Isothermal titration calorimetry (ITC) can provide detailed information on the thermodynamics of biomolecular interactions in the form of equilibrium constants, K-A, and enthalpy changes, Delta H-A. A powerful application of this technique involves analyzing the temperature dependences of ITC-derived K-A and Delta H-A values to gain insight into thermodynamic linkage between binding and additional equilibria, such as protein folding. We recently developed a general method for global analysis of variable temperature ITC data that significantly improves the accuracy of extracted thermodynamic parameters and requires no prior knowledge of the coupled equilibria. Here we report detailed validation of this method using Monte Carlo simulations and an application to study coupled folding and binding in an aminoglycoside acetyltransferase enzyme. (C) 2011 Elsevier B.V. All rights reserved.