Protein structural dynamics in solution closely correlate with its function. Although a great number of protein structures have been determined at atomic resolution, most of them are static or averaged structures and information is lacking on their structural dynamics. We have studied the thermodynamic properties of a DNA-binding protein, c-Myb R2R3, and their correlation with protein structure-function relationships using biophysical methods. The analysis of pH-dependent conformational stability showed that the α-helical content correlated with the enthalpy change. Under physiological pH conditions, c-Myb R2R3 exists in an enthalpically unstable but entropically stable state. The temperature-dependent NMR measurements showed that some signals were broadened with an increase in temperature even below 37^oC, indicating that extensive conformational fluctuations within the folded manifold are activated at a physiological temperature. We also analyzed the effects on the stability and function of different conformational states induced by the mutations. The thermodynamic analysis using DSC and ITC showed that the increased conformational fluctuation in the native state of protein results in a decreased entropy change for either unfolding or DNA-binding. The effects of the increased fluctuation could be detected not only by the binding but also by the folding thermodynamics, both of which well correlated.