9709418 Klebba This project addresses the transport mechanism of an Escherichia coli outer membrane (OM) protein. FepA transports the bacterial iron chelator (siderophore), ferric enterobactin (FeEnt), across the OM into the periplasm. This transport reaction is energy-dependent and TonB-dependent, involving two experimentally defined biochemical stages: binding and internalization. The molecular mechanisms comprising FeEnt binding to FepA will be elucidated; the siderophore binding site will be investigated by identifying residues that confer affinity and chemical specificity to the binding event. Using an abbreviated form of site-specific mutagenesis, termed double mutagenesis, two elements of the receptor-ligand interaction, ionic and hydrophobic bonds, will be explored. Using chemical analogs of FeEnt, chemical substitutions on the siderophore that impair or abrogate its binding to FepA will be identified. The PI will analyze conformational changes that occur in FepA during transport in vivo with electron spin resonance (ESR) spectroscopy, and study the proposed interaction between FepA and TonB with ESR by site-directed spin labeling (SDSL). Random and site-directed mutagenesis will be employed to identify regions in FepA that are important to ligand uptake after binding, and SDSL used to study the effects of mutations in these sites. Finally, the investigators will use a novel system of in vivo ESR to study biochemistry in the bacterial periplasm. These investigations will determine how an iron-containing molecule adsorbs to a receptor protein in the outer membrane of a bacterial cell. It will define the parts of the metal complex and the parts of the membrane protein that interact together during the passage of the iron through the cell membrane. Since all Gram-negative bacteria obtain iron using similar systems, the results of these studies will establish fundamental principles of binding and transport that are applicable to many different uptake systems in many diffe rent organisms. Electron spin resonance analysis of a spin labeled membrane protein (FepA) will augment general knowledge of membrane protein structure, and provide information about how such receptors function to allow entry of metal complexes. In many ways this process is akin to the opening and closing of a door on the outside of a cell, that allows the entry of certain molecules but not others; the proposed research will define the mechanisms underlying the operation of the membrane "doors."
