Structure and dynamics of retinal in rhodopsin elucidated by deuterium solid state NMR

Abstract

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.