The long term goal of this research is to determine the molecular mechanism of light -> energy and light -> information transduction in bacterial rhodopsins. Although the generation and utilization of a trans-membrane proton concentration gradient is a fundamental element in bioenergetics, we do not know the structure or mechanism of any ion pumps. Bacteriorhodopsin (BR) is an intrinsic membrane protein found in Halobacterium halobium that functions as a light-driven proton pump, and halorhodopsin (HR) is a light- driven chloride ion pump. Both of these proteins contain a retinal prosthetic group which undergoes a 13-trans ->13-cis photoisomerization that drives ion transport. Resonance Raman vibrational spectroscopy will be used to study the chromophore and protein structural changes in the photocycles of BR and HR to determine their molecular mechanism. This work should help us to understand the mechanism of other ion pumps and of chemiosomotically-driven enzymes. Sensory rhodopsin (SR) is a similar retinal-containing protein found in halobacteria that functions as a phototactic receptor. Visible resonance Raman spectroscopy will be used to determine the structure of the chromophore in the parent pigment(s) and its intermediates. The comparison of these SR results with the mechanism of light-sensing in visual rhodopsin and of ion pumping in BR and HR should help to define the basic functional concepts of these intrinsic membrane proteins. This work will also be important in developing our general knowledge of receptor structure and function. Specific proposed experiments are: (1) Time-resolved, visible resonance Raman vibrational spectra will be obtained of the BR and HR intermediates using a two-color, pump-probe configuration as a function of pH, salt and temperature. The kinetic amplitudes vs. time will be modeled to determine the photocycle scheme. (2) Time-resolved UV resonance Raman spectra will be obtained of the different amino acid side chain and amide components of the protein to define the protein structural changes in the photocycle. BR grown on isotopic amino acids and BR site specific mutants will be used to assign and interpret these data. (3) Time-resolved visible Raman experiments will be performed on BR site specific mutants to determine how point mutations perturb the chromophore structure and interactions. This will help to define the location, orientation and interactions of the chromophore in the various intermediates. (4) Visible Raman vibrational spectra of the K, L, M, N and O intermediates will be analyzed in detail to more thoroughly define the structure of the chromophore and chromophore-protein interactions. Retinal isotopic derivatives will be used to define these assignments and to refine force field calculations. (5) Resonance Raman spectra of the SR-I and SR-II pigments and their intermediates will be obtained and used to determine the chromophore structure in each species with isotopic retinal derivatives.
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