Solid-state NMR investigations of the bacteriorhodopsin photocycle

Abstract

Bacteriorhodopsin (bR) is a heptahelical integral membrane protein with a retinylidene prosthetic group from H. salinarum. The protein creates a proton gradient across the cell membrane by harvesting light energy. Proton transfer events can be segregated into transfers between the Schiff base of the chromophore and the cytoplasmic and extracellular half-channels. Critical to pump function is the switch in accessibility between the two half-channels. Models of the accessibility switch invoke protein or chromophore structural changes. This thesis describes the application of solid-state Nuclear Magnetic Resonance (NMR) to investigate each of the competing models in the native purple membrane environment. Conformational changes of the chromophore have been characterized by correlation of 13C-1H and 5N-1H dipolar interactions to determine chromophore dihedral angles in multiple photocycle intermediates. The resting state of the protein is found to contain a pre-loaded chromophore, with distortion about the Schiff base linkage and the neighboring C15-C14 bond. Changes of the dihedral about the C15-C14 were characterized during the photocycle, although the timing of the changes precludes their direct involvement in the accessibility switch. Putative protein conformation changes at the X-Pro peptide bonds were probed via the peptide 1 N and 13C chemical shifts. Proposed roles of the X-Pro bonds discriminate between structural and dynamic roles during the photocycle.

(cont.) Analysis of the three membrane-embedded X-Pro bonds indicates only slight differences between the resting state of the protein and the two species with a deprotonated chromophore, indicating that the buried prolines serve a principally structural role. General application of the proton-dependent dihedral angle measurement techniques to protein structure determination is hampered at sites with methylene carbons due to the pair of directly bonded protons. A general method is described for the preparation of stereoselectively deuterated glycine, which can be subsequently incorporated into peptides and proteins. This stereoselectively labeled glycine is expected to enable more precise determination of protein secondary structure.