Energetics of folding and DNA binding of the homeodomains

The thermodynamics of protein-DNA interaction is far from fully understood. We investigated the thermodynamics of folding and DNA binding of Antp(C39S), des(1-6)Antp(C39S) and engrailed HDs using isothermal titration calorimetry, differential scanning calorimetry, fluorescence titration, circular dichroism, ultracentrifugation and mass spectrometry. The temperature induced unfolding/folding processes for Antp(C39S), des(1-6)Antp(C39S) and engrailed HDs consist of three components. The third component is associated with the cooperative unfolding/folding process of the homeodomain core. The first and second transitions are most likely due to the thermal fluctuation of homeodomain molecules. ITC measurements showed that the DNA bindings of Antp(C39S), des(1-6)Antp(C39S) and engrailed HDs exhibit the large negative heat capacity changes and the corrections for heat capacity changes were small. The negative heat capacity change indicates that the hydrophobic interaction plays an important role in the DNA binding of these three homeodomains. The temperature dependence of the binding constants and the thermodynamic parameters of the DNA binding of these three homeodomains, i.e. ΔG, ΔH, ΔS and ΔC_p, were obtained.; The salt concentration dependence of the binding constants for the DNA binding of Antp(C39S) and des(1-6)Antp(C39S) HDs were measured. Hence, the observed overall thermodynamic parameters of the association were decomposed into the non-electrostatic and electrostatic components. The results show that both electrostatic and non-electrostatic interactions make favorable and significant contributions to the interactions of DNA with Antp(C39S) and des(1-6)Antp(C39S) HDs. The quantities of the contributions of electrostatic interactions to the DNA binding of Antp(C39S) and des(1-6)Antp(C39S) HDs were experimentally determined. The non-electrostatic interactions determine the specific protein-DNA interaction. Both enthalpy and entropy are favorable for the formation of the DNA complexes of Antp(C39S), des(1-6)Antp(C39S) and engrailed HDs at physiological temperatures. Enthalpy and entropy of the association compensate to each other to make the free energy almost insensitive to the temperature.; The overall thermodynamic parameters and their non-electrostatic and electrostatic components of the N-terminal arm binding to the minor groove of DNA were obtained. The negative free energy shows that the binding of N-terminal arm to the minor groove of DNA increase the affinity of Antp(C39S) HD binding to DNA. The DNA binding of the N-terminal arm of Antp(C39S) HD is enthalpically driven over the temperature range between 15°C and 30°C. Both electrostatics and hydrogen bonding play important role in the DNA binding of the N-terminal arm.; Free Antp(C39S) and free engrailed HDs have similar mechanism of the temperature-induced folding/unfolding. The thermodynamic parameters of the DNA binding of Antp(C39S) and engrailed HDs are comparable. Both enthalpy and entropy are favorable for the formation of the DNA complexes of Antp(C39S) and engrailed HDs.