Extended Data Fig. 3: Biochemical characterization of USP30 chimeras.
a, Protein stability assessment of USP30 constructs with thermal shift assays. Mean (N = 3 independent replicates). b, Ubiquitin probe reactivity assay. Samples were analyzed by SDS-PAGE and Coomassie staining. c, Changes in protein stability upon binding to Ub-PA. ΔTm was calculated as Tm (Ub-PA-bound) subtracted from Tm (apo protein). Mean ± s.d. (N = 3 independent replicates). d, Quantification of enzyme activity. Varying concentrations of USP30 proteins were incubated with Ubiquitin-RhoG substrate and fluorescence was recorded. e, Observed rate constants derived from plots in d were plotted over enzyme concentrations (upper panel) to derive catalytic efficiencies (lower panel). Mean ± s.e.m. (derived from curve fitting, with data for each concentration recorded as N = 3 independent replicates). f, Inhibitory potencies of Compound 39 and NK036. Compounds were pre-incubated with USP30 constructs for 1.5 h, and remaining activities were determined from Ub-RhoG cleavage assays. Mean ± s.d. (N = 3 independent replicates). g, IC50 values of assays shown in f. Mean ± s.e.m. (derived from curve fitting). h, Assessment of binding of Compound 39 and NK036 to indicated USP30 constructs by thermal shift assays. ΔTm was calculated as Tm of the inhibitor-bound sample subtracted from the Tm of the apo protein. Mean ± s.d. (N = 3 independent replicates).
