This proposal focuses on investigating the mechanism(s) utilized to regulate the autokinase activity of CheA, a central component of the signal transduction pathway in the chemotaxis system of Escherichia coli. Responses to chemotactic stimuli are mediated by receptor/transducer proteins that regulate CheA's autokinase activity. In vitro biochemical experiments and rapid-reaction kinetic studies will be used to evaluate how this regulation is accomplished at a kinetic level. The specific goals of these experiments are: to analyze the detailed kinetic properties of transducer-coupled CheA; to quantify the effects of transducer methylation on CheA auto-phosphorylation kinetics; to monitor the adjustments of protein phosphorylation levels and transducer methylation levels that are generated when the reconstituted signaling and adaptation systems are subjected to a pH-jump stimulus; to probe the kinetics of signal transduction reactions by monitoring fluorescence changes. The results provided by these experiments will address the following hypotheses: (A) CheA operates by the same basic kinetic mechanism regardless of whether it is coupled to transducers and regardless of whether the transducers are unmethylated, partially methylated or fully methylated; (B) Regulation of CheA auto-kinase activity by transducers and by transducer methylation are accomplished by modifying the rate constant(s) of specific step(s) within the basic kinetic mechanism defined for auto- phosphorylation of CheA in the absence of transducer. In addition to these kinetic analyses, the nature of the CheA active site will be explored by testing various combinations of site-directed, kinase- deficient CheA mutants for their abilities to accomplish trans- complementation. A separate initiative to isolate CheA mutants with altered activity and/or aberrant regulatory properties will also be undertaken with the long-range goal of defining segments of CheA that play a role in determining and regulating autokinase activity. Analysis of mutant proteins will test the hypothesis that CheA has a modular organization in which a distinct segment mediates its regulation by the transducer proteins. Overall, the proposed studies will provide fundamental insight into a signal transduction pathway that senses as a paradigm for a wide variety of related prokaryotic and eukaryotic two- component systems, several of which play direct roles in microbial pathogenesis.
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