Bacteria exhibit sophisticated sensory behaviors which are useful models for exploring the molecular basis of sensory signaling. In Escherichia coli many chemotactic responses are mediated by a family of inner membrane proteins known as """"""""methyl-accepting chemotaxis proteins"""""""" or MCPs, which detect attractant and repellent stimuli in the periplasm and in turn modulate cytoplasmic signals that regulate the pattern of flagellar rotation. Intracellular signaling is thought to involve MCP-mediated control over the catalytic activities of CheA, an autophosphorylating protein kinase. Another intracellular protein, CheW, plays an essential, but poorly understood, role in coupling CheA to chemoreceptor control. The goal of this project is to determine how MCP molecules modulate the enzymatic properties of CheA and what role CheW plays in this process. Experiments will focus on three aspects of the chemoreceptor signaling problem: (1) Protein-protein interactions among CheA, CheW, and Tsr, the serine chemoreceptor -- Mutant proteins with altered interaction affinities will be isolated as conformational suppressors and dominant inhibitors. Their ability to bind to various target proteins will be assessed by several methods, including co-immunoprecipitation and affinity chromatography. Proteins with demonstrable changes in affinity for their targets will be analyzed with a coupled in vitro phosphorylation system to determine their effects on chemoreceptor signaling. (2) Conformational studies of MCP signaling activities -- Tsr mutants that are """"""""locked"""""""" in a clockwise or counterclockwise signaling mode will be examined by 19F NMR, by native and denaturing gel electrophoresis, and by proteolytic digestion to see if their conformations are detectably different. If unique conformational signatures are seen for the two Tsr signaling modes, their interconversion rates and equilibrium proportions will be measured in mutants with various in vivo and in vitro signaling properties to identify the parameters most important for coupling efficiency. The conformational changes induced in Tsr molecules upon interaction with CheA and CheW molecules will also be examined. (3) The mechanism of CheA control -- Parts of the CheA molecule that play a role in controlling its activity will be identified by isolating mutants that are no longer sensitive to locked chemoreceptors or which have high catalysis rates in the absence of MCPs or CheW. The phosphorylation and binding properties of the mutant CheA proteins will be examined to determine whether CheW and MCPs contribute directly to the catalytic mechanism of CheA.
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