Solution and Solid State C-13 and N-15 NMR Studies of Visual Pigments and Related Systems: Rhodopsin and Bacteriorhodopsin^1a

Abstract

This paper constitutes a chronological review of C-13 and N-15 solution and solid state NMR investigations of bovine rhodopsin and the purple membrane of Halobacterium halobium. Model retinylideneimines and their protonated species yielded rather informative variation in C-13 chemical shifts, as a function of structure and conformation, Retinals, specifically enriched with C-13 at key atom positions, were incorporated in rhodopsin and bacteriorhodopsin during the bleaching-regeneration process. The labeled pigments exhibited C-13 signals which were interpreted in terms of the state of protonation and conformation of the Schiff base linkage between the chromophore and opsin (15-anti in rhodopsin and light adapted bacteriorhodopsin) and chromophore-protein interactions. Peaks arising from both all-trans and 13-cis-retinylidene moieties were observed in dark adapted bacteriorhodopsin. Upon protonation, the Schiff base nitrogen of model compounds displayed dramatic (up to 146 ppm) high field N-15 shifts (an order of magnitude larger than C-13). Hydrogen bonding also led to important (up to 50 ppm) nitrogen shielding. H. halobium grown on a medium containing ɛ¹⁵N-lysine incorporated, with high yield, the labeled amino acid in all of the seven lysine sites of bacteriorhodopsin. Of these, one forms the shift could be observed only in lyophilized preparations. It appears that the degree of protonation of the Schiff base that the degree of protonation of the Schiff base linkage depends on the degree of hydation of the linkage depends on the degree of hydration of the specimen. Double labeling (¹³C=¹⁵N) is expected to provide valuable information concerning the structure and conformation of this moiety. The six lysine residues not involved in retinal binding yielded, in a micellar (octyl-β-glucoside) solution, N-15 signals of various linewidths, spread over a narrow (2–4 ppm) range. This could be interpreted in terms of lysine residue interactions within the protein. Preliminary results indicate the feasibility of in vivo measurements which avoid alterations inherent to extraction, purification, and/or lyophilization procedures.