Macromolecular juggling for a prokaryotic Fe-S assembly system
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With this award, The Chemistry of Life Processes Program in the Chemistry Division is funding Dr. David Barondeau from Texas A&M University to investigate the enzymology of iron-sulfur (Fe-S) cluster biosynthesis. Iron-sulfur (Fe-S) clusters are one of the most versatile auxiliary cofactors used by proteins to facilitate biological processes, and there are literally hundreds of different examples. Many of these Fe-S proteins have unknown functions. Remarkably, despite tremendous diversity in the structures of these proteins, conserved sets of assembly proteins serve to build and deliver Fe-S clusters to all Fe-S containing proteins. These elaborate multi-component systems have evolved to protect organisms from the toxic effects of free iron and sulfide ions while promoting the efficient biosynthesis of these cofactors. One of the major limitations in studying Fe-S cluster biosynthesis has been the lack of specific probes that allow real-time analysis of cluster synthesis and transfer reactions. To overcome this limitation, labeling methods were developed that directly report on Fe-S content for labeled proteins in a background of unlabeled Fe-S proteins and other species. This methodology facilitates advanced time dependent experiments with complex Fe-S assembly assays, which are required for the rigorous evaluation of the role of protein components in Fe-S cluster biosynthetic pathways. This project will train graduate and undergraduate students in biochemical methods, protein labeling, and enzymology. High school students will also gain exposure to science. Overall, the proposed studies outlined in this grant are designed to provide insight into the chemistry of this important assembly pathway and generate robust tools that will be useful in investigating the function of other Fe-S containing systems. Currently, there is limited mechanistic information about how this system synthesizes different types of Fe-S clusters, identifies and discriminates protein targets for cluster transfer, and mediates ligand exchange from the assembly complex to target proteins. Fluorescent tagging approaches were developed in which the fluorescence is sensitive to different cluster types, ligand environments, and cluster oxidation states. This fluorescent reporter technology will be instrumental in untangling the intricate network of transfer pathways and identifying factors affecting flux through branch points. High sensitivity, suitability with high-throughput methodology, and compatibility with anaerobic techniques are additional benefits of this approach. Here a complex biosynthetic assay will be developed and used to test specific hypotheses for the roles of accessory proteins, chaperones, and regulatory proteins in Fe-S cluster biosynthesis. Fluorophore labels also permit anisotropy experiments that monitor protein association and dissociation during cluster assembly and transfer reactions. This fluorophore labeling methodology will contribute to the study of Fe-S cluster biosynthesis by tracking both the composition of the protein complex and the protein the Fe-S cluster is bound to during transfer reactions.