Arginine activity in the proton-motive photocycle of bacteriorhodopsin: Solid-state nmr studies of the wild-type and D85N proteins

Abstract

15N solid-state NMR (SSNMR) spectra of guanidyl-15N-labeled bacteriorhodopsin (bR) show perturbation of an arginine residue upon deprotonation of the retinal Schiff base during the photocycle. At the ε position, an upfield shift of 4 ppm is observed while the η nitrogens develop a pair of 'wing' peaks separated by 24 ppm. Proton-driven spin diffusion between the two 'wing' peaks indicates that they arise from a single Arg residue. An unusually asymmetric environment for this residue is indicated by comparison with guanidyl-15N chemical shifts in a series of arginine model compounds. The 'wing' peaks are tentatively assigned to Arg- 82 on the basis of the SSNMR investigations of the alkaline and neutral dark- adapted forms of the D85N bacteriorhodopsin mutant. Another, less asymmetric pair of η signals, that is not affected by Schiff base deprotonation or D85 mutation, is tentatively assigned to Arg134. The results are discussed in relation to existing models of bR structure and function.