Abstract
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.
Highlights
Majority of chemical reactions are associated with absorption or release of energy in the form of heat
Many membrane protein-ligand interactions has been probed by Isothermal titration calorimetry (ITC) (5)
In two studies on membrane transporters (12, 15), ITC was used to examine the thermodynamic linkage between ion and substrate binding to ion-driven transporters
Summary
Majority of chemical reactions are associated with absorption or release of energy in the form of heat. Over the last several decades, ITC has been applied to numerous systems. Majority of these studies focused on molecular interactions, such as between proteins, or between proteins and small ligands, DNA or other macromolecular systems. An incomplete list includes channels binding ions (6–8) and gating ligands (9, 10), secondary transporters binding substrates and coupled ions (11–26), and assembly of protein complexes (27, 28). ITC is routinely used to establish functionality and substrate specificity of channels and transporters. Other questions such as stoichiometry of binding (26) and ion-coupling mechanisms (12, 15) have been probed using this
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