Quantitative reactivity profiling predicts functional cysteines in proteomes

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Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quantitatively the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulphur protein biogenesis. We also demonstrate that quantitative reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.

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Figure 1: A quantitative approach to globally profile cysteine reactivity in proteomes.
Figure 2: Hyper-reactive cysteines are highly enriched in functional residues.
Figure 3: Functional characterization of the hyper-reactive cysteines in PRMT1.
Figure 4: Functional characterization of YHR122W/FAM96B.
Figure 5: Quantitative reactivity profiling predicts functional cysteines in designed proteins.

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We would like to thank T. Bartfai, I. Wilson and members of the B.F.C. laboratory for comments and critical reading of the manuscript, T. Ji for experimental assistance and J. Gallaher for expression of designed proteins. This work was supported by the National Institutes of Health (CA087660, MH084512), a Pfizer Postdoctoral Fellowship (E.W.), a Koshland Graduate Fellowship in Enzyme Biochemistry (G.M.S.), a National Science Foundation predoctoral fellowship (D.A.B.) and the Skaggs Institute for Chemical Biology.

Author information

B.F.C., E.W. and C.W. conceived the project and E.W. and C.W. performed MS experiments and yeast growth/Leu1 assays. C.W. and G.M.S. performed computational data analyses. S.K., F.R. and D.B. performed computational design of cysteine hydrolases and measured activity using a fluorogenic assay. D.A.B. purified PRMT1 and M.B.D.D. and K.M. performed PRMT1 activity assays. B.F.C., E.W., C.W. and G.M.S. analysed data and wrote the manuscript.

Correspondence to Benjamin F. Cravatt.

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Weerapana, E., Wang, C., Simon, G. et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790–795 (2010) doi:10.1038/nature09472

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