Folding thermodynamics of c-Myb DNA-binding domain in correlation with its α-helical contents

Abstract

The conformational and thermal stabilities of the minimum functional unit for c-Myb DNA-binding domain, tandem repeat 2 and 3 (R2R3), were analyzed under different pH conditions, ranging from 4.0 to 7.5, using circular dichroism and differential scanning calorimetry. Secondary structure analysis showed that the solution pH largely affects the conformational stability of the protein domain. Of all conditions analyzed, the α-helical content was maximal at pH 6.5, and the thermal stability was highest at pH 5.0. Thermodynamic parameters for thermal unfolding of R2R3 were determined using differential scanning calorimetry, and the origin of folding thermodynamics at the different pHs and its correlation with the α-helical content were further analyzed. It should be noted that the α-helical content correlates well with the enthalpy change in the pH range from 4.5 to 7.5, suggesting that the strength of hydrogen bonds and salt bridges needed for maintenance of helical structure is related to enthalpy in the native state. Under physiological pH conditions, c-Myb R2R3 exists in the enthalpically unstable but entropically stable state. Due to loss of rigid structure and high stability, the protein can now obtain structural flexibility, befitting its function.