Fig. 5. Bivalent binding of DM to folded CVN2L0. Isothermal calorimetric titration of DM by CVN2L0 at 298 K. (A) Aliquots (2 μL) of ligand (>600 μM) were injected into CVN2L0 (50 μM) in 100 mM PBS, pH 7.0, in excess of ligand (DM) for the saturation of binding sites. Two sets of binding sites fitting model was used to calculate KD values for the 2 low-affinity and 2 high-affinity carbohydrate binding sites on each CVN2L0 molecule. (B) The ratio between CVN2L0 = 50 μM and DM was chosen to be below 1 : 12 for CVN2L0, and 1 : 6 for other variants. Heats of binding were measured as delta (Δ)H, and one set of sites fitting model used to determine KD for the binding affinity of DM to soluble CVN2L0.
