Background

Macromolecular interactions are critical cellular events as they form the basis for signal transduction pathways. Thus, macromolecular interactions are an essential field of research, as they allow a deeper understanding of the molecular mechanisms which underlie both physiological and pathophysiological processes, and the rational design of drugs able to modulate disease-causing macromolecular binding events.

In this context, Isothermal Titration Calorimetry (ITC) is a powerful technique for the characterization of macromolecular interactions. ITC determines the heat change that occurs upon the binding of two molecules. Heat can be absorbed (endothermic reaction) or released (exothermic reaction). ITC monitors such heat changes by determining the differential power, provided by heaters of the instrument to both the reference and the sample cells, needed for counteracting any temperature difference between the two cells during the binding reaction such that no difference in temperature arises between the reference and sample cells (Figure 1).


Figure 1. Principle of Isothermal Titration Calorimetry (ITC). A. Cartoon representation of an isothermal titration calorimetry instrument composed of: a reference cell filled with MilliQ water; a sample cell containing a biomolecule; and an automated injection syringe containing the other binding molecule (ligand) used to titrate the ligand into the sample cell. The sample and reference cells are surrounded by an adiabatic jacket. The system is able to detect temperature differences between the reference and sample cells and to maintain an absence of temperature difference between them (ΔT = 0) by supplying power to both the reference and the sample cell via two heaters. The output of the instrument is the power (μcal/sec) required to maintain ΔT = 0 between the reference and sample cells. B-D. The temperature difference between the reference and the sample cell, induced by the ligand–biomolecule binding, is converted into the power needed to bring the two cells back to the same temperature during the binding reaction. As the titration proceeds, the biomolecule in the sample cell becomes saturated with the ligand, so that less interactions occur and consequently the heat change decreases (C) until the biomolecule is fully saturated and the instrument detects only heat change due to the dilution of the ligand (D).

ITC provides important information about the nature of the macromolecular interaction: the binding stoichiometry (N); the thermodynamic parameters of the binding reaction (enthalpy, ∆H, entropy, ∆S, and Gibbs free energy, ∆G); the strength of the interaction (the equilibrium association constant K_A, from which the more commonly used equilibrium dissociation constant K_D can be derived).

Among the methods used to characterize macromolecular interactions, ITC has two major advantages: i) the biomolecules are free to move in solution and are not labelled, which insures a direct characterization of the binding event, unbiased by labelling and/or by limitation on molecule motions due to their immobilization on a surface; ii) ITC is the only method that allows a detailed characterization of the binding event by providing not only the binding affinity, but also other critical information, i.e., the binding stoichiometry and the thermodynamic parameters. This information can help significantly in the understanding of the molecular mechanism of the binding reaction, even when no structural data are yet available. Furthermore, they can be used as complementary data to validate structural results.

Recently, I presented crystallographic and functional data showing that the K⁺ inward rectifier KAT1 (K⁺ Arabidopsis thaliana 1) channel is regulated by the direct binding of 14-3-3 proteins (Saponaro et al., 2017). In particular, I identified a 14-3-3 mode III binding site at the very C-terminus of KAT1 and co-crystallized it with tobacco 14-3-3 proteins (14-3-3c) to describe the protein complex in atomic detail. The structural results were complemented/supported by measuring, through ITC, the interaction between a synthetic KAT1 C-terminal phosphopeptide (CPP) and 14-3-3c. ITC was employed to quantify the stoichiometry, the equilibrium binding affinity and the thermodynamic parameters of the 14-3-3c-CPP binding reaction.

The aim of this protocol is to provide a detailed description of the setting procedure of an ITC experiment, highlighting the crucial steps and related concerns, and providing, at the same time, a well-established strategy to overcome such problems. Moreover, the present protocol describes the analysis of an ITC measurement of the single binding event in a 14-3-3c/CPP interaction.