The research will examine the mechanism of ferric enterobactin passage through its Escherichia coli outer membrane receptor,FepA. Binding, in which the siderophore or colicin associates with specific residues of FepA that are located on the external surface of the bacterial cell; Uptake, in which the receptor, by interaction with TonB, undergoes conformational change or other mechanisms, facilitates the movement of ferric enterobactin through the OM bilayer. A variety of biochemical, molecular genetic, and immunological approaches to these problems will be utilized, including random and site-specific mutagenesis, genetic selection for FepA proteins that function independently of TonB, and immunochemical purification of cell envelope proteins that may physically associate with FepA during ferric enterobactin uptake. These investigations will enhance understanding of the mechanism by which two distinct secreted proteins, colicins B and D, and a siderophore, ferric enterobactin, interact with a single receptor, FepA. They will augment knowledge of bacterial outer membrane protein structure, and provide specific information about the physiology of TonB-dependent outer membrane proteins. %%% The research will determine how a bacterial membrane receptor protein recognizes and transports small molecules into the cell. The membrane protein of interest, Escherichia coli FepA, is essential for the acquisition of iron. Theoretically, the structure of this receptor is akin to a barrel with a lid, that usually remains closed to protect the cell from noxious molecules in its environment, but can open to permit entry of iron-containing nutrients. To test this model experiments that subdivide the transport process into two stages have been designed: Binding, in which the receptor protein recognizes iron-containing molecules and holds them on the cell surface. Uptake, in which the iron is transferred across the cell membrane, presumably by moving through the 'lid' region of the receptor, and into the barrel domain. A variety of biochemical, molecular genetic, and immunological approaches to these problems will be utilized. The experiments will enhance understanding of the mechanism by which small molecules are recognized and captured by bacterial cells. They will be also improve knowledge of the structure of membrane proteins, and the transfer of energy across membranes.
