Fig. 5.

Kinetics of TDG binding to MIANS-labeled V331C LacY as a function of pH. (A) TDG concentration dependence of the rate of fluorescence change (k_obs) at given ambient pH values. Details of data collection and fitting are described in Fig. 3. Solid lines are linear fits to each dataset at pH 6 (●), 7.5 (▴), 8.5 (▾), 9.0 (■), 9.5 (♦), and 10.0 (⬣). ○, control experiment at pH 6.1 with labeled protein preincubated at pH 11.0 for 12 min. (B) Kinetic parameters of k_r (●) and k_f (■) estimated from A for each dataset are plotted as a function of pH. ○, □, k_f and k_r, respectively, for protein preincubated at pH 11.0 for 12 min and returned to pH 6.1 (in addition, see Fig. S6 for comparison of K_d^app values calculated from kinetic parameters and obtained from titration data).