Enthalpy of Binding (ΔH)
Isothermal Titration Calorimetry (ITC)
50 mM Tris, 150 mM KCl
ITC was performed using a nanoITC (TA Instruments). The protein concentration was adjusted to about 215, and 1500 mM ligand solutions were prepared in buffer from protein dialysis (50 mM Tris pH 8, 150 mM KCl). Measurements were performed at 20 C with a stirring speed of 300 rpm and spacings of 300 s between each 2 mL injection. The data was analyzed using NanoAnalyze (TA Instruments). Binding constants were determined from sigmoidal fits based on a one-site binding model. Heat of dilution baselines for ligand titrations into buffer were subtracted from the experimental data.