Isothermal titration calorimetry of ion-coupled membrane transporters

• Advantages and limitations of ITC in studies of ligand binding.
• Membrane protein sample preparation for ITC experiments.
• Analysis of thermodynamically linked binding processes in ion-coupled transporters.
• Measurements, analysis and interpretation of binding heat capacity.

Binding of ligands, ranging from proteins to ions, to membrane proteins is associated with absorption or release of heat that can be detected by isothermal titration calorimetry (ITC). Such measurements not only provide binding affinities but also afford direct access to thermodynamic parameters of binding – enthalpy, entropy and heat capacity. These parameters can be interpreted in a structural context, allow discrimination between different binding mechanisms and guide drug design. In this review, we introduce advantages and limitations of ITC as a methodology to study molecular interactions of membrane proteins. We further describe case studies where ITC was used to analyze thermodynamic linkage between ions and substrates in ion-coupled transporters. Similar type of linkage analysis will likely be applicable to a wide range of transporters, channels, and receptors.