Fig. 7.
Effect of pH on NPG dissociation rate (koff) for mutant G46W/G262W in DDM. Rates of displacement of bound NPG by excess of TDG were determined by measuring Trp→NPG FRET at given pH values. Protein preincubated with NPG was rapidly mixed with TDG, and rates of increase in Trp fluorescence measured by stopped-flow were obtained from single exponential fits (see traces in Fig. S7A). Final concentrations were: protein, 0.8–1.6 μM; NPG, 0.2–0.4 mM; and TDG 30–60 mM. Displacement rates (koff) plotted vs. pH and fitted with a sigmoidal equation (SigmaPlot 10.0) indicate a pKa of ∼10.5.