Low-Temperature Solid-State ¹³C NMR Studies of the Retinal Chromophore in Rhodopsin

Magic angle sample spinning (MASS) ¹³C NMR spectra have been obtained of bovine rhodopsin regenerated with retinal prosthetic groups isotopically enriched with ¹³C at C-5 and C-14. In order to observe the ¹³C retinal chromophore resonances, it was necessary to employ low temperatures (–15 → –35 °C) to restrict rotational diffusion of the protein. The isotropic chemical shift and principal values of the chemical shift tensor of the ¹³C-5 label indicate that the retinal chromophore is in the twisted 6-s-cis conformation in rhodopsin, in contrast to the planar 6-s-trans conformation found in bacteriorhodopsin. The ¹³C-14 isotropic shift and shift tensor principal values show that the Schiff base C=N bond is anti. Furthermore, the ¹³C-14 chemical shift (121.2 ppm) is within the range of values (120–123 ppm) exhibited by protonated (C=N anti) Schiff base model compounds, indicating that the C=N linkage is protonated. Our results are discussed with regard to the mechanism of wavelength regulation in rhodopsin.