The system of regulatory proteins that mediates chemotaxis in E coli and S typhimurium provides a useful model for understanding the biochemistry of signal transduction. Chemical stimuli induce conformational changes in membrane receptors at the cell surface that control a network of 6 interacting signal transduction proteins within the cytoplasm. CheY is a phosphorylated regulator that interacts with the motor to control swimming behavior, CheA is a protein kinase, CheZ is a phosphoprotein phosphatase, CheR is a protein methyltransferase, CheB is a protein methylesterase, and CheW is a component of the receptor-kinase complex. The objective of the proposed research is to determine how these 6 Che proteins interact with one another to transduce information from the receptors into an appropriate behavioral output. The enzymology of the Che proteins will be characterized under defined conditions with purified components. The mechanism of kinase activation by the receptor will be determined by investigating the kinetics of CheA autophosphorylation using a variety of stopped-flow and steady-state techniques. A coupled spectrophotometric assay will be used to follow the conversion of ATP to ADP in this reaction; and the rate of ATP/ADP exchange will also be measured by isotope exchange. The receptors are composed of an extracytoplasmic sensory domain connected via a transmembrane linker to a cytoplasmic signaling domain, and genetic studies indicate that the signaling domain can activate the kinase in constructs where the sensory and transmembrane linker regions have been deleted. Attempts will be made to use the purified signaling domain of the receptor to generate a soluble kinase complex, and conformational transitions associated with complex formation, nucleotide binding, and phosphorylation will be assessed using spectroscopic probes as well as by detecting alterations in protease sensitivity and monoclonal antibody binding. Stopped-flow fluorescence techniques will be used to analyze the CheY phosphotransfer mechanism, and efforts will be made to better define the interaction between CheY~P and the flagellar motor. Experiments will be undertaken with purified and reconstituted components to determine how CheZ activity is regulated, and understand how the methylating and demethylating enzymes function to cause adaptation. Specific structure-function issues will be addressed using molecular genetic techniques. Expression vectors will be engineered to produce defined subdomains, and site-directed mutagenesis will be used to investigate the roles of individual residues. These studies will parallel ongoing X-ray crystallographic research on these proteins.
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