The fundamental unsolved questions of membrane ion pumps can be studied to greatest advantage with bacteriorhodopsin and halorhodopsin, the light-driven electrogenic ion pumps in, Halobacterium halobium, the best characterized active transport proteins. Using the models we developed in the last few years for the photoreaction sequences and the energetics of ion transport in these proteins, we will describe the mechanism of specific steps of the transport cycles and the coupling between the chromophore reactions and the ion gradients created. By comparing two closely similar transport systems which transport different ions we expect to gain mechanistic and conceptual insights, and expect that these will be relevant to ionic pumps in general. A variety of approaches will be used to evaluate the role of specific amino acid residues in the structure and function of these small retinal proteins, most importantly by site-specific mutagenesis. Bacteriorhodopsins and balorhodopsins with single and multiple residue changes will be generated, using a recently developed shuttle vector and transformation system for Halobacterium halobium to introduce and express the cloned bop and hop genes. These homologously expressed proteins have different phenotypes from those expressed earlier in E. coli. The mutagenesis approach will be complemented by replacement of the retinal with retinal analogues with altered fit into the binding pocket. We will analyze the altered proteins with time-resolved optical multichannel and single wavelength spectroscopy, low temperature spectroscopy, and transport studies in cell envelope vesicles. Through collaborations FTIR and resonance Raman spectroscopy, photoelectric and electro-optical measurements, retinal analogues, and x-ray diffraction will be available to us when needed.
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