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Allostery has been studied for many decades, yet it remains challenging to determine experimentally how it occurs at a molecular level. We have developed an approach combining isothermal titration calorimetry, circular dichroism and nuclear magnetic resonance spectroscopy to quantify allostery in terms of protein thermodynamics, structure and dynamics. This strategy was applied to study the interaction between aminoglycoside N-(6')-acetyltransferase-Ii and one of its substrates, acetyl coenzyme A. It was found that homotropic allostery between the two active sites of the homodimeric enzyme is modulated by opposing mechanisms. One follows a classical Koshland-Némethy-Filmer (KNF) paradigm, whereas the other follows a recently proposed mechanism in which partial unfolding of the subunits is coupled to ligand binding. Competition between folding, binding and conformational changes represents a new way to govern energetic communication between binding sites.

Abstract: 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, ΔH A . A powerful application of this technique involves analyzing the temperature dependences of ITC-derived K A and ΔH 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. [Copyright &y& Elsevier]