We wish to understand the molecular basis of visual transduction. In particular, we intend to study in depth (1) the key light to chemical energy conversion step, the rhodopsin to bathorhodopsin transition, and (2) the first steps in the transducin chemical cascade reaction leading to rod cell hyperpolarization which is catalyzed by excited rhodopsin. In order to accomplish these goals, we propose here several experiments employing state-of- the-art spectroscopic techniques which yield very detailed molecular information. Resonance Raman and very fast (subpicosecond and picosecond) absorption and fluorescence techniques will be used to study the rhodopsin to bathorhodopsin photoreaction. The resonance Raman spectra of octopus rhodopsin and bathorhodopsin, and isotopically labelled derivatives, and the insect rhodopsin, Ascalaphus macaronius, will be obtained in order to see if the molecular concepts developed for the well studied bovine system can be generalized to different species. The rhodopsin to bathorhodopsin transition has not been kinetically resolved. Knowledge of whether or not proton translocation accompanies the rhodopsin to bathorhodopsin photoreaction and understanding the molecular dynamics of this step can only be obtained by kinetic measurements. We believe our 0.2 psec absorption spectrometer should be able to resolve this key step. The most novel work described in this proposal is the application of very sensitive classical Raman difference techniques, recently developed in our laboratory, to obtain the Raman spectra of GDP and GTP when bound to transducin, T. The exchange reaction T.GDP+GTP yields T.GTP+GDP, catalyzed by excited rhodopsin, is the first step in amplification of the light absorption event. The Raman spectra of bound GDP and GTP should provide a great deal of information on how the reaction takes place.
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