Effects of hydrostatic pressure on the kinetics reveal a volume increase during the bacteriorhodopsin photocycle

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Abstract

A protein structural change in the photocycle of the proton pump, bacteriorhodopsin, detected earlier in the M photointermediate by diffraction, consists mainly of changes at the cytoplasmic surface that include an outward tilt of the cytoplasmic end of helix F. Such a conformational rearrangement would result in greater exposure of the interhelical cavity to the medium, increased binding of water, and thus an increase in volume. In order to correlate the structural change with the kinetics of the photoreaction cycle, we measured the effects of hydrostatic pressure between 1 bar and 1 kbar on the rate constants of the photocycles of wild type bacteriorhodopsin and the D96N mutant. Combining the results provided all of the activation volumes and, therefore, the changes of volume in the various states after the K photointermediate is formed. There is an approximately 32 mL/mol volume increase after deprotonation of the retinal Schiff base to the extracellular side, during the M1→ M2 reaction, that is not reversed until well after its reprotonation from the cytoplasmic side. The magnitude of this volume increase is about as predicted by the increase of the lattice constant in the M state. It occurs in the photocycle at the proposed reprotonation switch, supporting the idea that this conformation change is what alters the accessibility of the Schiff base from one membrane side to the other. Additionally, we observe a large positive (approximately 50 mL/mol) activation volume for proton exchange between D96 and the Schiff base of the wild type protein. This is consistent with a requirement for increased hydration of the protein near D96 for the proton exchange to occur, as suggested earlier from the specific inhibition of this step by osmotically active solutes.

Original languageEnglish
Pages (from-to)12161-12169
Number of pages9
JournalBiochemistry
Volume34
Issue number38
Publication statusPublished - 1995

ASJC Scopus subject areas

  • Biochemistry

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