FepA is an 81 kDa outer membrane protein responsible for iron uptake in E. coli and many other gram-negative bacteria via the binding and transport of its cognate ligand, ferric enterobactin (FeEnt). Since the ability of microorganisms to compete effectively for limited supplies of iron in the host is a key factor in the development of pathogenicity, a detailed understanding of the components and mechanisms involved in iron uptake is of significant medical importance. Electron spin resonance (ESR) spectroscopy is able to provide information on the structure and dynamics of large membrane proteins such as FepA that is not accessible by other techniques. In particular, site-direct spin labeling ESR has emerged as a powerful technique for mapping protein structure and investigating functional dynamics. Based on previous observations of conformation changes in FepA upon ligand binding, highly cooperative unfolding of the ligand-binding surface loop, and spin-label mapping of an entire transmembrane beta- strand, the investigators hypothesize that FepA functions as a ligand-gated porin with surface loops that bind the ligand and regulate access to an underlying transmembrane channel and that interactions between surface loops and residues in the transmembrane domain play an important role in the structural organization of the receptor. To test this hypothesis, the investigators will 1) evaluate the proposal that FepA is a ligand- gated beta-barrel receptor utilizing cysteine-scanning mutagenesis and site-direct spin labeling, 2) analyze pairs of spin-labeled cysteine residues to characterize loop-channel interactions and determine strand proximity in the beta-barrel domain, 3) employ time-domain ESR methods to measure distances between an array of spin-labeled sites and bound FeEnt, 4) identify ligand-induced conformational changes in purified, reconstituted FepA, and 5) selectively label FepA in live bacteria and examine conformational changes that occur upon FeEnt binding and transport. This research will provide insights into the structure and location of the ligand-binding site in FepA, the structure of the transmembrane beta-barrel, and the conformational changes that accompany ligand binding. These studies may significantly advance understanding of the structure and dynamics of ligand-gated bacterial outer membrane receptors.