- Email: [email protected]
Phospholipid Monolayers
522a
Wednesday, February 15, 2017
2571-Pos Board B178 Stereochemistry and Phase Behavior in Hydroxycholesterol/Phospholipid Monolayers Vision B. Bagonza1, Blair E. Stewig1, Caroline Wochnik1, Joan C. Kunz2, Benjamin L. Stottrup3. 1 Augsburg College, Minneapolis, MN, USA, 2Chemistry, Augsburg College, Minneapolis, MN, USA, 3Physics, Augsburg College, Minneapolis, MN, USA. Hydroxycholesterols are cholesterol analogs formed either enzymatically or non-enzyamatically to have a second hydroxyl function group. In this work we study compositions of 24(R), 24(S), and 27 hydroxycholesterol monolayers mixed with 1,2-Dimysristoyl-sn-glycero-3-phosphocholine (DMPC). These sterols induce liquid-liquid phase separation which share similarities and distinct differences from commonly studied lipid raft model compositions with cholesterol. Previous work has identified these differences and the role that the position of the second hydroxyl group plays in determining phase behavior. Less well studied is the observation that unlike other liquidliquid phase separated lipid monolayers, hydroxycholesterols domains display distinct nucleation and growth behavior. We use fluorescence microscopy and Langmuir isotherms to test how the observed thermodynamic behavior matches with theory. A recently developed method of measuring line tension using domain size distribution is applied and results are compared to well-studied cholesterol/DMPC compositions. We have previously identified the important role optical activity plays in the differences between 22(R) and 22(S) hydroxycholesterol phase behavior. We attempt to develop a general model for these differences and present the results from studies of 24(R) and 24(S). 2572-Pos Board B179 Surfactant pKa Calculations using Molecular Dynamics Simulations W.F. Drew Bennett, Joan-Emma Shea, Frank L. Brown. Chemistry, University of California, Santa Barbara, Santa Barbara, CA, USA. The protonation state for ionizable surfactants is crucial for self-assembly, phase behavior, and morphology. For example, oleic acid forms vesicles at intermediate pH conditions (~7-9), micelles at high pH, and oil phases at low pH. The pKa for oleic acid shifts from ~4.8 as monomers in water to ~7.5 in model phospholipid vesicles. It remains challenging to accurately model surfactant self-assembly and protonation behavior with molecular dynamics simulations. We use constant pH molecular dynamics simulations and thermodynamic integration calculations for protonating a single fatty acid in different chemical environments. Long time scale Martini coarsegrained simulations show the two methods produce very similar titration curves for oleic acid in micelles. We use CHARMM36 atomistic simulations to calculate titration curves for oleic acid in different micelle environments and bilayers. The pKa for oleic acid in a DOPC bilayer is 7.2, which compares well to experiments. Cholesterol content is shown to decrease the pKa for oleic acid to 5.9 compared to a pure DOPC bilayer. Our calculations provide insight into the mechanism of oleic acid self-assembly and the effect of the local chemical environment on its pKa. 2573-Pos Board B180 Membrane Permeability of Ascorbic Acid Christof Hannesschlaeger, Peter Pohl. Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria. Vitamin C (ascorbic acid, AscH) is an essential nutrient for humans. Published values of the passive membrane permeability to AscH range over several orders of magnitude and hence appear to be contradictory. Since the characterization of AscH-carriers requires knowledge of AscH’s background membrane permeability, we used scanning electrochemical microscopy for its determination. Addition of AscH to only one of the compartments separated by a free standing planar lipid bilayer resulted in a transmembrane flux of the neutral (protonated) form of the acid. In the receiving compartment, the acid dissociated, giving rise to an increase in proton concentration within the unstirred layers in the immediate membrane vicinity [1]. We monitored the local pH distribution by moving a custom built pH-sensitive micro-electrode in micrometer steps towards and away from the membrane. By numerically solving the system of reactiondiffusion-equations [2] we calculated the membrane permeability PAscH=10 8 cm/s from the experimental pH profiles. We used this value to validate a new assay that is based on time lapse measurements of scattered light intensity of a vesicle suspension during AscH efflux. The new label-free approach calculates membrane permeability based on changes of (i) vesicle size and (ii) refractive index of the vesicle interior. [1] Saparov et al. Biophys.l J. 90.11 (2006): L86-L88. [2] Antonenko et al. Biophys. J. 64.6 (1993): 1701.
2574-Pos Board B181 Hallmarks of Reversible Phase Separation in Living, Unperturbed Cell Membranes Scott Rayermann, Glennis Rayermann, Alex Merz, Sarah Keller. University of Washington, Seattle, WA, USA. Controversy has long surrounded the question of whether micron-scale lateral phase separation can organize proteins and lipids within the membranes of unperturbed living cells. A clear answer hinges on observation of hallmarks of a reversible phase transition. Here, by directly imaging micron-scale membrane domains of yeast vacuoles in vivo, we demonstrate that the domains arise through a phase separation mechanism. The domains disappear above a distinct miscibility transition temperature, Tmix, and reappear below Tmix over multiple heating and cooling cycles. The domains are large, have smooth boundaries, and can merge, consistent with fluid phases. Hence, large-scale membrane organization in living cells under physiologically relevant conditions can be controlled by tuning a single thermodynamic parameter. 2575-Pos Board B182 Small-Reservoir Electrostatics Joel A. Cohen. Physics, Univ of Massachusetts, Amherst, MA, USA. The solution to the Poisson-Boltzmann (PB) equation for the mean-field electrostatic potential and ion distributions surrounding an isolated charged particle generally employs a boundary condition of thermodynamic equilibrium with a reservoir whose properties are specified, such as its bulk ion concentrations. The reservoir, by definition infinitely large, provides the reference state for the system. However no experimental reservoir is truly infinite. Here we consider a single macroion suspended in an electrolyte equilibrated with a finite reservoir of small enough volume that establishment of the macroion’s screening cloud depletes the reservoir’s concentration of counterions and enhances its concentration of coions. Such a ‘‘reservoir’’ no longer provides a useful reference, as its state depends on the macroion charge. In a semi-confined volume, such as inside an ion channel, a large-enough and/or close-enough reservoir may not be available, in which case reservoir-depletion effects may occur. An extreme example is the colloidal crystal, where no bulk phase exists and there is no bulk ion reservoir at all. For this system the usual solution of the PB equation does not apply, as electrostatic potentials are nowhere constant, hence there is no bulk phase by which to establish reference ion concentrations. In such a closed system the conserved quantities are the total ion numbers rather than bulk ion concentrations. Reformulation of the PB solution referenced to total ion numbers yields an expression having the form of a FermiDirac distribution. Comparisons between PB solutions referenced to reservoir ion concentrations and PB solutions referenced to total ion numbers are illustrated and compared to experimental data for colloidal-crystalline liposome suspensions. 2576-Pos Board B183 The Pathway of Singlet Oxygen Diffusion through the Membrane Governs Whether Double Bonds or Aromatic Rings of a Molecule are Damaged Valery Sokolov1, Oleg Batischev1, Sergey Akimov1, Anna Gavrilchik1, Arsenij Shcherbakov1, Vsevolod Tashkin1, Denus Knyazev2, Peter Oihl2. 1 Frumkin Institute of Physical Chemistry and Electrochemistry RAS, Moscow, Russian Federation, 2Institute of Biophysics, Johannes Kepler University, Linz, Austria. Photosensitizers (PS) are used to treat cancers and various skin diseases. Upon illumination, PS generate reactive oxygen species, e.g. singlet oxygen (SO). In turn, SO targets double bonds and aromatic residues of proteins, lipids and DNA, thereby damaging the adjacent cells. Although SO lives long enough to diffuse about 100 nm in a lipid membrane, damage does not occur randomly. The mechanism that governs the susceptibility of different chemical moieties is not known. Here we adsorbed aluminum phthalocyanines to only one interface of planar lipid bilayers (Sokolov, V. S. and P. Pohl. 2009. BJ: 96:77-85). Photo-excited SO damaged the aromatic ring of membrane-embedded dipolar molecules (ANEPPS) in a concentrationdependent manner as reported by changes of the membrane boundary potential 4b (Sokolov, V. S., / Y. G. Gorbunova. 2016. J Photochem Photobiol B: 161:162-169). However, 4b changed at a higher rate when the target and PS were added to different sides of the bilayer instead of being added to the same interface. This observation suggests that part of the SO must have been quenched by ANEPPS’s double bonds which in the latter case were closer to the PS than the aromatic ring. We confirmed the model by using PS molecules that release SO at various penetration depths from the membrane surface due to differences in their number of sulfonate groups. In all cases,
Wednesday, February 15, 2017
2571-Pos Board B178 Stereochemistry and Phase Behavior in Hydroxycholesterol/Phospholipid Monolayers Vision B. Bagonza1, Blair E. Stewig1, Caroline Wochnik1, Joan C. Kunz2, Benjamin L. Stottrup3. 1 Augsburg College, Minneapolis, MN, USA, 2Chemistry, Augsburg College, Minneapolis, MN, USA, 3Physics, Augsburg College, Minneapolis, MN, USA. Hydroxycholesterols are cholesterol analogs formed either enzymatically or non-enzyamatically to have a second hydroxyl function group. In this work we study compositions of 24(R), 24(S), and 27 hydroxycholesterol monolayers mixed with 1,2-Dimysristoyl-sn-glycero-3-phosphocholine (DMPC). These sterols induce liquid-liquid phase separation which share similarities and distinct differences from commonly studied lipid raft model compositions with cholesterol. Previous work has identified these differences and the role that the position of the second hydroxyl group plays in determining phase behavior. Less well studied is the observation that unlike other liquidliquid phase separated lipid monolayers, hydroxycholesterols domains display distinct nucleation and growth behavior. We use fluorescence microscopy and Langmuir isotherms to test how the observed thermodynamic behavior matches with theory. A recently developed method of measuring line tension using domain size distribution is applied and results are compared to well-studied cholesterol/DMPC compositions. We have previously identified the important role optical activity plays in the differences between 22(R) and 22(S) hydroxycholesterol phase behavior. We attempt to develop a general model for these differences and present the results from studies of 24(R) and 24(S). 2572-Pos Board B179 Surfactant pKa Calculations using Molecular Dynamics Simulations W.F. Drew Bennett, Joan-Emma Shea, Frank L. Brown. Chemistry, University of California, Santa Barbara, Santa Barbara, CA, USA. The protonation state for ionizable surfactants is crucial for self-assembly, phase behavior, and morphology. For example, oleic acid forms vesicles at intermediate pH conditions (~7-9), micelles at high pH, and oil phases at low pH. The pKa for oleic acid shifts from ~4.8 as monomers in water to ~7.5 in model phospholipid vesicles. It remains challenging to accurately model surfactant self-assembly and protonation behavior with molecular dynamics simulations. We use constant pH molecular dynamics simulations and thermodynamic integration calculations for protonating a single fatty acid in different chemical environments. Long time scale Martini coarsegrained simulations show the two methods produce very similar titration curves for oleic acid in micelles. We use CHARMM36 atomistic simulations to calculate titration curves for oleic acid in different micelle environments and bilayers. The pKa for oleic acid in a DOPC bilayer is 7.2, which compares well to experiments. Cholesterol content is shown to decrease the pKa for oleic acid to 5.9 compared to a pure DOPC bilayer. Our calculations provide insight into the mechanism of oleic acid self-assembly and the effect of the local chemical environment on its pKa. 2573-Pos Board B180 Membrane Permeability of Ascorbic Acid Christof Hannesschlaeger, Peter Pohl. Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria. Vitamin C (ascorbic acid, AscH) is an essential nutrient for humans. Published values of the passive membrane permeability to AscH range over several orders of magnitude and hence appear to be contradictory. Since the characterization of AscH-carriers requires knowledge of AscH’s background membrane permeability, we used scanning electrochemical microscopy for its determination. Addition of AscH to only one of the compartments separated by a free standing planar lipid bilayer resulted in a transmembrane flux of the neutral (protonated) form of the acid. In the receiving compartment, the acid dissociated, giving rise to an increase in proton concentration within the unstirred layers in the immediate membrane vicinity [1]. We monitored the local pH distribution by moving a custom built pH-sensitive micro-electrode in micrometer steps towards and away from the membrane. By numerically solving the system of reactiondiffusion-equations [2] we calculated the membrane permeability PAscH=10 8 cm/s from the experimental pH profiles. We used this value to validate a new assay that is based on time lapse measurements of scattered light intensity of a vesicle suspension during AscH efflux. The new label-free approach calculates membrane permeability based on changes of (i) vesicle size and (ii) refractive index of the vesicle interior. [1] Saparov et al. Biophys.l J. 90.11 (2006): L86-L88. [2] Antonenko et al. Biophys. J. 64.6 (1993): 1701.
2574-Pos Board B181 Hallmarks of Reversible Phase Separation in Living, Unperturbed Cell Membranes Scott Rayermann, Glennis Rayermann, Alex Merz, Sarah Keller. University of Washington, Seattle, WA, USA. Controversy has long surrounded the question of whether micron-scale lateral phase separation can organize proteins and lipids within the membranes of unperturbed living cells. A clear answer hinges on observation of hallmarks of a reversible phase transition. Here, by directly imaging micron-scale membrane domains of yeast vacuoles in vivo, we demonstrate that the domains arise through a phase separation mechanism. The domains disappear above a distinct miscibility transition temperature, Tmix, and reappear below Tmix over multiple heating and cooling cycles. The domains are large, have smooth boundaries, and can merge, consistent with fluid phases. Hence, large-scale membrane organization in living cells under physiologically relevant conditions can be controlled by tuning a single thermodynamic parameter. 2575-Pos Board B182 Small-Reservoir Electrostatics Joel A. Cohen. Physics, Univ of Massachusetts, Amherst, MA, USA. The solution to the Poisson-Boltzmann (PB) equation for the mean-field electrostatic potential and ion distributions surrounding an isolated charged particle generally employs a boundary condition of thermodynamic equilibrium with a reservoir whose properties are specified, such as its bulk ion concentrations. The reservoir, by definition infinitely large, provides the reference state for the system. However no experimental reservoir is truly infinite. Here we consider a single macroion suspended in an electrolyte equilibrated with a finite reservoir of small enough volume that establishment of the macroion’s screening cloud depletes the reservoir’s concentration of counterions and enhances its concentration of coions. Such a ‘‘reservoir’’ no longer provides a useful reference, as its state depends on the macroion charge. In a semi-confined volume, such as inside an ion channel, a large-enough and/or close-enough reservoir may not be available, in which case reservoir-depletion effects may occur. An extreme example is the colloidal crystal, where no bulk phase exists and there is no bulk ion reservoir at all. For this system the usual solution of the PB equation does not apply, as electrostatic potentials are nowhere constant, hence there is no bulk phase by which to establish reference ion concentrations. In such a closed system the conserved quantities are the total ion numbers rather than bulk ion concentrations. Reformulation of the PB solution referenced to total ion numbers yields an expression having the form of a FermiDirac distribution. Comparisons between PB solutions referenced to reservoir ion concentrations and PB solutions referenced to total ion numbers are illustrated and compared to experimental data for colloidal-crystalline liposome suspensions. 2576-Pos Board B183 The Pathway of Singlet Oxygen Diffusion through the Membrane Governs Whether Double Bonds or Aromatic Rings of a Molecule are Damaged Valery Sokolov1, Oleg Batischev1, Sergey Akimov1, Anna Gavrilchik1, Arsenij Shcherbakov1, Vsevolod Tashkin1, Denus Knyazev2, Peter Oihl2. 1 Frumkin Institute of Physical Chemistry and Electrochemistry RAS, Moscow, Russian Federation, 2Institute of Biophysics, Johannes Kepler University, Linz, Austria. Photosensitizers (PS) are used to treat cancers and various skin diseases. Upon illumination, PS generate reactive oxygen species, e.g. singlet oxygen (SO). In turn, SO targets double bonds and aromatic residues of proteins, lipids and DNA, thereby damaging the adjacent cells. Although SO lives long enough to diffuse about 100 nm in a lipid membrane, damage does not occur randomly. The mechanism that governs the susceptibility of different chemical moieties is not known. Here we adsorbed aluminum phthalocyanines to only one interface of planar lipid bilayers (Sokolov, V. S. and P. Pohl. 2009. BJ: 96:77-85). Photo-excited SO damaged the aromatic ring of membrane-embedded dipolar molecules (ANEPPS) in a concentrationdependent manner as reported by changes of the membrane boundary potential 4b (Sokolov, V. S., / Y. G. Gorbunova. 2016. J Photochem Photobiol B: 161:162-169). However, 4b changed at a higher rate when the target and PS were added to different sides of the bilayer instead of being added to the same interface. This observation suggests that part of the SO must have been quenched by ANEPPS’s double bonds which in the latter case were closer to the PS than the aromatic ring. We confirmed the model by using PS molecules that release SO at various penetration depths from the membrane surface due to differences in their number of sulfonate groups. In all cases,