Protein structural change at the cytoplasmic surface as the cause of cooperativity in the bacteriorhodopsin photocycle

Abstract

The effects of excitation light intensity on the kinetics of the bacteriorhodopsin photocycle were investigated. The earlier reported intensity-dependent changes at 410 and 570 nm are explained by parallel increases in two of the rate constants, for proton transfers to D96 from the Schiff base and from the cytoplasmic surface, without changes in the others, as the photoexcited fraction is increased. Thus, it appears that the pKa of D96 is raised by a cooperative effect within the purple membrane. This interpretation of the wild-type kinetics was confirmed 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 protein after deprotonation of the Schiff base that increases the area of the cytoplasmic surface, and the effects of high hydrostatic pressure and lowered water activity on the photocycle steps in question, we suggest that the pKa of D96 is raised by a lateral pressure that develops when other bacteriorhodopsin molecules are photoexcited within the two-dimensional lattice of the purple membrane. Expulsion of no more than a few water molecules bound near D96 by this pressure would account for the calculated increase of 0.6 units in the pKa.

A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin

“…It may be then the resulting abolition of the charge-pair at the active site, rather than the isomerization directly, that allows the protein to assume its alternative conformation and to facilitate the reprotonation of the Schiff base by Asp-96. Inasmuch as the pK a of Asp-96, and therefore the role of this residue as proton donor, depends on the conformation changes at the cytoplasmic surface (19,47), this seems reasonable. From different evidence, it has been argued, however, that deprotonation of the Schiff base and the reprotonation switch are both direct consequences of the isomerization (48).…”

Structure of the bacteriorhodopsin mutant F219L N intermediate revealed by electron crystallography

“…The effect is seen as an increase in the proportion of a slow‐decaying M species at increasing light intensities at the expense of a fast‐decaying species. It has been explained variously as resulting from heterogeneity in the photoexcited molecules or from cooperativity within a trimer or within the purple membrane (Hendler et al ., 1994; Shrager et al ., 1995; Váró et al ., 1996; Tokaji, 1998). The same effect is seen when a second light flash is given within a few milliseconds after the first (Tokaji and Dancsházy, 1991).…”

Mechanism of Ion Transport across Membranes

“…Reversal of the tilt of helix F restores the initial high pK a of Asp 96 (66), and this residue is then reprotonated from the cytoplasmic surface. The increase of the lateral cross-section of the protein in the bilayer during this process (50) may be the cause of the cooperativity in the two-dimensional lattice of the purple membrane upon photoexcitation (67).…”