Characterizing Protein-Protein interactions by iTC

Proteins operate as integral members of complex networks that precisely regulate physiological processes through a combination of signaling pathways and feedback. These regulatory functions, including signal transduction, protein trafficking, transcription and translation, all rely on the ability of proteins to rapidly target and form specific non-covalent complexes with other proteins in response to chemical signals. Predicting and understanding how these interactions occur and are controlled (or, in the case of many diseases, how an interaction misfunctions) is complicated by the dynamic events controlling recognition and binding. Each interaction results in protein structural changes and involves specific electrostatic and van der Waals interactions, the burial of solvent-exposed surfaces, and the breaking and formation of hydrogen bonds. It has been estimated that each protein in an organism forms interactions with an average of five other proteins (Piehler, 2005), and it is known that the same protein can form different interactions depending on its location or concentration in the cell (Liddington, 2004). Additionally, aside from such intermolecular interactions, protein-protein interactions also occur intramolecularly at interfaces between domains and between subunits within a protein. This note examines the application of isothermal titration calorimetry (ITC) for quantitatively measuring the thermodynamic properties driving protein-protein interactions, and also describes how ITC can be used to quantify protonation/deprotonation events occurring upon binding. Because ITC involves the titration of one component into another, ITC is generally limited to studying bimolecular interactions.