ITC analysis of ligand binding.

Thermograms of measurements performed at three different temperatures (A) 15°C, (B) 25°C and (C) 40°C are shown. At each temperature, core-bradavidin was first titrated with Brad-tag (1), followed by competitive titration with biotin (2). As a control measurement, core-bradavidin was titrated with biotin only (3). In order to prove that the intrinsic Brad-tag decreases the affinity towards biotin, wt bradavidin was also titrated with biotin (4). In addition, core-bradavidin was titrated with Brad-tag–EGFP at 15 and 25°C (5). (D) Comparison of the binding enthalpies of all measurements at different temperatures. Brad-tag had a clear effect on the binding enthalpy of the competitive titration with biotin at 15°C (endothermic Brad-tag binding) and 40°C (exothermic Brad-tag binding). At 25°C, the enthalpy of competitive titration was equal to that of titration with biotin only (no detectable binding of Brad-tag to core-bradavidin).