Differential Scanning Calorimetry (DSC), allows us to measure changes in the heat capacity of a solution as it is heated. When a protein, or protein domain unfolds, there is a peak in the heat capacity function representing the extra heat required to drive the unfolding reaction. This is typically done to determine the stability and subunit composition of a macromolecule, but can also be done as a function of ligand concentration to determine binding constants in the case of extremely tight binding. With the Pressure Perturbation accessory (PPC) we can also measure the relationship between heat and pressure, the coefficient of thermal expansion. From this measurement over a range of temperature, we can determine the changes in volume that accompany a reaction. The third kind of heat measurement we can make is the heat of interaction between molecules, using an Isothermal Titration Calorimeter. From the binding isotherm we can calculate the affinity, enthalpy and entropy of the reaction. By making this measurement over a range of temperatures, we can make a complete thermodynamic characterization of the reaction, allowing us to make inferences about the mechanism.
The calorimeters available complement other techniques aimed at studies of structure and protein-protein interactions, such as analytical ultracentrifugation. Specifically, it will be of value in studies designed to:
(i) characterize stability of proteins and effects of modifications. This will help in the design of suitable mutants for functional and crystallographic studies.
(ii) measure binding constants and characterize the thermodynamics of binding equilibria. ITC is more useful than fluorescence titrations, because it avoids the problem of perturbation caused by the presence of the label; ITC is more quantitative than ELISA assays of binding; and because a calorimetric titration isotherm corresponds to what is effectively the derivative of the binding curve from other methods, experiments may be done at more convenient and relevant concentrations under stoichiometric binding conditions.
For more details and consultation on calorimetry contact calorimetry@analyticalbiotechservices.com for a price quotation and availability of services.
