NMR Investigations Of Cell Membrane Structure

National Institute On Alcohol Abuse And Alcoholism

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Abstract

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. We extensively studied the effects of detergents, lipids and cannabinoid ligands on stability of the recombinant cannabinoid receptor CB2. The effort resulted in guidelines for preparation and handling of the fully functional receptor suitable for a wide array of downstream applications. We demonstrate that a concerted action of an anionic cholesterol derivative, cholesteryl hemisuccinate (CHS) and high affinity cannabinoid ligands CP-55,940 or SR-144,528 are required for efficient stabilization of the functional fold of CB2 in dodecyl maltoside (DDM)/ CHAPS detergent solutions. Similar to CHS, the negatively charged phospholipids with the serine headgroup (PS) exerted significant stabilizing effects in micelles while uncharged phospholipids were not effective. The purified CB2 reconstituted into lipid bilayers retained functionality for up to several weeks enabling high resolution structural studies of this GPCR at physiologically relevant conditions. 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 GTPgammaS radioactive 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. 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. Membranes with a high content of polyunsaturated phosphatidylethanolamines (PE) facilitate formation of metarhodopsin-II (MII), the photointermediate of bovine rhodopsin that activates the G protein transducin. We determined whether MII-formation is quantitatively linked to the elastic properties of PEs. Curvature elasticity of monolayers of the polyunsaturated lipids 18:0-22:6n-3PE, 18:0-22:5n-6PE and the model lipid 18:1n-9-18:1n-9PE were investigated in the inverse hexagonal phase. All three lipids form lipid monolayers with rather low spontaneous radii of curvature of 26-28 Angstrom. Negative curvature elastic stress in membranes containing high concentrations of polyunsaturated PEs is very high. Release of even a small fraction of this stress from the layer of lipids surrounding the receptor is sufficient to shift the MI/MII equilibrium towards MII, the state that activates G protein. Furthermore, polyunsaturated bilayers have a hydrophobic thickness of about 27 A which has been determined to match the length of the hydrophobic transmembrane helices of rhodopsin. The data show that polyunsaturated lipids are important for class A GPCR activation, and we speculate that the rhodopsin model is particularly relevant for constitutive activity of GPCR and activation by weak agonists.