The proposed research biochemically, genetically, and immunologically examines the interaction between the FepA protein from the Escherichia coli outer membrane, and the molecules that utilize FepA for penetration into the bacterial cell. These include colicins B (colB) and D (colD), and the siderophore ferric enterobactin. It has three primary objectives: (1) using immunochemical methods, to physically define the structural domains that are involved in binding events between the FepA protein and colicins, and between FepA and ferric enterobactin, (2) using mutant proteins, to identify coding regions within the FepA and colB structural genes that are responsible for the binding events between the molecules, (3) to establish whether colicins B and D possess identical or different structures for binding to FepA. These studies involve the following experimental methods: (1) purification of FepA, colB, and colD by established biochemical and immunological protocols, (2) isolation of anti-FepA and anti-colB mouse monoclonal antibodies from hybrid B-cell lines, and construction of epitope/function maps for each protein, (3) genetic and immunochemical analysis of binding events between FepA and its ligands. The proposed investigations will lead to a knowledge of the protein structures that are involved in specific receptor-ligand recognition events in the bacterial envelope. They will enhance understanding of the mechanism by which two distinct secreted proteins, colicins B nand D, and a siderophore, ferric enterobactin, interact with a single receptor, FepA, and they will evaluate the utility of antibodies in the structural characterization of membrane proteins. These studies are medically important because they focus on the biochemistry of iron absorption, an element which is essential to both the virulence of microbial pathogens and the defense mechanisms of mammalian hosts against infection. The experiments furthermore define a model system for drug delivery to specific cells, and create a panel of monoclonal antibodies which may be clinically useful.
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