*5.4. Thermal Stability of MTase with Mg2+*

A thermal denaturation study of MTase was performed on a specific wavelength (222 nm) using far-UV CD experiments, further analysed based upon the two-state unfolding model. In the case of a single-step unfolding process, NU, where N refers to the native state and U to the unfolded state, and *K*<sup>u</sup> for the equilibrium constant with *f* <sup>u</sup> and *f* <sup>n</sup> are the molar fractions of U and N, respectively.

$$K\_{\mathbf{u}} = \frac{f\_{\mathbf{u}}}{f\_{\mathbf{n}}} \tag{2}$$

$$f\_{\mathbf{d}} = \frac{\left(Y\_{\text{obs}} - Y\_{\text{n}}\right)}{\left(Y\_{\text{u}} - Y\_{\text{n}}\right)} \tag{3}$$

where *Y*obs, *Y*n, and *Y*<sup>u</sup> reflect the observed property, property of the native state, and property of the unfolded state, respectively.
