Deuterium NMR Structure of Retinal in the Ground State of Rhodopsin

Abstract

The conformation of retinal bound to the G protein-coupled receptor rhodopsin is intimately linked to its photochemistry, which initiates the visual process. Site-directed deuterium ((2)H) NMR spectroscopy was used to investigate the structure of retinal within the binding pocket of bovine rhodopsin. Aligned recombinant membranes were studied containing rhodopsin that was regenerated with retinal (2)H-labeled at the C(5), C(9), or C(13) methyl groups by total synthesis. Studies were conducted at temperatures below the gel to liquid-crystalline phase transition of the membrane lipid bilayer, where rotational and translational diffusion of rhodopsin is effectively quenched. The experimental tilt series of (2)H NMR spectra were fit to a theoretical line shape analysis [Nevzorov, A. A., Moltke, S., Heyn, M. P., and Brown, M. F. (1999) J. Am. Chem. Soc. 121, 7636-7643] giving the retinylidene bond orientations with respect to the membrane normal in the dark state. Moreover, the relative orientations of pairs of methyl groups were used to calculate effective torsional angles between different planes of unsaturation of the retinal chromophore. Our results are consistent with significant conformational distortion of retinal, and they have important implications for quantum mechanical calculations of its electronic spectral properties. In particular, we find that the beta-ionone ring has a twisted 6-s-cis conformation, whereas the polyene chain is twisted 12-s-trans. The conformational strain of retinal as revealed by solid-state (2)H NMR is significant for explaining the quantum yields and mechanism of its ultrafast photoisomerization in visual pigments. This work provides a consensus view of the retinal conformation in rhodopsin as seen by X-ray diffraction, solid-state NMR spectroscopy, and quantum chemical calculations.

Structure, initial excited-state relaxation, and energy storage of rhodopsin resolved at the multiconfigurational perturbation theory level

“…1 the S 0 -Rh optimized structure shows a chromophore conformation remarkably close to the one observed in bovine Rh (4,22,23). [This structure is similar to the one obtained by Sugihara et al (24) via self-consistent-charge-density-functional tight-binding computations on a reduced Rh model comprising the entire PSB11 unit.]…”

Solid-state 2 H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin

“…Rhodopsin was regenerated with 11-cis-retinal 2 Hlabeled at the C5-, C9-, or C13-Me groups, and recombined with phospholipids followed by membrane alignment (32). Deletion of any of the methyl substituents transforms all-trans-retinal from a full agonist to a very weak partial agonist (1,33,34).…”

“…Bovine rhodopsin was regenerated with retinal 2 H-labeled at the C5-, C9-, or C13-Me groups (32) and was recombined with phospholipids by detergent dialysis (22). Membranes were aligned on planar glass substrates (40) …”

“…To connect changes in local retinal mobility with large-scale protein fluctuations, we introduce the concept of a dynamically active receptor. Induced fit of retinal to the binding pocket enjoins a highly twisted conformation upon the ligand (32). Light-induced isomerization entails changes in ps-ns scale mobility and methyl group dynamics within the ligand-binding cavity.…”

How a small change in retinal leads to G‐protein activation: Initial events suggested by molecular dynamics calculations

“…29 Bond orientation vectors with respect to the bilayer normal were computed for retinal's methyl groups bonded to C 5 , C 9 , and C 13 for both the dark-adapted state and post-photoisomerization. Considering the large instantaneous fluctuations in bond orientation and the limited statistical sampling achieved within the 40-ns dark-adapted simulation, results for the dark-adapted state are in good agreement with the NMR measurements ( Table I).…”