FUNCTIONAL-SIGNIFICANCE OF A PROTEIN CONFORMATION CHANGE AT THE CYTOPLASMIC END OF HELIX-F DURING THE BACTERIORHODOPSIN PHOTOCYCLE

The second half of the photocycle of the light-driven proton pump bact eriorhodopsin includes proton transfers between D96 and the retinal Sc hiff base (the M to N reaction) and between the cytoplasmic surface an d D96 (decay of the N intermediate). The inhibitory effects of decreas ed water activity and increased hydrostatic pressure have suggested th at a conformational change resulting in greater hydration of the cytop lasmic region is required for proton transfer from D96 to the Schiff b ase, and have raised the possibility that the reversal of this process might be required for the subsequent reprotonation of D96 from the cy toplasmic surface. Tilt of the cytoplasmic end of helix F has been sug gested by electron diffraction of the M intermediate. Introduction of bulky groups, such as various maleimide labels, to engineered cysteine s at the cytoplasmic ends of helices A, B, C, E, and G produce only mi nor perturbation of the decays of M and N, but major changes in these reactions when the label is linked to helix F. In these samples the re protonation of the Schiff base is accelerated and the reprotonation of D96 is strongly retarded. Cross-linking with benzophenone introduced at this location, but not at the others, causes the opposite change: t he reprotonation of the Schiff base is greatly slowed while the reprot onation of D96 is accelerated. We conclude that, consistent with the s tructure from diffraction, the proton transfers in the second half of the photocycle are facilitated by motion of the cytoplasmic end of hel ix F, first away from the center of the protein and then back.