The energetics of HMG box interactions with DNA: Thermodynamics of the DNAbinding of the HMG box from mouse Sox-5

The energetics of the Sox-5 HMG box interaction with DNA duplexes, containi ng the recognition sequence AACAAT, were studied by fluorescence spectrosco py, isothermal titration calorimetry (ITC) and differential scanning calori metry (DSC). Fluorescence titration showed that the association constant of this HMG box with the duplexes is of the order 4 x 10(7) M-1, increasing s omewhat with temperature rise, i.e. the Gibbs energy is -40 kJ mol(-1) at 5 degrees C, decreasing to -48 kJ mol(-1) at 32 degrees C. ETC measurements of the enthalpy of association over this temperature range showed an endoth ermic effect below 17 degrees C and an exothermic effect above, suggesting a heat capacity change on binding of about -4 kJ K-1 mol(-1), a value twice larger than expected from structural considerations. A straightforward int erpretation of ITC data in heat capacity terms assumes, however, that the h eat capacities of all participants in the association reaction do not chang e over the considered temperature range. Our previous studies showed that o ver the temperature range of the ITC experiments the HMG box of Sox-5 start s to unfold, absorbing heat and the heat capacities of the DNA duplexes als o increase significantly. These heat capacity effects differ from that of t he DNA/Sox-5 complex. Correcting the ITC measured binding enthalpies for th e heat capacity changes of the components and complex yielded the net entha lpies which exhibit a temperature dependence of about -2 kJ K-1 mol(-1), in good agreement with that predicted on the basis of dehydration of the prot ein-DNA interface. Using the derived heat capacity change and the enthalpy and Gibbs energy of association measured at 5 degrees C, the net enthalpy a nd entropy of association of the fully folded HMG box with the target DNA d uplexes was determined over a broad temperature range. These functions were compared with those for other known cases of sequence specific DNA/protein association. It appears that the enthalpy and entropy of association of mi nor groove binding proteins are more positive than for proteins binding in the major groove. The observed thermodynamic characteristics of protein bin ding to the A + T-rich minor groove of DNA might result from dehydration of both polar and non-polar groups at the interface and release of counterion s. The expected entropy of dehydration was calculated and found to be too l arge to be compensated by the negative entropy of reduction of translationa l/rotational freedom. This implies that DNA/HMG box association proceeds wi th significant decrease of conformational entropy, i.e, reduction in confor mational mobility. (C) 1999 Academic Press.