Bacteria elaborate iron chelators that scavenge iron from the environment, including their human and animal hosts. Iron acquisition is a determinant of pathogenicity. One such iron chelate, the siderophore enterobactin, enters gram-negative bacteria through the FepA protein of the outer membrane. FepA is a ligand-gated porin, in that binding of ferric enterobactin triggers transport through its transmembrane pore. This high affinity multispecific, multicomponent, energy dependent transport process is a paradigm of prokaryotic membrane biochemistry. Based on the FepA crystal structure, the proposed research will use molecular biological, biochemical, and biophysical methods to investigate the mechanism of ferric enterobactin uptake. The experiments will address two stages of the transport event, binding and internalization. Dr. Klebba will study the specificity of the initial recognition event by binding experiments on both wild type FepA and site-directed mutants, containing alterations to residues in either the external loops of the top loops of the N-terminal globular domain. He will similarly characterize the ligand internalization reaction by mutagenesis of target residues that are conserved among other Gram-negative bacterial ligand-gated porins. Mutant proteins of interest, those with impaired ligand binding or ligand internalization phenotypes, will be crystallized and studied by X-ray diffraction Finally, he will perform biophysical analyses of conformation changes that occur in FepA during ligand transport.
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