EVIDENCE FOR A CONTROLLING ROLE OF WATER IN PRODUCING THE NATIVE BACTERIORHODOPSIN STRUCTURE

The experiments reported in this paper, based on reconstitution of bac teriorhodopsin (bR) from apomembrane at varying environmental conditio ns, demonstrate that the presence of water is a controlling factor in generating a native wild-type bR conformation. If water is lacking dur ing this reconstitution process, then a non-native bR structure is for med that exhibits altered M formation and decay kinetics, as well as d ifferent behavior following extensive dehydration, It is shown that mu tants affecting the ability of bR to form appropriate structures of wa ter in specific protein cavities also affect the ability to generate a native bR conformation, The results suggest that aspartic acid 96 pla ys a major role in anchoring the appropriate water structure conformat ion associated with bR. It is also demonstrated that the glutamic acid 204 residue is pivotal in controlling the protein/water affinity. Thi s water affinity can be further controlled by modifying the charge env ironment of the protein with altered pH. These data, based on kinetic absorption spectroscopy and Fourier transform infrared spectroscopy, h ighlight the central role of water in this protein.