Fig. 2: Quantification of ubiquitin and Fubi C-terminal hydrolase activity of USP DUBs with a fluorogenic Fubi-RhoG substrate.
From: Molecular basis for ubiquitin/Fubi cross-reactivity in USP16 and USP36
a, Schematic representation of the semisynthesis of the fluorogenic substrate Fubi-RhoG and its enzymatic turnover; 2G-Rho, 2G-Rhodamine; NHS, N-hydroxysuccinimide. b, Purification of Fubi-RhoG through optimized anion exchange chromatography. Fubi-RhoG-containing fractions are indicated in light purple and were evident from the 254-nm UV absorbance signal as Fubi lacks tryptophan and tyrosine residues; AU, arbitrary units. c, Structure and intact protein MS analysis of purified Fubi-RhoG; Calc., calculated. d–i, Quantification of ubiquitin/Fubi C-terminal hydrolase activity. Ubiquitin-RhoG or Fubi-RhoG (at 100 nM) were incubated with catalytic domains of the indicated purified enzymes, and fluorescence emission was recorded for USP16 (d), USP36 (f) and USP7 (h). Data are shown as the averages of three replicates. Observed rate constants were then plotted over enzyme concentrations to determine catalytic efficiencies for ubiquitin-RhoG and Fubi-RhoG processing by USP16 (e), USP36 (g) and USP7 (i). Data are shown as the results of curve fitting with associated s.e.m. Results were confirmed in three independent experiments.
