Dynamics of Methyl Groups in Membrane Proteins Studied by Deterium Solid State NMR Relaxation

Sunday, February 28, 2016 the two GP wavelengths (2). Lifetime measurements can isolate the two effects measured by Laurdan GP: the polarity of the environment (reported mainly by the intensity at 440 nm) and the rate of dipolar relaxation of water molecules that can reorient around laurdan’s dipole during its lifetime (monitored at 490 nm) We used sucrose monoester of myristic acid, b-D-Fructofuranosyl-6-O-myristyl-a-D-Glucopyranoside (MMS) and Laurdan (6-lauroyl,1-2-dimethylamino naphthalene) to study the changes promoted in membrane heterogeneity in erythrocytes with different cholesterol content. We followed the hemolysis, the changes in size and the changes in fluidity on the membrane of erythrocytes treated with increasing amount of MMS. Our results indicate that the insertion of MMS molecules in the membrane produces a decrease in polarity of the bilayer (reported mainly by the intensity at 440 nm) together with an increase on dipolar relaxation of water molecules around Laurdan (reported at 490 nm). Financial support: Fondecyt #1140454 (S.S.), Fondecyt# 1120196 (G.G.) and Beca de Doctorado Conicyt 21120554 (C.S.). Technical Support: CMABioBio-UdeC. [1] Parasassi T, et al. Biophys J. 57; 1179, 1990. [2] Golfetto O, et al. Biophys J. 104, 1238-1247, 2013 381-Pos Board B161 Update on Mechanical Moduli and Tilt Theories of Lipid Bilayers John F. Nagle1, Dmitry I. Kopelevich2, Stephanie Tristram-Nagle1. 1 Physics, Carnegie Mellon University, Pittsburgh, PA, USA, 2Chemical Engineering, University of Florida, Gainesville, FL, USA. The classical Helfrich-Canham membrane continuum mechanics model is being fruitfully enriched by the inclusion of molecular tilt, even in the fluid phase of lipid bilayers. Different enrichment theories largely agree, but it has been noticed that there is considerable disagreement in one prediction; one theory postulates that the average length of the hydrocarbon chain tails increases strongly with increasing tilt and another predicts no increase. Our analysis of an all-atom simulation favors the latter theory, but it also shows that the overall tail length decreases slightly with increasing tilt. We show that this deviation from continuum theory can be reconciled by consideration of the average shape of the tails, which is a descriptor not obviously includable in continuum theory. The inclusion of tilt in the analysis of our diffuse xray data increases our values of the bending modulus by roughly 20%. A recent concern about the bending modulus is that it may depend strongly on the inclusion of sugars used in various other experimental methods. However, we find negligible effect of fructose, glucose, sucrose, trehalose, and maltose on our x-ray determination of the bending modulus or the tilt modulus of DOPC, SOPC, POPC and DMPC. 382-Pos Board B162 Flexible String Model Analytical Description of Main Phase Transition in Lipid Bilayers Sergei I. Mukhin, Boris B. Kheyfets, Timur R. Galimzyanov. Theoretical Physics and Quantum Technologies, Moscow Institute for Steel and Alloys NUST ‘‘MISIS’’, Moscow, Russian Federation. Flexible string model [1,2,3] expanded with van der Waals attraction [4]: VdW= -g/a^{5/2}, between hydrophobic chains, occupying each an average area a (in dimensionless units) in bilayer plane, is shown to provide analytical description of the main phase transition in lipid bilayers. Single lipid is modeled as a flexible string with a given bending rigidity and a finite incompressible area. Entropic repulsion between fluctuating lipid tails is represented by a parabolic potential, its stiffness parameter, b, is found self-consistenly: vF/vb = n$(Oa-1)2, where n1 is small parameter of the theory. The free energy with added van der between adjacent hydrocarbon P Waals interaction chains: F = log(b) þ log(1 þ b/c$4) þ VdW. Here b is dimensionless entropic repulsion parameter, and c coefficients are characteristics of energies of lipid’s elementary modes. The theory, being microscopic, allows to study analytically an evolution of membrane properties in the vicinity of the main transition such as temperature dependence of the lateral lipid self-diffusion coefficient. It is also possible to study thermodynamic fluctuations of the lateral pressure near phase transition temperature in the random-phase approximation. [1] S.Mukhin, S.Baoukina. 2005. Analytical derivation of thermodynamic characteristics of lipid bilayer from a flexible string model. Phys. Rev. E. 71: 061918. [2] S. Mukhin, B. Kheyfets. 2010. Analytical Approach to Thermodynamics of Bolalipid Membranes. Phys. Rev. E. 82: 051901. [3] S. Mukhin, B. Kheyfets. 2014. Pore formation phase diagrams for lipid membranes. JETP Lett. 99: 358-362. [4] L. Salem. 1962. Attractive Forces between Long Saturated Chains at Short Distances. J. Chem. Phys. 37: 2100-2113.

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383-Pos Board B163 Dynamics of Methyl Groups in Membrane Proteins Studied by Deterium Solid State NMR Relaxation Xiaolin Xu1, Andrey V. Struts2,3, Aswini Kumar Giri2, Trivikram R. Molugu2, Charitha Guruge4, Samira Faylough4, Carolina L. Nascimento4, Nasri Nesnas4, Victor J. Hruby2, Michael F. Brown1,2. 1 Department of Physics, University of Arizona, Tucson, AZ, USA, 2 Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA, 3Laboratory of Biomolecular NMR, St. Petersburg State University, St. Petersburg, Russian Federation, 4Department of Chemistry, Florida Institute of Technology, Melbourne, FL, USA. NMR relaxation is a valuable tool to study membrane protein dynamics which are important for their functions. Dynamical parameters (correlation times and activation barriers) can provide important information about intraand intermolecular interactions. Here we present our study of the dynamics of the rhodopsin-bound cofactor retinal and a high-affinity transducin peptide (GaCT2) bound to opsin. To observe how the NMR relaxation and molecular dynamics depend on the local environment, we introduced a deuterated methyl group at positions C5, C9, or C13 of retinal [1], and in GaCT2 (ILENLKDVGLF) at leucine positions 2, 5, and 10. Selectively labeled retinals were studied in the dark, preactive Meta-I, and active Meta-II states of rhodopsin. High-affinity peptides were studied as the dry powder, in water, and bound to the opsin apoprotein. We measured deuterium NMR relaxation rates of Zeeman and quadrupolar order (T1Z and T1Q) in a wide temperature range from 15 to 120  C. The temperature dependences were fitted using two approaches: first, a model-free approach [2] and second by utilizing various models of molecular motion (3-site jump and rotational diffusion models). Jump rates (k) and rotational diffusion coefficients (corresponding to methyl group spinning and reorientation) showed site-specific methyl differences [1]. Plots of relaxation rates versus temperature for both retinal and the high-affinity GaCT2 peptide indicated that methyl group dynamics are mostly determined by intramolecular interactions. The protein environment can also affect the dynamics, either directly or through structural changes (e.g. changes of the torsion angle between the beta-ionone ring and the polyene chain) as shown by relaxation measurements. [1] A.V. Struts et al. (2011) PNAS 108, 8263. [2] X. Xu et al. (2014) eMagRes 3, 275-286. 384-Pos Board B164 Detection and Mechanical Characterization of Small Multilamellar Vesicles using Atomic Force Microscopy Margherita Marchetti, Daan Vorselen, Wouter Roos, Gijs Wuite. VU University Amsterdam, Amsterdam, Netherlands. Multilamellar vesicles (MLVs) have beneficial properties over currently used unilamellar vesicles (LUVs) for drug delivery, such as more hydrophobic volume for hydrophobic drugs. Moreover, they may have altered mechanical properties, which are suggested to influence cellular uptake. We investigated the mechanics of small MLVs using atomic force microscopy (AFM). We quantified the mechanical response of single liposomes by AFM nano-indentation. Analyzing the total distance of breaks in force-indentation curves we were able to determine the degree of lamellarity (1-5) of individual vesicles. This allowed us to characterize the influence of multilamellarity on morphological and mechanical properties. We found that MLVs, upon adhesion with a surface, stay in a more spherical shape respect to ULVs. Furthermore, the stiffness increases linearly with the addition of each lipid bilayer (0.0027 N/m added stiffness). We speculate that the added osmotic pressure, due to the inner and less-deformed vesicles, together with their bending, cause the observed stiffening. These results suggest that small MLVs might have beneficial properties for cellular uptake. 385-Pos Board B165 Line Tension and Phase Separation of a Four-Component Phospholipid Bilayer Wen-Chyan Tsai, Gerald W. Feigenson. Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA. When phospholipid bilayers have coexisting liquid disordered (Ld) and liquid ordered (Lo) phases, Ld/Lo line tension has strong control over the size of the coexisting liquid domains. In order to minimize light-induced artifacts that involve lipid reactivity, we chose a lipid mixture with minimal double bonds, DSPC, diphytanoyl-PC, DLPC, and cholesterol. Giant unilamellar vesicles (GUVs) were generated using the electroswelling technique