A basic feature of living cells is the ability to respond to specific, external chemical signals. Like eukaryotic cells that respond to hormones or neurotransmitters, bacteria contain specific receptors, exposed on the cell surface, that recognize relevant compounds. These cells are chemotactic as the result of the functioning of a sensory-response system that links receptors to flagella. The long-term goal of this laboratory is to contribute to a detailed molecular biological description of the chemotactic system. One important class of transmembrane receptors contains two hydrophilic domains connected by a minimal amount of hydrophobic, membrane-spanning sequence. Receptors for insulin, epidermal growth factor and bacterial chemoattractants are prominent members of this group. These receptor proteins contain multiple sites for covalent modification, implicated in sensory adaptation or """"""""desensitization"""""""". Understanding the mechanisms by which any of these receptors function will require answers to fundamental questions about how ligand binding or covalent modification at one place on a protein can affect the activity of a distant domain, how alterations at different sites on a protein can interact cooperatively or antagonistically, and how information is passed across a membrane between domains connected by the narrowest of links. It is with these goals in mind that we propose to continue our investigations of bacterial taxis. The proposal involves physiological, genetic and biochemcial approaches to the study of transmembrane receptor protein involved in chemotaxis by Escherichia coli and other bacteria. These proteins, called transducers, are central to both the excitation and adaptation phases of chemotactic behavior. Adaptation involves covalent modification of transducers, specifically protein carboxyl methylation at multiple glutamyl residue sites. Some of those glutamyl residues are present because glutamines are enzymatically deamidated to create glutamates. The functional significance of multiple methylation and deamidation will be examined by characterizing mutant proteins in which modified residues have been altered by oligonucleotide-directed, site-specific mutagenesis. The collection of mutant proteins with substitutions at modification sites and elsewhere will be purified by a procedure developed in the previous grant period and examined spectroscopically and biochemically for conformational differences that correlate with their functional states. A program of comparative biochemistry of transducers will be initiated by determining the nucleotide sequences of genes from Halobacterium halobium that were isolated by homology with E. coli transducer genes and similar approach will be used to identify, isolate and sequence transducer genes from a variety of bacterial species. An extensive project will be initiated aimed at producing crystals of transducers or transducer segments suitable for analysis by X-ray diffraction. Indications that transducer proteins share a common binding site for a small molecule will be pursued with the hope of identifying a functionally relevant biochemical activity.
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