title: Rhodopsin-lipid interactions studied by NMR.
abstract: The biophysical properties of the lipid matrix are known to influence function of integral membrane proteins. We report on a sample preparation method for reconstitution of membrane proteins which uses porous anodic aluminum oxide (AAO) filters with 200-nm-wide pores of high density. The substrate permits formation of tubular, single membranes that line the inner surface of pores. One square centimeter of filter with a thickness of 60μm yields on the order of 500cm(2) of solid-supported single bilayer surface, sufficient for NMR studies. The tubular bilayers are free of detergent, fully hydrated, and accessible for ligands from one side of the membrane. The use of AAO filters greatly improves reproducibility of the reconstitution process such that the influence of protein on lipid order parameters can be studied with high resolution. As an example, results for the G protein-coupled receptor of class A, bovine rhodopsin, are shown. By (2)H NMR order parameter measurements, it is detected that rhodopsin insertion elastically deforms membranes near the protein. Furthermore, by (1)H saturation-transfer NMR under conditions of magic angle spinning, we demonstrate detection of preferences in interactions of rhodopsin with particular lipid species. It is assumed that function of integral membrane proteins depends on both protein-induced elastic deformations of the lipid matrix and preferences for interaction of the protein with particular lipid species in the first layer of lipids surrounding the protein.
authors: Laboratory of Membrane Biochemistry and Biophysics, NIAAA, National Institutes of Health, Bethesda, Maryland, USA. gawrisch@helix.nih.gov
authors: Olivier
authors: O
authors: Soubias
authors: Klaus
authors: K
authors: Gawrisch
mesh_terms: D000537:Aluminum Oxide / Q000737:chemistry; D000818:Animals; D002417:Cattle; D004281:Docosahexaenoic Acids / Q000737:chemistry; D004548:Elasticity; D005374:Filtration; D008051:Lipid Bilayers / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010716:Phosphatidylinositols / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D016062:Porosity; D015203:Reproducibility of Results; D012243:Rhodopsin / Q000737:chemistry*
chemical_list: D008051:Lipid Bilayers; D010714:Phosphatidylethanolamines; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D004281:Docosahexaenoic Acids; C483858:phosphatidylethanolamine; D012243:Rhodopsin; D000537:Aluminum Oxide
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