*5.3. Secondary Structure Analysis Using CD Spectroscopy*

Circular dichroism (CD) spectra were performed on a Jasco J-1500 model spectropolarimeter. For the instrument calibration, (+)-10-camphor sulfonic acid was used. All CD experiments were performed at standard temperature (25 ◦C), which was thermostatically controlled by the Jasco Peltier PTC-423S/15 attached to the cell holder with a precision of ±0.1 ◦C. The change in the secondary structure of MTase under native conditions and the complex form with magnesium (Mg2+) ions were observed to be in the range of 200–250 nm by using a 0.1 cm cell path length. The HT voltage of the scans was kept below 600 V, and the reference signal spectrum was subtracted for each scan. The scan speed of 100 mm/min

and the response time of 1 s was set for each scan measurement; each spectrum was an average of three scans. Furthermore, the secondary structure content of MTase was calculated using online DichroWeb software and Chen et al. method [24]. The spectra were smoothed by the Savitzky–Golay method with 15 convolution widths. The results were expressed as mean residual ellipticity (MRE) in deg. cm<sup>2</sup> dmol−<sup>1</sup> , which is defined as:

$$\text{MRE} = \frac{\theta\_{\text{obs}}(m \text{deg})}{10 \times n \times \text{C} \times l} \tag{1}$$

where *θ*obs is the observed ellipticity in degrees, *C* is the molar fraction, *n* is the number of amino acid residues (321 − 1 = 320), and *l* is the length of the light path in centimeters.
