The objectives of this project are to: (i) study structure, dynamics, and domain formation in membranes composed of lipids with polyunsaturated fatty acids such as docosahexaenoic acid (DHA) 22:6n-3, (ii) study the interaction of the polyunsaturated lipid matrix with G-protein coupled membrane receptors (GPCR), and (iii) investigate structure and function of selected GPCR with relevance for alcoholism in reconstituted membrane systems. ? ? (i) It was explored if the surface of the GPCR rhodopsin should be viewed as homogeneous and the surrounding membrane as a continuum, or if specific interactions, in particular with polyunsaturated lipids may play a role in rhodopsin activation. The association of rhodopsin with poly- and monounsaturated lipids was studied by 1H MAS NMR with magnetization transfer from rhodopsin to lipid. Experiments were conducted on bovine rod outer segment (ROS) disks and on recombinant membranes containing bovine rhodopsin and lipids with polyunsaturated, docosahexaenoyl (DHA) and monounsaturated oleoyl chains. Rhodopsin was photoactivated in the spinning MAS rotor and magnetization transfer followed as a function of time after photoactivation. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids, but highest rates were observed for meta-III rhodopsin. Rates of magnetization transfer from protein to DHA are lipid headgroup dependent and increased in the sequence PC, PS, PE. In experiments conducted as a function of DHA content, it was shown that poly- and monounsaturated lipids interact specifically with different sites on the rhodopsin surface. Rhodopsin-associated lipids are in fast exchange with lipids in the bulk of the matrix on a timescale of milliseconds or shorter. Deciphering of the functional consequences of specific lipid-rhodopsin associations requires further investigations. ? We characterized the properties of liquid ordered phases related to formation of so-called rafts in biomembranes. Although biochemical evidence for existence of rafts is strong, detection of the structural equivalent of a raft in biomembranes has proven to be extremely difficult. In experiments on well-characterized model membranes it was shown that 1H MAS NMR spectra of lipids in liquid ordered domains (lo) have a distinctly different linewidth from liquid disordered (ld) and solid ordered (so) membrane regions. According to results of 1H NMR dipolar echo spectroscopy, the broadening of MAS resonances in the lo phase results from an increase in effective strength of intramolecular proton dipolar interactions between adjacent methylene groups, most likely because of a lower probability of gauche/trans isomerization in lo. In spectra recorded as a function of temperature, the onset of lo domain (raft) formation is seen as a sudden onset of line broadening. The 1H MAS NMR method of lo phase detection does not require labeling and may be applied to rafts in cell membranes. Experiments on biological membranes are underway.? ? (ii) We developed reagents and methods for formation of tubular single lipid bilayers containing high concentrations of GPCR that cover the pore walls of porous anodic aluminum oxide (AAO) filters. One square centimeter of the filter material with a thickness of 60 micrometers yields 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). The GPCRs rhodopsin, purified from natural sources, as well as the recombinant peripheral cannabinoid receptor, CB2, expressed in E. coli were incorporated into the tubular bilayers in functional form. Reconstitution of bovine rhodopsin into AAO filters did not interfere with rhodopsin function. Photoactivation of rhodopsin in AAO pores, monitored by UV-Vis spectrophotometry, was indistinguishable from rhodopsin in unsupported unilamellar liposomes. The rhodopsin in AAO pores is G-protein binding competent as shown by a [35S]GTP-gamma-S binding assay. Lipid-rhodopsin interaction was investigated by 2H NMR on sn-1-, or sn-2 chain perdeuterated 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospholine as matrix lipid. Rhodopsin incorporation increased mosaic spread of bilayer orientations and contributed spectral density of motions with correlation times in the range from nano- to microseconds, detected as significant reduction of spin-spin relaxation times. The change of lipid chain order parameters due to interaction with rhodopsin was insignificant.? ? (iii) Human peripheral-type cannabinoid receptor (CB2) was expressed in E. coli as a fusion with the maltose-binding protein and thioredoxin. Successful expression of the full-length fusion was confirmed by Western-blot analysis and mass spectroscopy. Functional activity and structural integrity of the receptor in bacterial protoplast membranes was confirmed by extensive binding studies with a variety of natural and synthetic cannabinoid ligands. Agonist stimulation of urea-treated E. coli membranes expressing recombinant CB2 resulted in an activation of the G proteins in the in vitro coupled assay. For purification of the protein, Strep-tags and decahistidine-tags were introduced at various locations in the polypeptide chain. The fusion-CB2 was successfully cleaved at Tev-protease cleavage sites and the CB2 purified by two steps of affinity chromatography. The purified CB2 was successfully reconstituted into lipid bilayers and retained, at least partially, its ligand binding properties. The protocol allows for expression and purification of milligram quantities of the recombinant receptor for structural studies by NMR and scattering techniques.
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