THE LAST PHASE OF THE REPROTONATION SWITCH IN BACTERIORHODOPSIN - THETRANSITION BETWEEN THE M-TYPE AND THE N-TYPE PROTEIN CONFORMATION DEPENDS ON HYDRATION

In order to elucidate the mechanism of the reprotonation switch of bac teriorhodopsin, the protein conformation of the M intermediate of the D96N mutant was examined at various hydration conditions by X-ray diff raction and FTIR spectroscopy. We observed two distinct protein confor mations at different levels of hydration. One is like in the N photoin termediate, although in this case with an unprotonated Schiff base. It is stabilized in highly hydrated samples. The other is a protein conf ormation identical to that in the normal M intermediate of wild-type b acteriorhodopsin, which is stabilized in partially dehydrated samples. The hydration dependence of the structural transition between the M-t ype and the N-type conformations suggests that there is a change in th e binding of water at the cytoplasmic surface. Thus, more water molecu les bind in the N-type structure than in the M-type. This is consisten t with the idea that the conformational change from the M-type to the N-type corresponds to the opening of the proton channel to the cytopla smic surface by tilt of the cytoplasmic end of helix F, and that this is required for proton transfer from Asp-96 to the retinal Schiff base .