Calorimetry to Quantify Protein-Ligand Binding

Abstract

Isothermal titration calorimetry (ITC) is the preferred method used to study biochemical reactions like protein-ligand binding due to its sensitivity, accuracy, and precision. ITC measures directly the heat absorbed or released (∆H) associated with a given binding process. A typical ITC experiment allows the dissection of the binding energy of a reaction into ligand-enzyme association constant (Ka), change in enthalpy (∆H), change in entropy (∆S), change in Gibbs-free energy (∆G), and the stoichiometry of association (N). The change in heat capacity (∆Cp) is obtained from the measurements of binding enthalpy over a range of temperatures. The magnitude and signs of the thermodynamic parameters that were obtained provide insight into the nature of interactions involved in the binding process. The strength of interaction is thermodynamically favorable is determined by the Gibbs free energy. ∆G is an important thermodynamic descriptor of a binding reaction since it dictates the binding affinity and is in turn defined by the enthalpy and entropy changes expressed in the following equation: ∆G = ∆H-T∆S. Up-close, this reflects the contradistinc-tions of two thermodynamic effects at a molecular level-the propensity to drop to lower energy (bond formation, negative ∆H), counterbalanced by the innate thermal Brownian motion's destructive characteristic (bond breakage, positive ∆S).