Metal-binding proteins are ubiquitous and often play an integral role in cellular function, from the iron-sulfur clusters of eukaryotic DNA polymerases to the complex metallocofactors of bacterial nitrogenases and hydrogenases. While current spectroscopic techniques can report on diverse properties of metal-protein interactions, including metal identity and oxidation state, a major limitation is that in order to obtain site-specific information, these studies must be performed in vitro. As these metal-protein interactions are non-covalent and can be transient and unstable, it is important to be able to assess these interactions in their native cellular context. By taking advantage of the fact that many of these metal cofactors are bound to proteins through cysteine ligation, we have developed a platform to use cysteine reactivity as a proxy for metal binding. Coupling cysteine-reactive chemical probes, such as iodoacetamide-alkyne, with quantitative mass spectrometry (MS) it is possible to globally interrogate the metal-binding cysteine proteome, monitoring a loss of cysteine labeling as a readout of metal binding. We have recently been able to demonstrate that IA-alkyne labeling of E. coli under iron-replete or iron-depleted conditions allows for the proteomic identification and quantification of Fe and Fe-S cluster ligating cysteine residues. This is the first method to directly assess global site-specific protein-metal ligation in an endogenous system. The platform is capable of resolving ambiguity at metal binding sites, as well as identifying potentially novel sites of metal-ligation. Additionally, the examination of gene knock-outs has provided information on the iron-sulfur maturation pathway in E. coli. This platform will be a powerful tool in the field of bioinorganic chemistry to study and assign in vivo metal ligation.

Chemoproteomic interrogations of endogenous bacterial metal-ligation

256th National Meeting (2018)

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