Figure 5.

(a) Box and whisker plots of L. lactis strains binding to Fn as determined by AFM. Whisker ends 9^th and 91^st percentiles. Box ends 25^th and 75^th percentiles. Median is marked with horizontal line. Blue- or red-colored boxes represent median force values that are, respectively, less than or greater than the wild type strain (L. lactis A). P-values calculated using Mann-Whitney, where p < 0.001 is indicated by ***. (b) Dependence of binding or dissociation force (f; pN) on the logarithm of the loading rate, (r; pN s⁻¹) between FnBPA expressed on the surface of the L. lactis E652D/H782Q and Fn. This dependence is described by the Bell model f = (k_BT/x_β) × (In (r x_β/k_off k_B T)), where r(pNs⁻¹) is the product of the retraction velocity (nm s⁻¹) and the slope of the force-distance curve at binding (pN nm⁻¹), k_off is the dissociation rate constant in the absence of the applied force, x_β is the position of the activation barrier, and k_BT is the thermal energy where k_B is Boltzmann constant and T is temperature (4.1 pN nm at room temperature). AFM measurements were conducted in PBS at retraction velocities between 0.05 and 18.8 µm/s. AFM tips were coated with protein by incubating tips in 100 µg/mL of Fn or Fg prior to force measurements.