We will continue our studies of the light-driven chloride ion pump, halorhodopsin, and attempt to elucidate its structure, photochemical reactions, and the mechanism of the ion translocation. Our central hypothesis at this time is that the two chloride-dependent equilibria detected in the photocycle scheme correspond to the uptake and release sites on the two sides of the protein, across the membrane barrier. We intend to critically test this hypothesis, elaborate it into a general model for the chloride pump, and provide experimental evidence for stating it in more molecular terms. A structural model for the protein (MW 25,000), based on the recently determined amino acid sequence, will be refined, as to the location of helices and loops. We will attempt to localize the three spectroscopically detected chloride binding sites relative to protein residues and the retinal, and to establish the role of these sites in the transport. The chloride translocation event in the photocycle will be identified, in relation to the configurational changes of the retinal. Conformational changes in the protein will be sought, with possible relevance to chloride binding and release. The role of oligomeric structure in functional properties will be analyzed.
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