NMR Investigations of Cell Membrane Structure

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The objectives of this project are to: (1) study structure and dynamics of membranes composed of lipids with polyunsaturated fatty acids such as docosahexaenoic acid (DHA) 22:6n-3, (2) study the interaction of the polyunsaturated lipid matrix with G-protein coupled membrane receptors (GPCR) and (3) investigate structure and function of selected GPCR with relevance for alcoholism in reconstituted membrane systems. (1) Our recent experiments indicate that polyunsaturated fatty acids (PUFA) are highly flexible molecules existing in a multitude of conformations with rapid conformational transitions. The mobility of PUFA near the glycerol group is similar to other chains. However, the correlation times decrease and the motional amplitudes increase from double bond to double bond, reaching correlation times of the order of 10 ps at the methyl terminal end of the PUFA chain. Despite the rigidity of the cis-locked double bonds PUFA have faster motions and larger motional amplitudes. The underlying cause for this flexibility is an extremely low potential barrier for rotations about the C-C bonds between the double bonds. The low potentials permit PUFA to rapidly change conformation without significant energetic penalty. We have detected significant differences in the distribution of PUFA chain density between the omega-3 docosahexaenoic acid (22:6n3, DHA) and the omega-6 docosapentaenoic acid (22:5n6, DPA) along the bilayer normal. The DHA tends to have higher density near the lipid water interface compared to the DPA as derived from differences in chain order parameters, in motional correlation times with resolution for every carbon atom along the chain, in the electron density profiles of lipid bilayers obtained by x-ray diffraction experiments (collaboration with Dr. Tristram-Nagle), and in the simulations (collaboration with Dr. Feller). Our observations clearly point toward a difference in biophysical properties between membranes rich in DHA or DPA. We speculate that the differences in the distribution of lipid hydrocarbon chains alter lateral pressure density profiles of membranes which alter the probability of GPCR to activate upon ligand binding. (2) The mechanism by which rhodopsin and other membrane proteins control the lipid composition of their local environments, e.g. through the formation of lipid rafts, has attracted considerable attention. We are developing magic angle spinning (MAS) NMR approaches to characterize lateral distribution of membrane constituents. By MAS NMR with simultaneous application of pulsed field gradients, a novel experimental approach, we measured rates of lateral diffusion of lipids and membrane associated substances such as drugs or endogenous ligands of GPCR, e.g. the polyunsaturated anandamide. The novel MAS NMR techniques are also applied to study specific interactions between polyunsaturated lipids and GPCR. Experiments on reconstituted membranes containing bovine rhodopsin as well as molecular simulations by our collaborators suggest a deeper penetration of DHA chains into the transmembrane region of the GPCR compared to saturated chains. (3) Work has begun to express GPCR as fusion proteins for functional and structural studies on reconstituted membranes.