Figure 4.

Binding of α-NPG to C154G LacY as detected by Trp → α-NPG FRET. Measurements were carried out in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl containing 0.05% DDM at a protein concentration of 5 μM; excitation was at 295 nm. (A) Quenching of Trp fluorescence at different concentrations of α-NPG. (B) Affinity of C154G LacY for α-NPG measured by displacement with TDG. The relative fluorescence change after addition of 10 mM TDG is plotted as a function of α-NPG concentration. Fluorescence after TDG addition was taken as 100% at each α-NPG concentration. Solid lines represent Receptor binding – Saturation (One site - Total binding) (A) and Michaelis-Menten (B) fits of the data. In all cases, the experiments were repeated four times, and the data represent mean values ± SD.