Figure 5: HM binding and release by the isolated FimH lectin domain FimHL.
Analysis of FimHL·HM interactions based on competition between HM and the synthetic fluorescent GN-FP-4 ligand. (a) HM binding to FimHL analysed by displacement of GN-FP-4 from FimHL variants as indicated. An equimolar mixture of FimHL and GN-FP-4 (1 μM each) was incubated with different HM concentrations (10 nM–3.2 mM) for >18 h. GN-FP-4 displacement is monitored by a decrease in fluorescence polarization at 528±20 nm (excitation at 485 nm). Data were fitted (solid lines) according to a mechanism in which two ligands compete for the same binding site, with fixed Kd values for GN-FP-4 binding (cf. Table 2). (b) Kinetics of HM dissociation from FimHL. A solution with equimolar concentrations of FimHL and HM (3 μM each, guaranteeing >95% occupancy with HM) was mixed with excess GN-FP-4 (10 μM), and the decrease in GN-FP-4 fluorescence at 520 nm as a consequence of HM dissociation and GN-FP-4 binding was recorded (Supplementary Fig. 5f–j). The obtained first-order kinetics are independent of the GN-FP-4 concentration and thus directly monitor HM dissociation.
