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) We developed methods to use magic spinning NMR with application of pulsed field gradients (PFG MAS NMR) for the study of lipid lateral diffusion in biomembranes. This approach measures diffusion on the micrometer lengthscale without the use of perturbing labels. The excellent resolution of resonance signals afforded by magic angle spinning permited diffusion measurements for individual membrane constituents as well as for membrane incorporated drugs, e.g. the hydrophobic ligands of G-protein coupled membrane receptors. Lateral diffusion was studied for a large number of model membrane systems. The samples consisted of purified lipids comparing the headgroups phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine; contrasting chain lengths of fourteen, sixteen, and eighteen carbons; and comparing degrees of unsaturation from one to six double bonds along a chain. Highly unsaturated lipids like those with DHA hydrocarbon chains had significantly higher rates of lateral diffusion and lower thermal activation energies. We linked the high diffusion rates to the surprisingly large flexibility and the rapid conformational transitions of polyunsaturated chains which we had reported recently. The rates of lateral diffusion of matrix lipids, water, and membrane dissolved substances are intrinsically linked to membrane organization, including the presence of domains or rafts. Domain formation was studied in mixtures of biologically relevant phosphatdiylcholines, phosphatidylethanolamines, and cholesterol. (2) We developed reagents and methods for formation of tubular single lipid bilayer membranes containing high concentrations of membrane receptors inside porous solid supports. The tubular bilayers are ideal for use in biosensors and for structural studies. One square centimeter of the filter material with a thickness of 60 micrometers may yield up to 500 cm2 of oriented membranes, which is sufficient for multinuclear solid state NMR studies on the lipid matrix and on incorporated, isotopically labeled protein. By solid state NMR methods we determined that the membranes are separated from the support by a closed and stable aqueous cushion. The inner surface of the lipid tubules is freely accessible from an outside solution. The aluminum oxide-based support provides the advantage of high flow rates to exchange solutions, efficient particle retention, rigid, uniform surface, and transparency (when wet). Using this technology, G-protein coupled membrane receptors (GPCR), purified from natural sources, as well as recombinant receptors expressed in E-coli were incorporated into the bilayer in functional form. The setup is ideal for ligand binding studies, including drug testing. The technology may be applied to a broad variety of membrane receptors but appears to be particularly useful for GPCR. The use of single lipid bilayers greatly reduces nonspecific interactions of ligands with the substrate therefore enhancing sensitivity and reproducibility of binding studies. The water layer between the membrane and the solid support prevents perturbation of receptor function. The substrates are compatible with signal detection by fluorescence, radiotracers, NMR, and other methods. Considering the ease of preparation of such systems, protein containing biomembranes supported by the porous aluminum oxide filters have considerable promise for use in NMR structural studies as well as in biosensors. (3) Human peripheral-type cannabinoid receptor (CB2) was expressed iin E. colii as a fusion with maltose-binding protein (at the N-terminal end) and thioredoxin followed by ten histidine residues (at the C-terminal end). Successful expression of the full-length fusion was confirmed by Western-blot analysis. The expressed receptor was localized in the inner-membrane fraction of E. coli cells. The fusion-CB2 protein was purified to 85-90% by immobilized-metal affinity chromatography followed by ion-exchange chromatography in the presence of detergents. The protocol allows for expression and purification of milligram quantities of the recombinant receptor. N- and C-terminal tags can be removed from the fusion protein by action of a specific TEV protease. The functional activity of CB2 expressed in E. coli was confirmed by filter-binding assays with synthetic cannabinoid ligands. Agonist stimulation of the urea-treated E. coli membranes expressing recombinant CB2 resulted in an activation of the G proteins in the in vitro coupled assay. Preliminary results suggest the possibility of successful reconstitution of the recombinant receptor into a lipid matrix, and deposition onto a solid support suitable for subsequent structural characterization by nuclear magnetic resonance spectroscopy and neutron scattering techniques.
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