Extended Data Fig. 5: Ubiquitin-primed CRL substrate ubiquitylation enables estimation of the rate of poly-ubiquitin chain formation kobs.
From: Mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases
a, Fluorescence-scanned gel showing ubiquitylation reactions comparing wild-type (WT) and S138D UBE2R2 activity with Hif1α peptide substrate (see methods). Notice that, while S138D UBE2R2 is highly defective at poly-ubiquitin chain formation, it can prime substrate, facilitating production of highly pure Sil1-ubiquitin substrate. The scan is representative of duplicate technical replicates. UB, ubiquitin. b, Schematic of the production strategy for substrate-ubiquitin synthesis. c, Coomassie-stained SDS-PAGE gel showing purification of Sil1-ubiquitin. Following the ubiquitylation reaction (R), Sil1-ubiquitin was first separated from unreacted peptide owing to ubiquitin’s N-terminal Histidine tag (Ni) followed by gel filtration chromatography. The gel is representative of triplicate technical replicates. SEC, size exclusion chromatography. d, Illustration of the Rapid Quench Flow (RQF) device (Kintek) used to estimate the rates of poly-ubiquitin chain formation (kobs) on ubiquitin-primed CRL substrates by UBE2R2. Timepoints are computer-controlled according to the speed of a plate pushing against the syringe plungers, first mixing the reactants followed by introduction of the quench buffer. e, Autoradiogram showing a time course from quench flow, pre-steady state kinetic ubiquitylation reactions with neddylated CRL2FEM1C. The gel migrations of radiolabeled, ubiquitin-primed Sil1 substrate (indicated by *) and ubiquitylated product are shown (top). The graph shows the fraction of remaining substrate as a function of time in the presence of WT proteins (bottom). The autoradiogram is representative of triplicate technical replicates. f, Graphs showing the fraction of ubiquitin-primed Sil1 substrate remaining as a function of time with WT or mutant proteins. The data were fit to a single exponential decay model (GraphPad Prism software v9). Datapoints from triplicate technical replicates are shown. g, Bar graph comparing the ubiquitin transfer rates (kobs) for the indicated proteins in reactions that contained neddylated CRL2FEM1C, Sil1-ubiquitin substrate, and UBE2R1. The value of each bar represents the estimated value of kobs based on n = 3 technical replicates. UBD, donor ubiquitin; UBA, acceptor ubiquitin.
