The purpose of the proposed research is to describe in molecular detail the chemical and stereochemical mechanism of G-protein mediated hormone signal transduction. Specifically, X-ray crystallographic techniques will be used to determine the three-dimensional structures of G-protein alpha subunits, the active components of the transduction circuit, which, in the GTP bound state, interact with and activate specific effector molecules. The proteins of interest will be produced and purified from recombinant E. Coli expression systems. Crystals of selenomethionylated Gi-alpha1 have been prepared in order to employ multiwavelength anomalous dispersion techniques in combination with heavy atom methods in x-ray phase determination. The research will focus primarily on Gi-alpha, the G- protein alpha subunit that activates cardiac potassium channels in response to muscarinic receptors. Three-dimensional structures shall be determined of the inactive, GDP bound conformation of this subunit as well as the active GTP bound form stabilized by nonhydrolyzable guanine nucleotide analogs. Structures shall also be determined of mutants of Gi- alpha that lack intrinsic GTPase activity or that are unable to undergo certain conformational changes. The goal of these studies will be to identify the conformational change from the inactive to the active state. Structural studies of oncogenic muteins may aid in the design of pharmacotherapeutic agents. These studies will define the interaction between G-proteins and receptors that triggers signaling.
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