A molecular understanding of the role of periplasmic binding proteins in active transport and chemotaxis in bacteria requires a detailed pictures of this family of proteins. Our long range goal is to determine the structure and function of several binding proteins which serve as initial receptors for transport and chemotaxis, two important biological process. Accordingly, an x- ray structural analysis of seven different binding proteins - at least two from each of the major groups with specificity for carbohydrates, inorganic anions, and amino acids - has been initiated. This goal is attainable because considerable success in solving the three-dimensional structures of six binding proteins specific for L-arabinose, D-galactose, D-maltose, sulfate, leucine/isoleucine/valine and leucine has recently been achieved. The phosphate-binding protein has recently been crystallized. These structural analyses have also revealed novel features of the mode of binding of sugars, sulfate anion and amino acid zwitterion. The major goal of the proposed research is extend the structural analysis of four binding proteins to better than 2 sugar A resolution and the solve the structure of the phosphate - binding protein. The binding of different sugar substrates and sugar derivatives will be examined in detail by high resolution structure analysis. Other correlative studies will also be undertaken. The contribution of each sugar hydroxyl interaction to the overall affinity of protein-sugar complexes will be assessed with various deoxy/fluoro substituted monopyranosides as probes. Binding of these probes will be assessed by crystallographic, equilibrium and kinetic binding, and theorteical techniques. With the three- dimensional structures providing a solid foundation, unique structural and functional features in the binding proteins will be further explored by site-directed mutagenesis to generate proteins with specific alterations at desired points in the primary sequence of the protein. Mutant proteins produced will be isolated and characterized extensively with regard to their binding activity, physical and chemical properties. Structures of mutants will also be determined for direct comparison with the parent wild-type structures.
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