Structures and Mechanisms of Iron-Sulfur Proteins in Redox Control and Stress Response

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PROJECT SUMMARY Iron-sulfur (Fe-S) clusters are ancient cofactors composed of multiple iron and sulfur atoms. Owing to the rich, tunable redox reactivity and selectivity of the cluster, Fe-S proteins play multifaced roles in redox control under both physiological and stress conditions, which is required for maintaining normal cellular functions and cell survival. Thus, Fe-S proteins are tightly linked to health and diseases (such as cancer, diabetes, and bacterial infection) in humans. The parent Maximizing Investigators’ Research Award (MIRA; R35 GM138157) project addresses the critical gaps in structural and mechanistic understanding of Fe-S proteins in the cellular control of redox homeostasis, including: i) the structural basis of redox reactivity and ligand selectivity in Fe-S clusters; ii) the mechanism by which the redox state and integrity of the Fe-S cluster allows these proteins to sense redox states and regulate transcription; iii) the structural biochemistry of Fe-S cluster biosynthesis and regulation; and iv) the role of non- proteinaceous thiols in modulating Fe-S cluster-mediated redox control. The research goals and approaches of the parent project are demonstrated in the three selected Fe-S protein-mediated mechanisms for redox control and stress response in mycobacteria: i) redox sensing and transcriptional regulation by the WhiB-like (Wbl) family proteins; ii) assembly, transfer, and repair of Fe-S clusters by the sulfur utilization factor (SUF) system; and iii) mycothiol in Fe-S cluster homeostasis. This administrative supplement requests funds for the the acquisition of a size-exclusion chromatography/ multiple-angle light scattering-dynamic light scattering (SEC/MALS-DLS) instrument to identify and characterize how Fe-S proteins interact with their functional partners to regulate redox homeostasis and respond to stress. This instrument will be used to screen and characterize the interactions between Fe-S proteins and the functional partners, and to evaluate and optimize sample preparation for three-dimensional structural characterization by X-ray crystallography and single-particle cryo-electron microscopy. The implementation of this instrument within the scope and experimental workflow of the parent MIRA project is expected to provide unprecedented molecular insights and greatly expedite the mechanistic understanding the Fe-S proteins-mediated cellular control of redox homeostasis.