Conformational change of helix G in the bacteriorhodopsin photocycle: Investigation with heavy atom labeling and X-ray diffraction

According to the current structural model of bacteriorhodopsin, lle(222) is located at the cytoplasmic end of helix G. We labeled the single cysteine of the site-directed mutant lle(222) --> Cys with p-chloromercuribenzoic ac id and determined the position of the labeled mercury by x-ray diffraction in the unphotolyzed state, and in the M-N photointermediate accumulated in the presence of guanidine hydrochloride at pH 9.5. According to the differe nce Fourier maps between the M-N intermediate and the unphotolyzed state. t he structural change in the M-N intermediate was not affected by mercury la beling. The difference Fourier map between the labeled and the unlabeled 12 22C gave the position of the mercury label. This information was obtained f or both the unphotolyzed state and the M-N intermediate. We found that the position of the mercury at residue 222 is shifted by 2.1 +/- 0.8 Angstrom i n the M-N intermediate. This agrees with earlier results that suggested a s tructural change in the G helix. The movement of the mercury label is so la rge that it must originate from a cooperative conformational change in the helix G at its cytoplasmic end, rather than from displacement of residue 22 2. Because lle(222) is located at the same lever on the z coordinate as Asp (96), the structural change in the G helix could have the functional role o f perturbing the environment and therefore the pK(a) of this functionally i mportant aspartate.