Rhodopsin is a seven transmembrane helix GPCR found which mediates dim light vision, in which the binding pocket is occupied by the ligand 11- cis-retinal. A site-directed ²H-labeling approach utilizing solid-state ²H NMR spectroscopy was used to investigate the structure and dynamics of retinal within its binding pocket in the dark state of rhodopsin, and as well the MetaI and MetaII. 11-cis-[5-C²H₃]-, 11-cis-[9-C²H₃]-, and 11-cis-[13-C²H₃]-retinal were used to regenerate bleached rhodopsin. Recombinant membranes comprising purified rhodopsin and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were prepared (1:50 molar ratio). Solid-state ²H NMR spectra were obtained for the aligned rhodopsin/POPC recombinant membranes at temperatures below the order-disorder phase transition temperature of POPC. The solid-state NMR studies of aligned samples, give the orientations of the ²H nuclear coupling tensor relative to the membrane frame, which involve both the conformation and orientation of the bound retinal chromophore. Theoretical simulations of the experimental ²H NMR spectra employed a new lineshape treatment for a semi-random distribution due to static uniaxial disorder. The analysis gives the orientation of the ²H-labeled C-C²H₃ methyl bond axes relative to the membrane plane as well as the extent of three-dimensional alignment disorder (mosaic spread). These results clearly demonstrate the applicability of site-directed ²H NMR methods for investigating conformational changes and dynamics of ligands bound to rhodopsin and other GPCRs in relation to their characteristic mechanisms of action.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/280741 |
| Date | January 2004 |
| Creators | Salgado, Gilmar F. |
| Contributors | Brown, Michael F. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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