Fig. 3.

TDG binding to DACM-labeled or MIANS-labeled V331C LacY. (A) Dependence of fluorescence on TDG concentration as percentage of initial emission level before TDG addition for each fluorophore. Titration traces were recorded as a function of time at 0.4 μM protein with excitation and emission wavelengths of 397 and 440 nm for DACM-labeled or 330 and 415 nm for MIANS-labeled LacY, respectively. Solid lines are hyperbolic fits to the data with estimated K_d^app values of 21.4 ± 0.4 and 1.2 ± 0.1 μM for DACM- and MIANS-labeled LacY, respectively. ●, DACM; ▾, MIANS. (B) Kinetics of TDG binding to DACM-labeled LacY. Concentration dependence of the rates (k_obs) of the fluorescence changes was measured at 0.4 μM protein in 50 mM NaP_i (pH 7.5), 0.02% DDM. Data were collected after rapid mixing of equal volumes of TDG and labeled protein by stopped-flow. Final concentrations of TDG are shown. Rates were estimated from single exponential fitting to time traces. Each point is an average of five to seven measurements. Linear fit to the data is shown as a solid line. The intercept with the y axis is k_r (1.0 ± 0.1 s⁻¹), and the slope is k_f (28.66 ± 0.02 mM⁻¹s⁻¹). Estimated K_d^app (k_r/k_f) is 34.9 ± 3.5 μM. (C) Kinetics of TDG binding to MIANS-labeled LacY. Measurements were done as described above. Data were collected by stopped-flow at TDG concentrations from 0.05 to 4 mM and from time traces at TDG concentrations from 0.45 to 15 μM. Estimated parameters are k_r = 0.011 ± 0.001 s⁻¹; k_f = 10.81 ± 0.03 mM⁻¹s⁻¹; and K_d^app = 1.0 ± 0.1 μM.