Thermodynamics of specific and non-specific DNA binding by the c-Myb DNA-binding domain.

The thermodynamics of the c-Myb DNA-binding domain (R2R3) interaction with its target DNA have been analyzed using isothermal titration calorimetry and amino acid mutagenesis. The enthalpy of association between the standard R2R3, the Cys130 mutant substituted with Ile, and the cognate DNA is -12.5 (+/- 0.1) kcal mol-1 at pH 7.5 and at 20 degrees C, and this interaction is enthalpically driven throughout the physiological temperature range. In order to understand the DNA recognition mechanism, several pairs of interactions were investigated using single and multiple-base alterations with single and multiple-amino acid substituted mutants. The interactions between the standard R2R3 and many non-cognate DNAs were accompanied by binding enthalpy changes and heat capacity changes, although their affinities were reduced. The roles of the electrostatic interactions in binding to the cognate and the non-cognate DNAs were also analyzed from the dependency of the thermodynamic parameters on the salt concentration. The heat capacity change was found to be significantly dependent upon the salt concentration. Several mutant proteins bound to the multiple-base altered DNA with very small enthalpy changes, although they bound to the cognate and the single-base altered DNAs with detectable enthalpy and heat capacity changes. From the thermodynamic cycles derived from the DNA binding of the amino acid substituted R2R3 to the base substituted DNA duplexes, the individual thermodynamic mechanisms of the specific DNA recognition of R2R3 were dissected. The local folding mechanism was highlighted by the substitution of Pro with either Gly or Ala at the linker between R2 and R3. The characteristic thermodynamic features of specific and non-specific DNA binding are discussed.