*5.5. Fluorescence Quenching Measurements*

Mg2+ binding with MTase was performed by fluorescence measurements and was performed on Fluorolog TCSPC Horiba FL-1057 spectrofluorometer attached to a temperaturecontrolled water bath with an accuracy of ±0.1 ◦C. The change in fluorescence intensity of MTase was observed at 340 nm and further analysed by using the Stern–Volmer equation [36]

$$\frac{\text{F}\_{\text{o}}}{\text{F}} = K\_{\text{sv}}[\text{Q}] + 1 \tag{4}$$

where F<sup>o</sup> and F are the MTase fluorescence intensities in the absence and presence of Mg2+ (quencher), and *KSV* is the Stern–Volmer quenching constant was calculated from the equation

$$K\_{\rm sv} = k\_{\rm q} \cdot \tau\_{\rm o} \tag{5}$$

where *k*<sup>q</sup> is the bimolecular rate constant of the protein–ligand reaction process and *τ<sup>o</sup>* is the average integral fluorescence lifetime of Trp, which is ~5.78 <sup>×</sup> <sup>10</sup>−<sup>9</sup> s [37]. Therefore, binding constants and binding stoichiometry were calculated [38,39].

$$\log\left(\frac{F\_o}{F} - 1\right) = \log K\_\mathsf{b} + n \log[Q] \tag{6}$$

where *K*<sup>b</sup> is the binding constant and *n* is binding stoichiometry.
