Figure 2. Kinetics of GDP binding to the biosensor.

(A) Fluorescence time courses after mixing 0.1 μM 5-ATR–ParM with increasing concentrations of GDP in a stopped-flow apparatus. Micromolar GDP concentrations are indicated in the Figure. All concentrations are those in the mixing chamber. Fluorescence was excited at 546 nm and emission was detected after a 570 nm long-pass filter. Data were analysed by single exponential curve fitting yielding the observed rate constant k_obs. (B) Plot of k_obs against GDP concentration for 5-ATR– (open circles) and 6-ATR– (closed circles) ParM. Association (k_on) and dissociation (k_off) rate constants were obtained from linear regression analysis. For 5-ATR–ParM, k_on is 0.047 μM⁻¹·s⁻¹ and k_off is 4.8 s⁻¹; for 6-ATR–ParM, k_on is 0.029 μM⁻¹·s⁻¹ and k_off is 3.2 s⁻¹. Experiments were performed in 30 mM Tris/HCl (pH 7.5), 25 mM KCl, 3 mM MgCl₂ and 5 μM BSA at 20°C.