Functional reconstitution of rhodopsin into tubular lipid bilayers supported by nanoporous media.
We report on a novel reconstitution method for G-protein-coupled receptors (GPCRs) that yields detergent-free, single, tubular membranes in porous anodic aluminum oxide (AAO) filters at concentrations sufficient for structural studies by solid-state NMR. The tubular membranes line the inner surface of pores that traverse the filters, permitting easy removal of detergents during sample preparation as well as delivery of ligands for functional studies. 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]GTPgammaS binding assay. The 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 a matrix lipid. Rhodopsin incorporation increased mosaic spread of bilayer orientations and contributed to spectral density of motions with correlation times in the range of nano- to microseconds, detected as a significant reduction in spin-spin relaxation times. The change in lipid chain order parameters due to interaction with rhodopsin was insignificant.
Soubias
Olivier
O
Laboratory of Membrane Biochemistry and Biophysics, NIAAA, National Institutes of Health, Bethesda, Maryland 20892, USA.
Polozov
Ivan V
IV
Teague
Walter E
WE
Yeliseev
Alexei A
AA
Gawrisch
Klaus
K
D000537:Aluminum Oxide / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D005374:Filtration / Q000295:instrumentation; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008027:Light; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D053758:Nanoparticles / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry; D016062:Porosity; D011485:Protein Binding / Q000235:genetics; D011510:Proteolipids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D012542:Scattering, Radiation; D013056:Spectrophotometry, Ultraviolet
C409542:1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine; D008024:Ligands; D008051:Lipid Bilayers; D008823:Micelles; D010713:Phosphatidylcholines; D011510:Proteolipids; C020485:proteoliposomes; D012243:Rhodopsin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000537:Aluminum Oxide