SPR analysis of TD4 and Int280 binding to TirM.

A. Change in resonance units (RU) with time (sensograms) obtained with purified Int280_EHEC (at the indicated concentrations) passed through a streptavidin chip coated with biotinylated TirM_EHEC. Sensograms were used to calculate the kinetic constants of association (k_on) and dissociation (k_off), and the equilibrium dissociation constant (K_D) using the BiaEvaluation software. The curves were fitted to a model 1:1 Langmuir interaction (black lines) for the calculation. B. Sensograms obtained with purified TD4-HlyA (at the indicated concentrations) passed through a streptavidin chip coated with biotinylated TirM_EHEC. The absence of significant dissociation preclude the calculation of kinetic constant of dissociation (k_off). The RU shift near the steady state (at 400 s, indicated with a rectangle) were used to estimate an apparent constant of dissociation (K_D) of TD4-HlyA for TirM_EHEC representing the shift in RU values vs concentration of TD4-HlyA (graph on the right). C. Sensograms showing binding to TirM_EHEC by 40 nM of TD4 followed by HEPES buffer (green line) or 40 nM TD4 followed by 80 nM Int280_EHEC (red line). The blue line represents the sensogram obtained by 80 nM Int280_EHEC (without TD4) followed by HEPES buffer. The black arrows indicate the time of injection of HEPES buffer. The red arrow indicates the time of injection of Int280_EHEC in the chip in which TD4 was previously injected.