Isothermal Titration Calorimetry for Investigating Thermodynamics of Protein-Lipid Interactions

Isothermal titration calorimetry (ITC) is a remarkably versatile and powerful technique for studying the interactions between molecules. In ITC, intermolecular interactions are investigated by measuring heat changes (heat released/absorbed) occurring during the binding process. A very important feature of ITC is that it is a label-free technique and does not need any probe to be incorporated into the system. In view of this, it has become the method of choice for investigating the interaction of proteins with a wide range of species, including small ligands, other proteins, nucleic acids, drugs, nanoparticles, and metal ions. In recent years, ITC has gained popularity for studying protein interactions with lipids and lipid membranes as well. In this chapter, we provide a comprehensive overview of the ITC instrument and its practical application to determining the thermodynamic parameters characterizing the interaction between proteins and lipids. Employing ITC one can determine a variety of thermodynamic parameters, including enthalpy of binding (ΔH), binding stoichiometry (n), association constant (K_a), entropy of binding (ΔS), free energy of binding (ΔG), and change in heat capacity at constant pressure (ΔC_p) from a single calorimetric titration. Thus, ITC provides a complete thermodynamic description of the interaction, enhancing our understanding of the system. The ITC experimental system described in this chapter includes a well-known protein from bovine seminal plasma, PDC-109. This protein is chosen for its high affinity for choline-containing lipids, such as phosphatidylcholine, a major phospholipid component of the sperm cell membrane. Using this as a representative example, researchers can establish a standard protocol for investigating the thermodynamic parameters associated with the interaction of other soluble proteins with lipid membranes using ITC.