Figure 4: Kinetics of HM binding and release by full-length FimH.

(a) Fluorescence spectra (excitation at 280 nm) of FimH_L^F18 (2 μM; red lines) and FimH^F18·DsG (2 μM; black lines) in the absence (solid lines) or presence of 200 μM HM (dotted lines). (b) Equilibrium titration of FimH^F18·DsG (2 μM) with HM, recorded via the fluorescence increase at 320 nm. The total concentration of HM is plotted against the recorded fluorescence signal. Data were fitted (solid line) according to equation (2) (cf. experimental section) and yielded a K_d value of 9.9±1.5 μM. (c) Stopped-flow fluorescence kinetics of HM binding to FimH^F18·DsG (1.0 μM), recorded via the fluorescence change above 320 nm. The HM concentration was varied between 0 and 50 μM. Five representative traces are shown (HM concentrations are given in μM). The fluorescence traces were globally fitted according to a second-order binding and first-order dissociation reaction (solid lines; Table 2). (d) Amplitudes of the reactions monitored in c, plotted against the total HM concentration. Data were fitted (solid line) according to equation (2), yielding a K_d value of 12±1 μM.