Dynamic Structural Analyzes of the Photointermediates of Bacteriorhodopsin

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The major purpose of this researchi is the structural analysis of each photointermediate of bacteriorhodopsin (bR), in order to understand the molecular mechanism of its light-driven proton pump.Despite that D85N mutant bR does not generate the M-intermediate by photo-reaction, alkaline D85N has similar properties to the M-intermediate. The structure of D85N at alkaline pH was compared with the structure at neutral pH.In order to record the X-ray diffraction patterns at the same place of the same oriented specimen under different pH,we developed an ammonium treatment method. The changes in diffraction profiles from neutral to alkaline pH resemble to those from the grand state to the M-intermediate of wild type bR and D96N.The structural changes occurred at alkaline pH are remarkable around helices C,F and G by a difference Fourier synthesis. These structural changes are common for the structural changes in the M intermediate. Although the similar changes were observed for D85N/D96N,the … More extent of the changes were much smaller than that of D85N.X-ray diffraction profile of D85N/D96N at neutral pH clearly indicates that some portion of the double mutant take the M-like structure even at neutral pH.We conclude that the deprotonation of Schiff base stabilizes the M-type structure, and that the M-type structure stimulates the deprotonation of Schiff base. Schiff base proton can interact with the cytoplasmic surface only in the M-type structure, while the proton can interact with the extracellular surface only in the grand state structure.The M-decay processes at various pH were studied by kinetic spectroscopy and kinetic X-ray diffraction. The reversion of the structure from the M-type to grand state is closely correlated with the reprotonation of Schiff base. Using mercury-labeled cysteine substitution mutant bR,we revealed that the mercury position can be identified by X-ray diffraction. This technique will be utilized for the further quantitative description of the structural change in detail. Less