Proteins that bind metal cofactors, including iron-sulfur (Fe-S) clusters, are ubiquitous in nature and integral to proper cellular function, from DNA repair and tRNA maturation to cofactor biosynthesis and respiratory metabolism. Current in vitro spectroscopic techniques have provided fundamental insight into diverse aspects of metal ligation, including metal identity, oxidation state, and ligand field. Since metal-protein interactions are non-covalent and often transient and unstable, it is essential to them in a native cellular context, which to date has proven challenging. By taking advantage of the cysteine ligation sphere of Fe-S clusters, we developed a chemoproteomic platform that employs cysteine reactivity as a proxy for metal binding. A strategy coupling cysteine-reactive chemical probes, such as iodoacetamide-alkyne (IA-alkyne), with quantitative mass spectrometry (MS), resulted in the global interrogation of the bacterial Fe-S proteome. Specifically, by IA-alkyne labeling E. coli under conditions of both iron-depletion and inhibited Fe-S biosynthesis global quantification of Fe-S ligation within a native organism was achieved. This work enabled 1) resolution of ambiguity at known metal binding sites, 2) insight into the cluster transfer steps of iron-sulfur maturation in E. coli, and 3) identification of novel Fe-S proteins involved in tRNA modification, heme transport, and oxidative respiration. This is the first method to directly assess global site-specific protein-metal ligation in an endogenous system and will be a powerful tool in the field of bioinorganic chemistry to study and assign in vivo metal ligation.

Chemoproteomic strategies for probing endogenous protein-metal interactions

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.