The electron spin resonance site-directed spin labeling technique is a powerful method for the biophysical characterization of proteins. The ability to introduce cysteine labeling sites at desired locations, rather than relying on endogenous cysteines, makes the spin labeling approach both general and systematic. In this proposal we describe the application of site-directed spin labeling to the Escherichia coli ferric enterobactin receptor, FepA. FepA is an 81 kilodalton transmembrane protein that transports iron across the outer membrane against a large concentration gradient in a manner that is dependent on the proton motive force across the bacterial inner membrane, and the inner membrane protein TonB. Since the assimilation of iron by microorganisms is a key factor in their survival and the development of pathogenesis, a detailed understanding of the components and mechanisms of microbial iron uptake is of significant medical importance. This has been emphasized by the recent proliferation of antibiotic resistance in pathogenic bacteria. We have advanced the hypothesis that FepA functions as a gate porin, with a ligand-binding domain that functions both in the high affinity recognition of ferric enterobactin and in controlling access to an autonomous transmembrane channel. The proposed studies will further clarify this hypothesis by providing a structural characterization of the ligand-binding domain, and by examining ligand-induced conformational changes in the receptor. Specifically, we will 1) utilize site-directed mutagenesis to introduce cysteine residues into the putative extracellular ligand-binding domain, 2) evaluate the effects of these mutations on receptor function and global topology, 3) label the introduced cysteine residues with sulfhydryl-specific spin labels and use electron spin resonance spin labeling methods to examine the biophysical characteristics of the labeled sites, 4) examine conformational changes induced by the binding of the ligand, and 5) map distances from the bound spin labels to the bound paramagnetic ligand, Fe3+ -enterobactin. It is our expectation that the systematic application of this approach will allow the development of a comprehensive understanding of the ligand- binding domain of FepA, and will contribute to our knowledge of the basic mechanisms of ferric enterobactin uptake.
