Analysis of Receptor G-protein coupling in the signal Transduction System of Atomic Resolution.

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To elucidate the molecular mechanism of receptor G-protein coupling at atomic and/or submolecular resolution, we have investigated the structures of visual pigments and their interaction with G-proteins by means of various spectroscopic, biochemical and molecular biological techniques and the following results were obtained.1. Studies on the process of complex formation between visual pigments and G-proteins. From the low-temperature spectroscopy, we have identified rhodopsin two intermediate states that interacted with G-protein with different manners. The early intermediate can bind to G-protein but induces no GDP-GTP exchange reaction in G-protein, while the exchange reaction occurs at the later intermediate. The vibrational spectroscopy revealed the interaction site of rhodopsin with G-protein and the structural constraint of the interaction site. Using mutational rhodopsins, the protein region of rhodopsin that are essential for G-protein activation and for the selectivity of G-pr … More otein subtype, respectively, were identified.2. Structural analysis of visual pigments and analogs. The purification and crystallization conditions were examined to get a rhodopsin 3-dimensional crystal for X-ray crystallography and the diffraction pattern of the crystal was obtained. Using 2-dimensional crystal of bacteriorhodopsin in combination with electron cryo-microscopy, the 3-dimensional structure of bacteriorhodopsin was obtained. The large scale expression of pharaonis phoborhodopsin, the receptor protein for phototaxis in bacteria, were performed in E-coli and amino acid residues responsible for the spectral tuning and thermal behavior were identified.3. Identification of the novel photoreceptor proteins and functional analysis of the regulatory proteins in the signal transduction cascade. The photoreceptive proteins present in deep brain (hypothalamus) were newly identified by mean of cDNA cloning. The functional analysis of pinopsin, the photoreceptive protein in chicken pineal, was performed in comparison with those the visual pigments in retinas. Also, S-modulin, the regulator protein to kinase, and phosphodiesterase were characterized. Less