Retinal Chromophore Structure in Meta-II Rhodopsin Revealed by Solid-State 2H NMR and Molecular Modeling

Abstract

Knowledge of the structural dynamics of retinal bound to rhodopsin is crucial for understanding its activation mechanism [1]. Recent X-ray structures of rhodopsin mutants in the active state and opsin with bound all-trans retinal have reported different orientations of the chromophore. Here we investigate the structure of all-trans retinal bound to the active Meta-II photointermediate by 2H NMR spectroscopy. Rhodopsin was regenerated with retinal containing 2H-labeled methyl groups, and was reconstituted with POPC/DOPE bilayers, followed by alignment of membranes on glass slides. Rhodopsin was then bleached and trapped in the Meta-II state [2] at temperatures below -‍60 °C. Solid-state 2H NMR spectra were acquired for different tilt angles of the aligned samples. The methyl group orientations relative to the membrane normal were calculated by fitting the experimental 2H NMR spectra using a static uniaxial distribution [3] for the protein embedded within lipid bilayers. We found that the orientation of the C9-methyl group obtained from 2H NMR spectroscopy was similar to that from X-ray data. By contrast, the orientations of the C5- and C13- methyl groups were different versus the X-ray crystal structures. The retinal structure was analyzed using the three-plane model, as in previous studies of rhodopsin in the dark and the Meta-I states [4]. Moreover, a new approach was tested that combines 2H NMR and X-ray restraints for retinal together with the rhodopsin binding-pocket using simulated annealing. Our results yield new insights into formation of the activated state of the receptor in lipid membranes. [1] A.V. Struts et al. (2011) Nat. Struct. Mol. Biol. 18, 392-394. [2] A.V. Struts et al. (2015) Meth. Mol. Biol. 1271, 133-158. [3] A.A. Nevzorov et al. (1999) JACS 121, 7636-7643. [4] G.F.J. Salgado et al. (2006) JACS 128, 11067-11071.