This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Organisms causative of diseases such as respiratory tract infections (Haemophilus influenzae), enteric conditions (Shigella dysenteriae) and the opportunistic Pseudomonas aeruginosa have developed sophisticated mechanisms for sequestering iron from their host. This intense competition between invading pathogens and their host for the nutrient has led to the idea that new antimicrobials may target iron acquisition and homeostasis. To study this idea more closely, it is important to gain molecular-level understanding of the mechanisms by which pathogens manage iron, from acquisition and internalization to storage and utilization. Significant advances have improved our understanding of iron uptake by P. aeruginosa and many other pathogens. In comparison, little is known about the fate of internalized iron. One mechanism whereby iron toxicity is controlled is by storage of iron in ferritin and bacterioferritin, which are large proteins capable of storing up to 4,000 iron atoms in their internal cavities. Despite the importance of ferritins and bacterioferritins in regulating iron concentrations and preventing its toxic effects, little is known about the processes that deliver Fe2+ for storage or the signals that prompt its release for safe integration in metabolism. We have recently demonstrated that mobilization of Fe2+ from bacterioferritin A (BfrA) in P. aeruginosa requires electron transfer from a ferredoxin reductase (FPR). Thus the BfrA-FPR complex is an unprecedented opportunity to investigate how bacterioferritins recognize their physiological regulators and if binding modulates the dynamic properties of the bacterioferritin to facilitate iron release. To fill these gaps we plan to: (1) Investigate the dynamic properties of BfrA utilizing a strategy specifically tailored to study large proteins using hydrogen/deuterium H/D exchange coupled to NMR spectroscopy. (2) Investigate the dynamic properties of BfrA with the aid of computational methods and (3) Utilize computational and HD/NMR methods to investigate how BfrA binds to FPR and determine the effect that the inter-protein association exerts on the dynamic properties of BfrA.
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