Research will continue on the stereochemical basis of biologically important activity at the cell surface. Emphasis will shift from the interfacial catalysis by phopholipase A2 to mechanisms of signal transduction by a pathway whose main components are seven- transmembrane helical receptors (7TM), heterotrimeric G-protein (Gabg) and so-called 'effectors', which are the protein targets of the signal. These systems are triggered by light, oderants, hundreds of hormones, neurotransmitters and chemoattractants. The systems extend back in evolution to the most primitive of eukaryotes where they mediate the pheromone response in the mating of yeast and the intercellular signals of amoeba. The proteins involved form huge homologous superfamilies. For example, there are over 400 oderant receptors in the human olfactory system. The approach is to solve the three-dimensional structure of the relevant proteins and their assemblies and infer from their stereochemistry their mechanism of action. The applicant has already defined the stereochemical determinants that distinguish the inactive GDP-bound form of the Ga subunit from its active GTP-bound form and found these determinants to be canonical for the entire superfamily. The applicant also has elucidated the probable mechanism of GTP hydrolysis by which Ga-GTP returns to Ga-GDP, its inactive state. It is proposed to complete the crystallographic structure/function analysis of transducin (Gtabg), the heterotrimeric G-protein of the visual system, by visualizing the Gbg subunits, the Gabg assembly and the complexed formed by Ga-GTP with its downstream target,the g-subunit of the cGMP phosphodiesterase. It is further proposed to visualize the mechanism by which ligands (or light) trigger the 7TM response and how that response is transmitted to the G-protein. To this end, the applicant intends to produce two and three dimensional crystals containing the 7TM/G-protein complex and solve their three-dimensional structure by either cryo-electromicroscopy/ diffraction (2-D crystals) or by single crystal X-ray diffraction (3-D crystals). The signalling systems to be addressed are the light sensing system (rhodopsin/transducin); the intercellular system of the amoebic slime mold, Dictyostelium discoideum (cAR1/G2abg) and the yeast alpha- mating factor response system (ste2/Gabg).
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