INVESTIGATING STRUCTURAL BASIS OF SIGNAL TRANSDUCTION

点击翻译按钮获取中文摘要

We are studying the structure and function of signal transduction proteins, specifically heterotrimeric GTP-binding proteins (G proteins) and G protein-coupled receptors (GPCRs). When an agonist such as a hormone or neurotransmitter binds its receptor, exchange of GTP for GDP bound to the G protein is catalyzed. The GTP-bound alpha subunit of the G protein separates from the beta-gamma subunit complex, and each of these can go on to stimulate downstream effectors. The hydrolysis of GTP to GDP by the alpha subunit and subsequent hterotrimer reassembly turns off the signal. While the structures of some G protein subunits have been solved, atomic-level structures have not been determined for the receptors. Our specific goals are to understand the structural basis of function by designing and evaluating mutant forms of the G proteins and receptors. One category of mutants will contain two or more histidines placed so that they could be bridged by a metal ion. Such a link could cause metal-dependent activation or inactivation of the protein. In known structures, this would enhance our knowledge of the activation process; in unknown structures, this would also yield information on the proximity of regions of the protein. Another category of mutants arises from random mutagenesis. Experiments include purified protein assays, functional assays in mammalian cell lines, and a yeast growth assay where the "readout" is a large number of receptor sequences capable of signalling. The Computer Graphics Laboratory facilities are crucial for many steps of this research: viewing crystallographic or modelled structures to design mutants, to rationalize the properties of the mutants, to develop hypotheses about protein-ligand and protein-protein interactions, and to develop hypotheses of conformational changes involved in activation and deactivation.