Bacteriorhodopsin and halorhodopsin are the simplest ion pumps, and prototypes of the ubiquitous seven-transmembrane-helical proteins. We have made unusual progress in the past years and now propose to test and extend the resulting detailed step-by-step hypothesis for the mechanism of proton transport. It includes not only the proton transfer steps in the protein but also the thermodynamic and structural rationales for the unidirectionality and the vectoriality of the ion translocation. All aspects of this comprehensive hypothesis are now open to critical examination, and in the proposed work we will pay particular attention to the numerous mutant phenotypes not predicted by its present version. We will continue to use our present approach of combining sitespecific mutagenesis, time-resolved spectroscopy, and high-resolution protein x-ray crystallography to investigate i) structural questions at the proton release step, ii) how the pKs of dissociable groups are modulated, iii) the nature and causes of conformational coupling, iv) the mechanism of proton transport in the absence of asp-85 and asp-96, and v) the lessons to be learned from halorhodopsin and the D85T bacteriorhodopsin mutant, that both transport chloride ions and in the opposite direction from protons. The goal of this proposal is to establish, finally, a full and explicit model for the molecular mechanism of this kind of ion pump.
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