*5.6. Binding Affinity Using Isothermal Titration Calorimetry (ITC)*

The binding affinity of MTase and Mg2+ at 25 ◦C was determined using isothermal titration calorimetry (ITC). MTase and Mg2+ were dissolved in 50 mM phosphate buffer, 50 mM NaCl, pH 7.4, and gently degassed. Forty microliters of water was added to a sample cell containing 280 µL of MTase. There was a total of 20 injections, with each infusion of 2 µL of 17 mM Mg2+ ion titrated into the sample cell containing 30 µM of MTase simultaneously. Intervals of 150 s separated each injection to allow the signal to return to baseline. A constant stirring speed of 750 rpm was maintained to ensure proper

mixing after each infusion. Control experiments were performed under similar conditions by titrating Mg2+ into the buffer and were subtracted to correct for the heat of dilution. Thermodynamic parameters were obtained by fitting the data to a two-set site model using Origin software (7.0, OriginLab Corporation, Northampton, MA, USA).
