Supplementary Figure 4: Kinetics of Atto532-tUI binding to Ub.
From: High-affinity free ubiquitin sensors for quantifying ubiquitin homeostasis and deubiquitination
a, To determine the dissociation rate of the Atto532-tUI•Ub complex, Atto532-tUI (1.0 nM) was pre-incubated at 25 °C with 1.0 nM Ub, and then excess unlabeled tUI (1 µM) was rapidly added and the fluorescence was measured every 0.25 s using a stopped-flow fluorimeter. Three independent reactions were performed, and all the data were fit with a single-exponential decay model to determine koff. The experimental data (blue) are shown superimposed with the fitted curve (red). b, The change in fluorescence intensity of 50 pM Atto532-tUI immediately after addition of 0.5, 1.0, or 2.0 nM Ub was monitored at 25 °C. The fluorescence was measured every 0.25 s. From each curve, an observed rate (kobs) was determined from the best-fitting exponential curve, and from these kon was determined from the equation kobs = kon * [Ub] + koff. Each reaction to generate the association data was performed once. c, The experimentally determined association and dissociation rates ± s.d. for the Atto532-tUI•Ub complex are shown together with the Kd ± s.d. calculated from their ratio ± s.d.
