PROTEIN STRUCTURAL-CHANGE AT THE CYTOPLASMIC SURFACE AS THE CAUSE OF COOPERATIVITY IN THE BACTERIORHODOPSIN PHOTOCYCLE

The effects of excitation light intensity on the kinetics of the bacte riorhodopsin photocycle were investigated. The earlier reported intens ity-dependent changes at 410 and 570 nm are explained by parallel incr eases in two of the rate constants, for proton transfers to D96 from t he Schiff base and from the cytoplasmic surface, without changes in th e others, as the photoexcited fraction is increased. Thus, it appears that the pK(a) of D96 is raised by a cooperative effect within the pur ple membrane. This interpretation of the wild-type kinetics was confir med by results with several mutant proteins, where the rates are well separated in time and a model-dependent analysis is unnecessary. Based on earlier results that demonstrated a structural change of the prote in after deprotonation of the Schiff base that increases the area of t he cytoplasmic surface, and the effects of high hydrostatic pressure a nd lowered water activity on the photocycle steps in question, we sugg est that the pK(a) of D96 is raised by a lateral pressure that develop s when other bacteriorhodopsin molecules are photoexcited within the t wo-dimensional lattice of the purple membrane. Expulsion of no more th an a few water molecules bound near D96 by this pressure would account for the calculated increase of 0.6 units in the pK(a).