Functional Reconstitution of Rhodopsin into Tubular Lipid Bilayers Supported by Nanoporous Media
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- 作者：Olivier Soubias ; Ivan V. Polozov ; Walter E. Teague ; Alexei A. Yeliseev ; Klaus Gawrisch
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：December 26, 2006
- 年：2006
- 卷：45
- 期：51
- 页码：15583 - 15590
- 全文大小：192K
- 年卷期：v.45,no.51(December 26, 2006)
- ISSN：1520-4995
文摘
We report on a novel reconstitution method for G-protein-coupled receptors (GPCRs) thatyields detergent-free, single, tubular membranes in porous anodic aluminum oxide (AAO) filters atconcentrations sufficient for structural studies by solid-state NMR. The tubular membranes line the innersurface of pores that traverse the filters, permitting easy removal of detergents during sample preparationas well as delivery of ligands for functional studies. Reconstitution of bovine rhodopsin into AAO filtersdid not interfere with rhodopsin function. Photoactivation of rhodopsin in AAO pores, monitored byUV-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
S binding assay.The lipid-rhodopsin interaction was investigated by 2H NMR on sn-1- or sn-2-chain perdeuterated1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospholine as a matrix lipid. Rhodopsin incorporation increasedmosaic spread of bilayer orientations and contributed to spectral density of motions with correlation timesin 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.
S binding assay.The lipid-rhodopsin interaction was investigated by 2H NMR on sn-1- or sn-2-chain perdeuterated1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospholine as a matrix lipid. Rhodopsin incorporation increasedmosaic spread of bilayer orientations and contributed to spectral density of motions with correlation timesin 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.