The energetics of HMG box interactions with DNA: thermodynamic description of the target DNA duplexes

Abstract

The thermal properties and energetics of formation of 10, 12 and 16 bp DNA duplexes, specifically interacting with the HMG box of Sox-5, have been studied by isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). DSC studies show that the partial heat capacity of these short duplexes increases considerably prior to the cooperative process of strand separation. Direct extrapolation of the pre and post-transition heat capacity functions into the cooperative transition zone suggests that unfolding/dissociation of strands results in no apparent heat capacity increment. In contrast, ITC measurements show that the negative enthalpy of complementary strand association increases in magnitude with temperature rise, implying that strand association proceeds with significant decrease of heat capacity. Furthermore, the ITC-measured enthalpy of strand association is significantly smaller in magnitude than the enthalpy of cooperative unfolding measured by DSC. To resolve this paradox, the heat effects upon heating and cooling of the separate DNA strands have been measured by DSC. This showed that cooling of the strands from 100 °C to −10 °C proceeds with significant heat release associated with the formation of intra and inter-molecular interactions. When the enthalpy of residual structure in the strands and the temperature dependence of the heat capacity of the duplexes and of their unfolded strands have been taken into account, the ITC and DSC results are brought into agreement. The analysis shows that the considerable increase in heat capacity of the duplexes with temperature rise is due to increasing fluctuations of their structure (e.g. end fraying and twisting) and this effect obscures the heat capacity increment resulting from the cooperative separation of strands, which in fact amounts to 200(±40) JK −1 (mol bp) −1. Using this heat capacity increment, the averaged standard enthalpy, entropy and Gibbs energy of formation of fully folded duplexes from fully unfolded strands have been determined at 25 °C as −33(±2) kJ (mol bp) −1, −93(±4) J K −1 (mol bp) −1 and −5.0(±0.5) kJ (mol bp) −1, respectively.