Structure and function of bacteriorhodopsin.

Abstract

Bacteriorhodopsin (bR) is a powerful light-driven proton pump. We have developed a procedure to prepare bR-containing membrane vesicles in which a pH gradient as large as 4 pH units can be generated and maintained in the light. Using such a system, we have demonstrated that bR exhibits a high proton pump activity in a wide pH region; it works well at least between pH 4 and 9.5. For the large light-induced pH change in the external medium, the presence of a high concentration (approximately 0.1 M) of magnesium or transition metal ion is required. It is suggested that the influx of magnesium ion, electrically coupled with the proton release, takes place in the light. The three-dimensional structure of bR was studied by fluorescence energy transfer techniques. It was shown that the retinal chromophore is located 10 A below a surface of purple membrane. The in-plane location and orientation of retinal was also determined; it exists in a pocket surrounded by the helices 3, 4, 5, and 6. The position of a fluorescent probe labeled to Lys 41 was determined to be near the helix 7. Based on the results obtained, we propose a model of the bR structure. The dynamic structure of bR was investigated by fluorescence depolarization techniques. It was shown that the retinal chromophore is tightly buried in a pocket within the protein. In the presence of detergents like octylglucoside, its tertiary structure can be stable near the electric isosbestic point. The rate of dissociation/association process of bR molecules is sensitive to the pH of the medium. Dimeric and/or trimeric bR can exist stably if the concentration of detergent and other solvent conditions are adequately controlled. The photoreaction of bR in purple membrane, including the dark/light adaptation, the trans photocycle and the primary photoreaction, was reported. With respect to the trans photocycle, we found that, at alkaline pH, an M-like photoproduct (NM) is generated by excitation of a long-lived photoproduct N560 which has a major absorption maximum near 560 nm. We suggest that, at alkaline pH, the overall photoreaction of bR under steady illumination is approximated by the two-photon cycle: bR570 approximately greater than M412----N560 approximately greater than NM----bR570. With respect to the primary photoreaction, we found that the fluorescence quantum yield of the near-infrared emission of bR was greatly enhanced at acidic pH (approximately pH 2).(ABSTRACT TRUNCATED AT 400 WORDS)