We propose an organochemical approach to determine molecular recognition elements that the visual photoreceptors employ to trigger the cascade of biochemical events, which culminate in neural responses. We are specifically interested in: 1) the nature of molecular events at the retinal chromophore's binding site in rhodopsins that enables the ultrafast photoisomerization and wavelength regulation, 2) the mechanisms of conformational changes of the rhodopsin, initiated by retinal isomerization, that in turn create a binding domain in the tertiary structure of rhodopsin, structurally optimized for photoactivated rhodopsin - G protein (transducin) interactions. Concerted efforts of several disciplines have been successful in elucidating many aspects of this information processing system but the tertiary structures of rhodopsin and its photoactivated form are not known. Knowledge of these tertiary structures are however, a prerequisite for understanding the mechanism of the sequence of events in a structural perspective, at the molecular level, 3) the structural make-up of the G protein binding site that requires guanine nucleotides and hitherto was difficult to investigate due to the lack of appropriately labeled nucleotide analogs. We propose to achieve our goals by incorporation of synthetic probes into rhodopsins and G proteins followed by studies of the properties of functional rhodopsin and G protein analogs generated. In the case of rhodopsin, novel type of spacer-armed retinals will be employed, and in the case of G protein, isotopically labeled and site specifically modified GDPs will be used as probes of the protein's structure. The structural information obtained from employing the probes described in this project will open up a whole new area of exploratory studies. For instance, development of domain-specific rhodopsin-immunoconjugates and site-specific rhodopsin-peptide conjugates will be possible. It will also pave the way to site-specific conjugation of rhodopsins to effect their crystallization and to effect their site-specific cleavage. In the case of the G protein, Raman spectroscopic investigation employing isotopically labeled GDP analogs will yield insight into the solution structure of the interaction domain(s) of the G?? with the protein. The organochemical approach in this project will thus have a major impact in the area of sensory transduction, where molecular level investigations can make unique contributions towards the elucidation of the cellular events in precise structural terms, by identifying the molecular domains and chemical mechanisms involved in the activation of excitable membranes.
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