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Probing the Ripple Phase of Lipid Bilayers using Molecular Simulations
86a
Sunday, February 28, 2016
441-Pos Board B221 Improved Methods for Preparing Asymmetric Vesicles using MethylAlpha-Cyclodextrin Johnna St Clair, Qing Wang, Erwin London. Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA. Many living cell membranes display lipid asymmetry, with a distinct difference between the phospholipids of the inner and outer leaflets of the lipid bilayer. Although artificial lipid vesicles have long been an important tool to study biological membranes, they are limited due to their lack of lipid asymmetry. We have recently developed methods to prepare asymmetric lipid vesicles using cyclodextrins, and improved methods using alphacyclodextrins. These hexasaccharide rings can promote the exchange of lipids between vesicles, but do not transport sterols. This allows the efficient and selective replacement of the outer leaflet lipids of lipid vesicles while at the same time tightly controlling sterol content. As a result, asymmetric vesicles can now be constructed using a range of lipids which closely mimic the natural asymmetry and content of mammalian plasma membranes. Here we show that methyl-alpha-cyclodextrin is an efficient tool for constructing asymmetric vesicles with asymmetric phospholipid distribution closely resembling that of natural mammalian plasma membranes. Progress in using methyl-alphacyclodextrin to prepare ‘‘inside-out’’ asymmetric vesicles, in which the lipids that normally face the cytosolic side of plasma membranes (PE and PS) are in the outer leaflet while those normally on the extra-cellular leaflet (SM and PC) are in the inner leaflet, and in developing improved assays for lipid asymmetry, will also be described. 442-Pos Board B222 Study of Self-Association of Amphotericin B and its Synthetic Derivatives using UV-Vis Spectroscopy Rosmarbel Morales-Nava1, Arturo Galva´n-Herna´ndez2, Mario Ferna´ndez-Zertuche3, Ivan Ortega-Blake2. 1 Facultad De Ciencias Quı´micas, Beneme´rita Universidad Auto´noma De Puebla, Puebla, Puebla, Mexico, 2Instituto De Ciencias Fisicas, Universidad Nacional Autonoma De Me´xico, Cuernavaca, Mexico, 3Centro De Investigaciones Quı´micas, Universidad Auto´noma Del Estado De Morelos, Cuernavaca, Mexico. Polyene antibiotics are the principal therapeutic agents as antimycotics drugs. Mainly, Amphotericin B (AmB) is the most potent known antimycotic and has been used for a long time, though its use is limited by its large collateral toxicity. Due to this there have been many attempts to produce AmB derivatives with a reduced collateral damage (US Patent Application 20090186838; J. Med. Chem. 2009, 52, 189-196; J. Am. Chem. Soc. 2013, 135, 8488-8491). In this work the spectroscopic properties of AmB and its derivatives were determined by the presence of the heptaenic chromophore and the self-association between monomeric and dimeric forms of polyenes in solutions has been characterized (Biochimica et Biophysica Acta 1528, 2001, 15-24). In our group, we have been testing the idea advanced by Huang et al., that the self-association of polyenes is a component in the mechanism that determines the difference in activity on membranes with cholesterol or ergosterol. Here, we are studying AmB derivatives self-association compared with that presented by AmB using UV-Vis spectroscopy absorption spectra in PBS solutions. To know the threshold where dimeric forms start to appear different concentrations of polyenes were used to determine the ratio between the bands at 409 nm (monomeric form), and 347 nm (aggregated form). Plotting this absorbance ratio as a function of the polyene concentration we determine the threshold and compare these values with the activity that derivatives present in single channel studies of ergosterol or cholesterol containing membranes, henceforth testing the hypothesis. 443-Pos Board B223 Determining the Pivotal Plane of Fluid Lipid Membranes in Simulations Xin Wang, Markus Deserno. Physics, Carnegie Mellon University, Pittsburgh, PA, USA. Each leaflet of a curved lipid membrane contains a surface at which the area strain vanishes, the so-called pivotal plane. Its distance z0 from the bilayer’s mid-plane arises in numerous membrane structure related contexts, for instance the connection between monolayer and bilayer moduli, stress-profile moments, or area-difference elasticity theories. Experimentally, the pivotal plane is known to lie close to the glycerol backbone of a lipid, but only very few simulations have tried to locate it. Here we propose two precise methods for determining z0, both of which rely on monitoring the lipid imbalance across a curved bilayer. The first method considers the ratio of lipid number between the two leaflets of cylindrical or spherical vesicles and requires lipid flip-flop for equil-
ibration. The second method looks at the leaflet difference across local sections cut out from a buckled membrane and is free of this limitation. Our simulations rely on two different coarse-grained lipid models. First, the generic three-bead solvent-free Cooke model, which is amenable to both methods and gives results for z0 that agree at the percent level. And second, a ten-bead representation of dimyristoylphosphocholine (DMPC) with the explicit solvent MARTINI model, which can only be analyzed with the buckling method. For the latter, the obtained value z0 = 0.850(11) nm lies about 0.4 nm inwards of the glycerol backbone, essentially in the middle of the leaflet, and is hence unexpectedly small. We attribute this to limitations of the coarse-grained description, suggesting that the location of the pivotal plane might be a novel indicator for how well lipid models capture the microscopic origins of curvature elasticity, especially the relative contributions of head and tail regions to the overall curvature modulus. 444-Pos Board B224 Lo/Ld Phase Coexistence and Interaction in Model Membranes with IPC Lipids Viviana Monje-Galvan, Jeffery B. Klauda. Chemical and Biomolecular Engineering, University of Maryland, College Park, MD, USA. New computational resources, such as Anton, have allowed the study of Lo/Ld phase coexistence in equimolar mixtures of inositol phosphoceramide (IPC), ergosterol (ERG), and phosphatidylinositol (PI) or phosphatidylcholine (PC) lipids. These models are preliminary studies of representative models for the trans-Golgi network and plasma membrane of yeast (Saccharomyces cerevisiae). Our past research on yeast membrane models lacked IPC lipids because at the time ceramide lipids were not fully parameterized for the CHARMM36 lipid force field. Such parameters have been published for sphingolipids (BJ, 107:134-145) and we have extended their use for various ceramides. Phase segregation was shown in IPC/PI/ERG mixtures obtained from separated total lipid extracts of yeast membranes (JBC, 285:30224-30232). Moreover, IPC/PI/ERG had more order than IPC/PC/ ERG mixtures, but the reasoning for this is not known. We present the results of two 6ms trajectories of all-atom simulations for each ternary system to probe the formation of lipid domains as well as the increase in order effect of PI vs. PC lipids. Our studies expand on the work of Sodt et al. (JACS, 136:725-732) from simple model systems (DPPC/DOPC/cholesterol) to understanding the effects of head group on the phase behavior and longchained IPC lipids on potential interleaflet coupling (BJ, 103:2311-2319). We examined lipid diffusion time scales, deuterium order parameters, electron density profiles, lipid head group interaction mechanisms such as hydrogen bonding, and sterol flip-flop timescales. 445-Pos Board B225 Probing the Ripple Phase of Lipid Bilayers using Molecular Simulations Pouyan Khakbaz, Jeffery Klauda. University of maryland college park, College Park, MD, USA. Lipid bilayers without sterols can exist in the gel, ripple, and liquidcrystalline phase depending on temperature. The ripple phase is the mysterious transition phase between gel and liquid-crystalline phases. For this work, a set of molecular dynamics (MD) simulations were carried out for pure 1,2-dimyristoyl-3-sn-phosphatidylcholines (DMPC), pure 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC), and their mixture at different temperatures. For some mixture and pure DMPC conditions, simulations were also performed in larger system size (144 lipids for each leaflet), which was four times bigger than small system (36 lipids for each leaflet), to determine its influence on ripple phase structure. Simulations were performed for 300ns, and the CHARMM 36 force field was used (J. Phys. Chem. B., 114, 7830-7843). For pure DMPC and DPCC (small system), ripple phase was obtained at 275 and 293K, respectively. For small system size of the DMPC-DPPC mixture, which had %75 DMPC, the ripple phase was obtained at slightly higher temperature at 298K. Ripple conformations were fairly stable at the last 100 ns of simulations. Interestingly, for large system of pure DMPC simulation, ripple phase was obtained at 291K, which is the temperature that ripple phase was observed based on X-ray scattering technique (Soft Matter, 11, 918-926). In case of DMPC-DPPC mixture with large system size, ripple phase was still obtained in a fairly stable conformation. Moreover, in large system set up for both pure DMPC and DMPC-DPPC mixture, the ripple length was increased, which is more in agreement with experiment. Form factor from simulations will be compared with 2D X-ray form factors from experiment (Soft Matter, 11, 918-926). In overall, CHARMM 36 force field can predict the ripple phase within reasonable range of temperatures and provide interpretation of experimental form factors.
Sunday, February 28, 2016
441-Pos Board B221 Improved Methods for Preparing Asymmetric Vesicles using MethylAlpha-Cyclodextrin Johnna St Clair, Qing Wang, Erwin London. Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA. Many living cell membranes display lipid asymmetry, with a distinct difference between the phospholipids of the inner and outer leaflets of the lipid bilayer. Although artificial lipid vesicles have long been an important tool to study biological membranes, they are limited due to their lack of lipid asymmetry. We have recently developed methods to prepare asymmetric lipid vesicles using cyclodextrins, and improved methods using alphacyclodextrins. These hexasaccharide rings can promote the exchange of lipids between vesicles, but do not transport sterols. This allows the efficient and selective replacement of the outer leaflet lipids of lipid vesicles while at the same time tightly controlling sterol content. As a result, asymmetric vesicles can now be constructed using a range of lipids which closely mimic the natural asymmetry and content of mammalian plasma membranes. Here we show that methyl-alpha-cyclodextrin is an efficient tool for constructing asymmetric vesicles with asymmetric phospholipid distribution closely resembling that of natural mammalian plasma membranes. Progress in using methyl-alphacyclodextrin to prepare ‘‘inside-out’’ asymmetric vesicles, in which the lipids that normally face the cytosolic side of plasma membranes (PE and PS) are in the outer leaflet while those normally on the extra-cellular leaflet (SM and PC) are in the inner leaflet, and in developing improved assays for lipid asymmetry, will also be described. 442-Pos Board B222 Study of Self-Association of Amphotericin B and its Synthetic Derivatives using UV-Vis Spectroscopy Rosmarbel Morales-Nava1, Arturo Galva´n-Herna´ndez2, Mario Ferna´ndez-Zertuche3, Ivan Ortega-Blake2. 1 Facultad De Ciencias Quı´micas, Beneme´rita Universidad Auto´noma De Puebla, Puebla, Puebla, Mexico, 2Instituto De Ciencias Fisicas, Universidad Nacional Autonoma De Me´xico, Cuernavaca, Mexico, 3Centro De Investigaciones Quı´micas, Universidad Auto´noma Del Estado De Morelos, Cuernavaca, Mexico. Polyene antibiotics are the principal therapeutic agents as antimycotics drugs. Mainly, Amphotericin B (AmB) is the most potent known antimycotic and has been used for a long time, though its use is limited by its large collateral toxicity. Due to this there have been many attempts to produce AmB derivatives with a reduced collateral damage (US Patent Application 20090186838; J. Med. Chem. 2009, 52, 189-196; J. Am. Chem. Soc. 2013, 135, 8488-8491). In this work the spectroscopic properties of AmB and its derivatives were determined by the presence of the heptaenic chromophore and the self-association between monomeric and dimeric forms of polyenes in solutions has been characterized (Biochimica et Biophysica Acta 1528, 2001, 15-24). In our group, we have been testing the idea advanced by Huang et al., that the self-association of polyenes is a component in the mechanism that determines the difference in activity on membranes with cholesterol or ergosterol. Here, we are studying AmB derivatives self-association compared with that presented by AmB using UV-Vis spectroscopy absorption spectra in PBS solutions. To know the threshold where dimeric forms start to appear different concentrations of polyenes were used to determine the ratio between the bands at 409 nm (monomeric form), and 347 nm (aggregated form). Plotting this absorbance ratio as a function of the polyene concentration we determine the threshold and compare these values with the activity that derivatives present in single channel studies of ergosterol or cholesterol containing membranes, henceforth testing the hypothesis. 443-Pos Board B223 Determining the Pivotal Plane of Fluid Lipid Membranes in Simulations Xin Wang, Markus Deserno. Physics, Carnegie Mellon University, Pittsburgh, PA, USA. Each leaflet of a curved lipid membrane contains a surface at which the area strain vanishes, the so-called pivotal plane. Its distance z0 from the bilayer’s mid-plane arises in numerous membrane structure related contexts, for instance the connection between monolayer and bilayer moduli, stress-profile moments, or area-difference elasticity theories. Experimentally, the pivotal plane is known to lie close to the glycerol backbone of a lipid, but only very few simulations have tried to locate it. Here we propose two precise methods for determining z0, both of which rely on monitoring the lipid imbalance across a curved bilayer. The first method considers the ratio of lipid number between the two leaflets of cylindrical or spherical vesicles and requires lipid flip-flop for equil-
ibration. The second method looks at the leaflet difference across local sections cut out from a buckled membrane and is free of this limitation. Our simulations rely on two different coarse-grained lipid models. First, the generic three-bead solvent-free Cooke model, which is amenable to both methods and gives results for z0 that agree at the percent level. And second, a ten-bead representation of dimyristoylphosphocholine (DMPC) with the explicit solvent MARTINI model, which can only be analyzed with the buckling method. For the latter, the obtained value z0 = 0.850(11) nm lies about 0.4 nm inwards of the glycerol backbone, essentially in the middle of the leaflet, and is hence unexpectedly small. We attribute this to limitations of the coarse-grained description, suggesting that the location of the pivotal plane might be a novel indicator for how well lipid models capture the microscopic origins of curvature elasticity, especially the relative contributions of head and tail regions to the overall curvature modulus. 444-Pos Board B224 Lo/Ld Phase Coexistence and Interaction in Model Membranes with IPC Lipids Viviana Monje-Galvan, Jeffery B. Klauda. Chemical and Biomolecular Engineering, University of Maryland, College Park, MD, USA. New computational resources, such as Anton, have allowed the study of Lo/Ld phase coexistence in equimolar mixtures of inositol phosphoceramide (IPC), ergosterol (ERG), and phosphatidylinositol (PI) or phosphatidylcholine (PC) lipids. These models are preliminary studies of representative models for the trans-Golgi network and plasma membrane of yeast (Saccharomyces cerevisiae). Our past research on yeast membrane models lacked IPC lipids because at the time ceramide lipids were not fully parameterized for the CHARMM36 lipid force field. Such parameters have been published for sphingolipids (BJ, 107:134-145) and we have extended their use for various ceramides. Phase segregation was shown in IPC/PI/ERG mixtures obtained from separated total lipid extracts of yeast membranes (JBC, 285:30224-30232). Moreover, IPC/PI/ERG had more order than IPC/PC/ ERG mixtures, but the reasoning for this is not known. We present the results of two 6ms trajectories of all-atom simulations for each ternary system to probe the formation of lipid domains as well as the increase in order effect of PI vs. PC lipids. Our studies expand on the work of Sodt et al. (JACS, 136:725-732) from simple model systems (DPPC/DOPC/cholesterol) to understanding the effects of head group on the phase behavior and longchained IPC lipids on potential interleaflet coupling (BJ, 103:2311-2319). We examined lipid diffusion time scales, deuterium order parameters, electron density profiles, lipid head group interaction mechanisms such as hydrogen bonding, and sterol flip-flop timescales. 445-Pos Board B225 Probing the Ripple Phase of Lipid Bilayers using Molecular Simulations Pouyan Khakbaz, Jeffery Klauda. University of maryland college park, College Park, MD, USA. Lipid bilayers without sterols can exist in the gel, ripple, and liquidcrystalline phase depending on temperature. The ripple phase is the mysterious transition phase between gel and liquid-crystalline phases. For this work, a set of molecular dynamics (MD) simulations were carried out for pure 1,2-dimyristoyl-3-sn-phosphatidylcholines (DMPC), pure 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC), and their mixture at different temperatures. For some mixture and pure DMPC conditions, simulations were also performed in larger system size (144 lipids for each leaflet), which was four times bigger than small system (36 lipids for each leaflet), to determine its influence on ripple phase structure. Simulations were performed for 300ns, and the CHARMM 36 force field was used (J. Phys. Chem. B., 114, 7830-7843). For pure DMPC and DPCC (small system), ripple phase was obtained at 275 and 293K, respectively. For small system size of the DMPC-DPPC mixture, which had %75 DMPC, the ripple phase was obtained at slightly higher temperature at 298K. Ripple conformations were fairly stable at the last 100 ns of simulations. Interestingly, for large system of pure DMPC simulation, ripple phase was obtained at 291K, which is the temperature that ripple phase was observed based on X-ray scattering technique (Soft Matter, 11, 918-926). In case of DMPC-DPPC mixture with large system size, ripple phase was still obtained in a fairly stable conformation. Moreover, in large system set up for both pure DMPC and DMPC-DPPC mixture, the ripple length was increased, which is more in agreement with experiment. Form factor from simulations will be compared with 2D X-ray form factors from experiment (Soft Matter, 11, 918-926). In overall, CHARMM 36 force field can predict the ripple phase within reasonable range of temperatures and provide interpretation of experimental form factors.