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. 2021 Apr 22;143(17):6423–6433. doi: 10.1021/jacs.0c10513

Figure 3.

Figure 3

Validation of covalent adduct between Rho-Ub-alkyne ABPs and catalytic Cys205 in USP16CD. (A) In-gel fluorescence (top) and Coomassie stain (bottom) of purified recombinant USP16CDWT or mutant USP16CDC205A incubated with ABP (Rho-Ub-Prg, Rho-Ub-2, or Rho-Ub-5). Adduct is formed with USP16CDWT, but preincubation with the thiol-alkylating reagent N-ethylmaleimide (NEM) prior to incubation with ABPs blocks adduct formation, indicating cysteine thiol is required for adduct formation. Adduct is not observed with USP16CDC205A, identifying catalytic Cys205 as the modified cysteine residue. (B) Deconvoluted mass from intact protein MS confirms the covalent adduct (+8.9 kDa) of USP16CDWT with Rho-Ub-Prg, Rho-Ub-2, and Rho-Ub-5. (C) Covalent adduct is not observed in deconvoluted mass from intact protein MS for catalytically inactive mutant USP16CDC205A with Rho-Ub-Prg, Rho-Ub-2, or Rho-Ub-5.