Mechanism of Ferric Enterobactin Uptake by FepA

University Of Oklahoma Norman Campus, Norman OK

Investigators

Abstract

Bacteria elaborate iron chelators that scavenge iron from the environment, and one such iron chelate, the siderophore ferric enterobactin (FeEnt), enters Gram-negative bacteria through the FepA protein of the outer membrane. This receptor protein is a paradigm of bacterial metal transporters, and its uptake reaction is a specific, high affinity process, accomplished by a multi-component, energy-dependent process that is prototypical of ligand-gated transport: FeEnt binding activates FepA to transport competence. On the basis of the FepA, FhuA, FecA and BtuB crystal structures, and in light of potential mechanisms of metal transport, the research will focus on a preeminent question about the uptake reaction: what is the function of the N-terminal globular domain within the transport protein's transmembrane channel? Although the molecular architecture of many membrane proteins is known from crystallography, in most cases their transport mechanisms are unresolved or controversial. The project will define the mechanism of FepA-mediated transport, that involve site-directed mutagenesis of potentially relevant target residues, genetic engineering to introduce biophysical probes, and spectroscopic characterizations of reaction kinetics and protein conformational motion. The studies will illuminate a currently obscure membrane transport process that is essential to the physiology of prokaryotic cells: the internalization of iron through the outer membrane, against a concentration gradient. In a broad sense, the project involves methodologies that permit observation of membrane transport reactions in living cells, and it thereby generally advances membrane biochemistry. It will accomplish this goal with novel biochemical and spectroscopic techniques, while integrating the teaching and training of student researchers with the overall scientific objectives. The participant students include male and female individuals at the undergraduate, pre-doctoral and post-doctoral levels at the University of Oklahoma, from a broad spectrum of ethnic and cultural backgrounds.