Purification of SUMO conjugating enzymes and kinetic analysis of substrate conjugation

Abstract

SUMO conjugation to protein substrates requires the concerted action of a dedicated E2 ubiquitin conjugation enzyme (Ubc9) and associated E3 ligases. Although Ubc9 can directly recognize and modify substrate lysine residues that occur within a consensus site for SUMO modification, E3 ligases can redirect specificity and enhance conjugation rates during SUMO conjugation in vitro and in vivo. In this chapter, we will describe methods utilized to purify SUMO conjugating enzymes and model substrates which can be used for analysis of SUMO conjugation in vitro. We will also describe methods to extract kinetic parameters during E3-dependent or E3-independent substrate conjugation.

Figure 1. Ubc9 mediated SUMO conjugation to human p53

(A) Time course for a single turnover assay for SUMO transfer from the E2-SUMO thioester to the C-terminal tetramerization domain of human p53 at various p53 concentrations after separation by SDS-PAGE and detection with an anti-SUMO-1 antibody. (B) Data in A depicted graphically to calculate the reaction rates at various p53 concentrations by linear regression analysis. (C) Reaction rates (Y-axis) plotted against various p53 concentrations (X-axis) and data fit to a rectangular hyperbola of the form y=ax/(b+x). The data points represent the mean of three independent experiments and error bars denote one standard deviation.

Figure 2. Titration of pH and analysis of Ubc9 mediated SUMO conjugation to human p53

(A) Time course for SUMO conjugation to p53 at 94 μM under single turnover conditions at various pH values at 4°C after separation by SDS-PAGE and detection with an anti-SUMO-1 antibody. (B) Data in A depicted graphically to calculate the reaction rates at various pH values. The reaction rates are calculated by linear regression analysis. (C) Initial reaction rates (Y-axis) plotted as a function of pH and data fit to a sigmoidal function (see text for details). The vertical lines mark the pH at half-maximal activity (pK of the titratable group). The data points represent the mean of two independent experimental trials and error bars denote one standard deviation.

Figure 3. Siz1-dependent Smt3-Alexa Fluor 488 conjugation to yeast PCNA

(A) Time course of a single turnover assay for Smt3 transfer from the E2~Smt3 thioester to the yeast PCNA mutant K127G at various PCNA concentrations after separation by SDS-PAGE and detection of the fluorescent signal. (B) Data in A depicted graphically to calculate the reactions rates at various substrate concentrations using linear regression analysis. (C) Initial reaction rates (Y-axis) plotted against various yeast PCNA concentration and data fit to a rectangular hyperbola of the form y=ax/(b+x). The data points represent the mean of three independent experiments and error bars denote one standard deviation.