X-RAY-DIFFRACTION STUDIES OF BACTERIORHODOPSIN - DETERMINATION OF THEPOSITIONS OF MERCURY LABEL AT SEVERAL ENGINEERED CYSTEINE RESIDUES

The single cysteine-containing bacteriorhodopsin mutants F27C, L100C, T170C, F171C and I222C were labeled with p-chloromercuribenzoic acid, which specifically reacts with sulfhydryl groups. These cysteines shou ld be located at the cytoplasmic ends of the transmembrane helices A, C, F or G. We determined the positions of the bound mercury atoms by X -ray diffraction of purple membrane films, with better than 1 Angstrom accuracy. The determined mercury positions were compared with the str uctural model from cryoelectron microscopy (N. Grigorieff, T. A. Ceska , K. H. Downing, J. M. Baldwin and R. Henderson, J. Mol. Biol. 259, 39 3-421, 1996). Given that the distance between the mercury and the C al pha atom of the cysteine in the xy plane must be shorter than 4.5 Angs trom and that the mercury atom is located at the delta position, the p ositions obtained for the mercury labels agree with their expected pos itions from the structural model. The present results give a rationale for detecting structural changes upon illumination as shifts occur in the mercury label position.