Combined solid state and solution NMR studies of α,ε-15N labeled bovine rhodopsin

Rhodopsin is the visual pigment of the vertebrate rod photoreceptor cell and is the only member of the G protein coupled receptor family for which a crystal structure is available. Towards the study of dynamics in rhodopsin, we report NMR-spectroscopic investigations of alpha,epsilon-N-15-tryptophan labeled rhodopsin in detergent micelles and reconstituted in phospholipids. Using a combination of solid state C-13, N-15-REDOR and HETCOR experiments of all possible C-13' (i-1) carbonyl/N-15(i)-tryptophan isotope labeled amide pairs, and H/D exchange H-1, N-15-HSQC experiments conducted in solution, we assigned chemical shifts to all five rhodopsin tryptophan backbone N-15 nuclei and partially to their bound protons. H-1, N-15 chemical shift assignment was achieved for indole side chains of Trp35(1.30) and Trp175(4.65). N-15 chemical shifts were found to be similar when comparing those obtained in the native like reconstituted lipid environment and those obtained in detergent micelles for all tryptophans except Trp175(4.65) at the membrane interface. The results suggest that the integrated solution and solid state NMR approach presented provides highly complementary information in the study of structure and dynamics of large membrane proteins like rhodopsin.