lipid composition and local/integral characteristics of the bilayers were delineated. In particular, anomalous dependence on the lipid composition was observed for mixed bilayers composed of zwitterionic/anionic lipids. Also, the effects of lipid-water environment on membrane peptides and their interactions were investigated [4]. The results permit deciphering of the factors determining microscopic structural and dynamic ‘‘portrait’’ of lipid bilayers and assess he role of heterogeneous and highly fluctuating membrane medium in structural organization and functioning of proteins. This makes possible rational design of lipids with modified properties (e.g., capacity of H-bonding), which could presumably affect local/integral parameters of membranes. Our preliminary data show that such lipids induce significant changes of some crucial properties of model membranes. This opens new avenues in goal-oriented design of artificial membranes with engineered properties. Acknowledgements: Authors thank Russian Science Foundation (14-1400871), RFBR, RAS MCB Program. [1] D.V. Pyrkova et al., Soft Matter 7 (2011) 2569. [2] N.A. Krylov et al., ACS Nano 7 (2013) 9428. [3] A.O. Chugunov et al., Scientific Reports 4 (2014) 7462. [4] A.A. Polyansky et al., J. Amer. Chem. Soc. 134 (2012) 14390. 2803-Pos Board B180 Pre-Transition Effects Mediate Forces of Assembly between Transmembrane Proteins: The Orderphobic Effect Shachi Katira1, Kranthi K. Mandadapu1, Suriyanarayanan Vaikuntanathan2, Berend Smit3, David Chandler1. 1 Chemistry, University of California, Berkeley, Berkeley, CA, USA, 2 Chemistry, University of Chicago, Chicago, IL, USA, 3Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA. We present a mechanism for a generic and powerful force of assembly and mobility for transmembrane proteins in lipid bilayers. This force is a pre-transition (or pre-melting) effect for the first-order transition between ordered and disordered phases in the host membrane. Using large scale molecular simulation, we show that a protein with hydrophobic thickness equal to that of the disordered phase embedded in an ordered bilayer stabilizes a microscopic order–disorder interface, and the stiffness of that interface is finite. When two such proteins approach each other, they assemble because assembly reduces the net interfacial free energy. In analogy with the hydrophobic effect, we refer to this phenomenon as the ‘‘orderphobic effect.’’ The effect is mediated by proximity to the order-disorder phase transition and the size and hydrophobic mismatch of the protein. The strength and range of forces arising from the orderphobic effect are significantly larger than those that could arise from membrane elasticity for the membranes we examine.
Membrane Dynamics 2799-Pos Board B176 Flip-Flop Promotion by Membrane-Spanning Sequences in the ER Membrane Proteins Hiroyuki Nakao1, Keisuke Ikeda2, Yasushi Ishihama1, Minoru Nakano2. 1 Kyoto University, Kyoto, Japan, 2University of Toyama, Toyama, Japan. Phospholipid flip-flop in the endoplasmic reticulum (ER) is very rapid and non-specific to phospholipid headgroups, which is necessary to maintain lipid homeostasis in the membrane. Much research suggested that the rapid flip-flop in the ER is mediated by membrane proteins. However, ‘‘flippases’’, which are responsible for the flip-flop, have not been identified yet. We have previously demonstrated that peptide sequences that contain a hydrophilic residue in the transmembrane region are effective to promote the flipflop and that such sequences can be seen in the transmembrane region of ER proteins predicted by SOSUI. Therefore, we hypothesized that ER proteins with hydrophilic residues in the transmembrane region may promote the flip-flop. In this study, we synthesized peptides with membranespanning sequences of several ER proteins and investigated their flip-flop promotion abilities by using fluorescent-labeled phospholipids. We found that the sequences of EDEM1 and SPAST promoted the flip-flop of fluorescent lipids non-selectively with respect to the glycerophospholipid structure. These two peptides contain both positively charged Arg and noncharged His near the center of the sequences. Thus, we next examined how the flip-flop promotion abilities were altered by substitution of hydrophobic Ala for Arg or His. The Arg to Ala mutation inhibited the flip-flop promotion abilities of each peptides completely, while the His to Ala mutation did partly. These results suggested that both Arg and His are responsible for the flip-flop promotion by the peptides and Arg plays a vital role in the activities. Considering the high activity of the EDEM1 peptide observed at significantly low peptide concentrations, EDEM1 protein might act as a ‘‘flippase’’. 2800-Pos Board B177 Molecular Dynamics Simulations of Inter-Leaflet Dependence in Asymmetric Lipid Membranes Michael D. Weiner1, Gerald W. Feigenson2. 1 Field of Physics, Cornell University, Ithaca, NY, USA, 2Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA. Different lipid compositions are found on the cytosolic and exoplasmic leaflets of eukaryotic plasma membranes. We study the effects of this compositional asymmetry through Molecular Dynamics simulations. In particular, we consider the influence between a phosphatidylcholine-rich exoplasmic leaflet’s phase, either liquid ordered or liquid disordered, and a cytosolic leaflet either rich in phosphatidylethanolamine or rich in both phosphatidylserine and cholesterol. Changes to lipid order and lateral pressure are assessed. We examine the flip-flop of cholesterol between leaflets over the course of several microseconds by tracking the net movement of cholesterol due to compositional asymmetry. We consider the time scale for equilibration of cholesterol movement and the relative cholesterol concentrations in each leaflet. 2801-Pos Board B178 Controlling Membrane Dynamics by Tuning the Hydrophobic Mismatch and Lipid Compostion Butler D. Paul1,2, Elizabeth Kelley1, Rana Ashkar3, Robert Bradbury1,4, Andrea Woodka5, Michihiro Nagao1,4. 1 NIST, Gaithersburg, MD, USA, 2Chemical and Biomolecularl Engineering Dept, University of Delaware, Newark, DE, USA, 3Biology and Soft Matter division, Oak Ridge National Laboratory, Oak Ridge, TN, USA, 4Center for Exploration of Energy and Matter, Indiana University, Bloomington, IN, USA, 5West Point Military Academy, West Point, NY, USA. Lipid membranes undergo an array of conformational and dynamic transitions, ranging from individual lipid motions to undulations of micron-sized patches of the membrane. However, the dynamics at intermediate length scales are largely unexplored due to experimental challenges in accessing the appropriate length and time scales. Here we use neutron spin echo spectroscopy (NSE) to provide unique insights into these elusive dynamics and measure both bending and collective thickness fluctuations in model lipid bilayers. We build on our previous direct measurements of thickness fluctuations in single component lipid vesicles and extend the use of NSE to study more complex two component systems. We show that hydrophobic mismatch between lipids with different acyl chain lengths tunes the thickness fluctuation amplitude and relaxation time in a way not achievable in single component
567a
systems. In the mixed lipid systems, the fluctuation amplitude is enhanced in the fluid phase and reaches approximately 20% of the bilayer thickness, presumably due to an increase in the bilayer compressibility. Meanwhile the fluctuation relaxation time is comparable to the result for single component bilayers in the fluid phase but slows upon gelation of the longer-tailed lipid. Interestingly, our results suggest a decoupling of the fluctuation amplitude with the relaxation time, implying the potential for independent control over the fluctuation space and time domains and providing new insights into the role of lipid diversity in controlling the rich dynamics of biomembranes. 2802-Pos Board B179 Stochastic but Fine-Tuned: Dualism in Cell Membranes’ Organization as Revealed by Computer Simulations Roman G. Efremov. Lab. of Biomolecular Modeling, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation. Model lipid bilayers exhibit atomic-scale structural/dynamic properties, which help to understand complex macroscopic phenomena observed in membranes of living cells. These are: local phase separation, clustering, mosaicity of water-lipid interface, water dynamics, ability to bind external molecules (e.g., proteins). Atomistic simulations are invaluable in exploration of such effects, which still resist easy characterization in experiments. We used molecular dynamics simulations to assess physicochemical parameters of a large set of hydrated bilayers composed of lipids with different polar heads and acyl chains. Special attention was given to molecular mechanisms determining dynamic clustering of lipids and fine-tuned interplay of interactions between membrane components [1-3]. Putative relationships between lipid composition and local/integral characteristics of the bilayers were delineated. In particular, anomalous dependence on the lipid composition was observed for mixed bilayers composed of zwitterionic/anionic lipids. Also, the effects of lipid-water environment on membrane peptides and their interactions were investigated [4]. The results permit deciphering of the factors determining microscopic structural and dynamic ‘‘portrait’’ of lipid bilayers and assess he role of heterogeneous and highly fluctuating membrane medium in structural organization and functioning of proteins. This makes possible rational design of lipids with modified properties (e.g., capacity of H-bonding), which could presumably affect local/integral parameters of membranes. Our preliminary data show that such lipids induce significant changes of some crucial properties of model membranes. This opens new avenues in goal-oriented design of artificial membranes with engineered properties. Acknowledgements: Authors thank Russian Science Foundation (14-1400871), RFBR, RAS MCB Program. [1] D.V. Pyrkova et al., Soft Matter 7 (2011) 2569. [2] N.A. Krylov et al., ACS Nano 7 (2013) 9428. [3] A.O. Chugunov et al., Scientific Reports 4 (2014) 7462. [4] A.A. Polyansky et al., J. Amer. Chem. Soc. 134 (2012) 14390. 2803-Pos Board B180 Pre-Transition Effects Mediate Forces of Assembly between Transmembrane Proteins: The Orderphobic Effect Shachi Katira1, Kranthi K. Mandadapu1, Suriyanarayanan Vaikuntanathan2, Berend Smit3, David Chandler1. 1 Chemistry, University of California, Berkeley, Berkeley, CA, USA, 2 Chemistry, University of Chicago, Chicago, IL, USA, 3Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA. We present a mechanism for a generic and powerful force of assembly and mobility for transmembrane proteins in lipid bilayers. This force is a pre-transition (or pre-melting) effect for the first-order transition between ordered and disordered phases in the host membrane. Using large scale molecular simulation, we show that a protein with hydrophobic thickness equal to that of the disordered phase embedded in an ordered bilayer stabilizes a microscopic order–disorder interface, and the stiffness of that interface is finite. When two such proteins approach each other, they assemble because assembly reduces the net interfacial free energy. In analogy with the hydrophobic effect, we refer to this phenomenon as the ‘‘orderphobic effect.’’ The effect is mediated by proximity to the order-disorder phase transition and the size and hydrophobic mismatch of the protein. The strength and range of forces arising from the orderphobic effect are significantly larger than those that could arise from membrane elasticity for the membranes we examine.