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Structural dynamics of c-Myb DNA-binding domain revealed by variable temperature and pH studies

S. Inaba and M. Oda, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, 606-8522, Japan.

A protein in solution generally equilibrates among multiple conformational sub-states. This is critical as dynamics behavior of protein conformational changes in solution closely correlated with its function. Although a great number of protein structures have been determined by X-ray, NMR, and Cyro-EM, most of them are static or averaged protein structures and lack information on their structural dynamics. We have studied the thermodynamic properties of a DNA-binding protein, c-Myb R2R3, and their correlation with structure and function using biophysical methods, and demonstrated the unique feature of fluctuation, shedding light on the structure-function relationship. We analyzed the properties of c-Myb R2R3 under physiological conditions (approximately 37°C and pH 7.3), considering that the protein would fold into its native and functional structure. Within a limited temperature range around 37°C, the temperature-dependent NMR measurements showed that some signals were broadened with an increase in temperature even below 37°C, which is much lower than the temperature of global unfolding transition (approximately 50°C). The results indicate that extensive conformational fluctuations within the folded manifold are activated at a physiological temperature (Inaba et al., 2015). We also analyzed pH-dependent conformational stability using circular dichroism and differential scanning calorimetry. The results showed that the α-helical content correlated with the enthalpy change at pH ranging from 4.5 to 7.5. Under physiological pH conditions, c-Myb R2R3 exists in an enthalpically unstable but entropically stable state. Owing to the loss of its rigid structure and high stability, the protein gains structural flexibility favoring its function. The conformational flexibility of the c-Myb R2R3 resulting from the reduction of intramolecular interactions, is necessary to its function, despite of its decreased stability (Inaba et al., 2016).

Inaba S, Maeno A, Sakurai K, Narayanan SP, Ikegami T, Akasaka K, Oda M. (2015) Functional conformer of c-Myb DNA-binding domain revealed by variable temperature studies. FEBS J 282, 4497-4514.

Inaba S, Fukada H, Oda M. (2016) Folding thermodynamics of c-Myb DNA-binding domain in correlation with its α-helical contents. Int J Biol Macromol 82, 725-732.