ThANNCs for kinetically optimizing ITC

Abstract

For decades, isothermal titration calorimetry (ITC) has been an indispensable tool for determining the thermodynamics of macromolecular binding equilibria. Stoichiometry, binding constants (affinity), free energy, enthalpy, and entropy differences between bound and free states can readily be obtained through the measurement of the heat of association. Most modern ITCs operate on the principle of power compensation between a reference cell and a sample cell containing the titrand (i.e., a protein) into which the titrant (i.e., a small-molecule ligand, inhibitor, or another macromolecule) is injected. Upon binding, a small amount of heat is released or absorbed, and the calorimeter compensates for the temperature change by applying or withdrawing heat to the sample cell in order to maintain a constant temperature throughout the reaction time. This gives rise to a differential power trace over time that is integrated to directly determine the enthalpy of binding. Subsequent analysis of a series of injections at different mole ratios of the interacting species in solution can provide binding affinities and the stoichiometry of the association.