NMR Investigations Of Cell Membrane Structure

National Institute On Alcohol Abuse And Alcoholism

Investigators

Linked publications, trials & patents

Abstract

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<phosphatidyl-serine<phosphatidylethanolamine. Experiments conducted on rod outer segment disk membranes from bovine retina with photoactivation of rhodopsin in the spinning rotor using a laser beam demonstrated that all photointermediates show some preference for interaction with DHA chains, but highest affinity was observed for Meta-III rhodopsin. The influence of direct lipid-rhodopsin interaction on the dynamics of DHA-containing phospholipids was studied by 13C-MAS NMR relaxation measurements. It was shown that the polyunsaturated DHA chains maintain their high flexibility near the protein. A mismatch between the hydrophobic length of the transmembrane helices of membrane proteins and the thickness of the hydrophobic core of the lipid matrix are thought to be critical for membrane protein function. For a series of PCs with a perdeuterated, saturated sn-1 hydrocarbon chain and a monounsaturated sn-2 chain, both with 14-20 carbons per chain, the MI-MII equilibrium shifts steadily toward MII with increasing acyl chain length. The lipids adjust to the length mismatch to rhodopsin by stretching or compressing hydrocarbons chains as detected by 2H NMR order parameter measurements. The crossover from stretching to compression occurs at a bilayer hydrophobic thickness of 27 Angstrom. Furthermore, an increase of the helicity of rhodopsin with increasing hydrocarbon chain length was detected by circular dichroism (CD) suggesting that the length of transmembrane helices adjusts to bilayer thickness. This is contrary to the widespread belief that hydrophobic matching involves primarily an adjustment of the lipid matrix to the protein. The observation is likely to be applicable to the entire class of G-protein-coupled membrane receptors. We express CB2 recombinantly in Escherichia coli as a fusion with maltose-binding protein and several affinity tags. The CB2-fusion protein is solubilized, purified, the fusion cleaved, and CB2 purified again from cleavage products. Finally, the pure CB2 is reconstituted into a lipid matrix of controlled composition for structural and functional studies. Reconstitution of functional CB2 at the level of milligrams, and concentration to a volume of 40 microliters, sufficient for structural studies by solid state NMR has been achieved. Functionality of the receptor was verified by ligand binding using radioactive ligands as well as deuterated ligands in combination with 2H-MAS NMR and by G protein activation studies using recombinantly produced G protein in a 35SGTP-&#61543;-S assay. Composition, size, and homogeneity of proteoliposomes were investigated by analytical NMR, fluorescence spectroscopy using labeled lipid and CB2, dynamic light scattering, and sucrose gradient centrifugation. The protein was successfully stabilized during purification and reconstitution by a proper mixture of detergents, lipids, as well as ligand. While the receptor proved to be vulnerable to degradation in micellar solution, the reconstituted CB2 in a lipid matrix has long-term stability that enables functional and structural studies. Exploratory NMR experiments conducted on a 2-mg sample of homogeneously 13C- and 15N labeled CB2 and comparison of experimental results with simulated spectra obtained from the atomic coordinates of a CB2 model have demonstrated feasibility of the experimental concept. Specific isotopic labeling schemes have been developed to achieve the desired spectral resolution for a structural analysis.