The long term goal of the project is to elucidate the mechanism of visual excitation in vertebrate photoreceptor cells.
The specific aim focuses on the coupling action of transducin, a GTP-binding protein that regulates the flow of information from photo-activated rhodopsin to the cGMP phosphodiesterase. The proposed studies are an outgrowth of pervious results obtained from the laboratory. (1) The molecular structure of transducin will be investigated. The locations of specific chemical modification sites on the T-alpha subunit of transducin will be identified by peptide sequencing. Distances between these sites will be estimated by fluoresence energy transfer measurements and by in situ cross-linking experiments. A topological structure of T-alpha will be constructed based on these measurements and compared to a proposed three-dimensional model of T-alpha [Hingorani and Ho, FEBS Letter 220, 15-22.(1987)]. (2) The role Mg2+ on the coupling action T-alpha will be examined. Mn2+ will be substituted into the Mg2+ binding site and the coordination of the bound Mn2+ will be investigated by electron spin resonance spectroscopy via the interactions with 17O labeled guanine nucleotides and 17O-enriched water molecules. (3) A major effort will be put into elucidating the activation mechanism of the cGMP phosphodiesterase. Non-inhibitory photoaffinity probes will be attached to the T-alpha-Gpp(NH)p complex and the Pgamma subunit of phosphodiesterase, transforming these proteins into a photoaffinity labeling molecule. The interacting sites between T-alpha- Gpp(NH); and Pgamma will be probed by cross-linking and peptide mapping studies. Furthermore, the changes of the hydrodynamic properties of the PDE-T-alpha-Gpp(NH)p complex associated with the activation process will be examined by sedimentation studies. The results should provide an understanding on the molecular mechanism of signal transduction mediated by GTP-binding proteins.
