Fig. 7.

Rates of sugar binding and closing of the cytoplasmic cavity. The data shown were obtained for bimane-labeled N137C/Q340W LacY from the stopped-flow measurements described in Figs. 3 B and C and 5 and Fig. S7. (A) Concentration dependence of NPG-binding rates (k_obs) measured by Trp151→NPG FRET with protein solubilized in DDM (blue ●), reconstituted into PL (brown ■), or after dissolving the PL in DDM (◇). The k_off values for protein in DDM micelles (○) or PL (□) are shown. Kinetic parameters for NPG binding in DDM are k_on = 0.3 μM⁻¹⋅s⁻¹ and k_off = 43 s⁻¹. Reconstituted protein binds sugar with k_obs = 6.2 ± 1.1 s⁻¹. (B) Rates of bimane fluorescence change measured in DDM after mixing protein with NPG (○ and □ with excitation at 295 nm and 380 nm, respectively) or after dilution of protein preincubated with NPG (pink ● and ■ for measurements with excitation at 295 and 380 nm, respectively). The estimated rate of opening/closing of the cytoplasmic cavity in DDM is 4.4 ± 2.0 s⁻¹. (C) Rates of bimane quenching measured after mixing of NPG with protein reconstituted into PL (□, excitation at 380 nm) or with the same PL dissolved in DDM (blue ◆). The estimated rate of cytoplasmic cavity closing for the reconstituted protein is 4.2 ± 1.3 s⁻¹.