Kinetic and Thermodynamic Studies Relevant to Biological Iron Transport

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This award in the Inorganic, Bioinorganic and Organometallic Chemistry program and the Molecular and Cell Biology Biochemistry program supports research by Provessor Alvin Crumbliss at Duke University to investigate mechanisms for biological Fe transport mediated by siderophores and two periplasmic proteins, ferric binding protein (FbpA) from Neisseria and periplasmic binding protein (PBP) from Pseudomonas. Specific aims are to: explore a redox switch hypothesis for Fe transport whereby Fe/ligand exchange is initiated by Fe3+ reduction, investigate the role of the outer membrane receptor TbpA/B in removing Fe from Fe2hTf , investigate the role of the TbpA plug domain in inserting Fe into FbpA, investigate the role of the synergistic anion (X) in inserting Fe into FbpA and in modulating the biophysical properties of FeFbpA-X, use an ex vivo method to determine the in vivo synergistic anion changes in FeFbpA-X with variations in cell growth environment, investigate Ga3+ sequestration by FbpA to probe the mechanism of periplasm-to-cytosol Fe transport, investigate the molecular interactions between the FpvAI-plug domain, pyoverdine, and a periplasmic binding protein that are relevant to Fe transport in Pseudomonas, investigate the relationship between Fe and Zn sequestration by brasilibactin A and the biological activity and signaling functions of this siderophore, investigate the coordination and redox chemistry of a siderophore conjugate designed to initiate intracellular Fenton chemistry, and directly correlate in vitro results with in vivo studies performed in biological collaborators laboratories. In vitro and ex vivo biophysical methods will be used to achieve the objectives. SUPREX analysis of MALDI-TOF mass spectra will be used to characterize metal-protein, protein-protein, protein-siderophore, and protein-anion interactions, and their influence on Fe/ligand exchange. Recombinant WT and mutant proteins, including plug domains from membrane receptors, will be used to explore individual steps in the transport process. This project enables mentoring minority students through Project SEED, high school teacher workshops, and four-year college seminars. Research results on Fe transport are used to illustrate principles taught in General Chemistry. This research program involves extensive collaboration with five US and international biological laboratories, which will enhance the interdisciplinary and global research experience of students in the PI's and collaborators' laboratories, thereby broadening contributions to education and scientific infrastructure.