A direct and quantitative analysis of the internal structure and dynamics of a polyunsaturated lipid bilayer composed of 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6n3-PC) containing 29 mol % cholesterol was carried out by neutron diffraction, 2H NMR and 13C-MAS NMR. The distribution of cholesterol segments as well as of sn-1 and sn-2 hydrocarbon chains of 18:0-22:6n-3-PC were obtained by conducting experiments with specifically deuterated cholesterol and phosphatidylcholine (PC). Cholesterol orients parallel to PC with the A-ring near the lipid glycerol and the terminal methyl groups 3 away from the bilayer center. Previously we had reported that density of polyunsaturated DHA chains was higher near the lipid water interface. Addition of cholesterol partially redistributes DHA density from near the lipid/water interface to the center of the hydrocarbon region. Cholesterol raises chain order parameters of both saturated stearic acid and DHA chains. The fractional order increase for stearic acid methylene carbons C8 to C18 is larger, reflecting the redistribution of DHA chain density toward the bilayer center. The correlation times of DHA chain isomerization are short and mostly unperturbed by the presence of cholesterol. The uneven distribution of saturated and polyunsaturated chain densities and the cholesterol-induced balancing of chain distributions may have important implications for function and integrity of membrane receptors, such as GPCR. We have shown that DHA is not only important because of the particular continuum elastic properties of membranes composed of lipids with polyunsaturated hydrocarbon chains, but also because of weak and transient DHA interactions with the GPCR that take place at the lipid-rhodopsin interface. For a quantitative assessment of the role of membrane elastic properties and lipid protein interactions at the lipid-protein interface, we added a series of six mono- and polyunsaturated lipids at increasing concentrations to a lipid matrix and followed the influence on rhodopsin activation measured as the metarhodopsin-I (MI)/metarhodopsin-II (MII) equilibrium. For all those lipids, monolayer elastic properties as characterized by the spontaneous radius of lipid monolayer curvature and the bending elastic moduli were measured by exposing inverted hexagonal lipid phases to controlled, reduced water activity and following changes in lipid monolayer curvature by x-ray diffraction. The experiments clearly indicate that the change of free energy due to a release of membrane curvature stress that is particularly high in phosphatidylethanolamines with polyunsaturated DHA chains is not the sole determinant of the conformational energetics of the MI-MII equilibrium. We indentified additional energetic contributions that were tentatively assigned to hydrogen bonding between lipid headgroups and rhodopsin and to direct interactions between lipid DHA chains with the receptor. Existence of such transient interactions between lipids and rhodopsin was confirmed by NMR. For the study of lipid-protein interaction in membranes we adapted the Saturation-Transfer Difference NMR experiment that is widely used for protein-ligand binding studies in solution NMR to solid state 1H-MAS NMR. The experiments indicate that DHA interacts with preferentially with a limited number of sites on rhodopsin. Rates of magnetization transfer from protein to DHA are lipid headgroup-dependent and increase in the sequence phosphatidylcholine

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National Institute on Alcohol Abuse and Alcoholism
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Yeliseev, Alexei; Gawrisch, Klaus (2017) Expression and NMR Structural Studies of Isotopically Labeled Cannabinoid Receptor Type II. Methods Enzymol 593:387-403
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