Zn 2+ /Mn 2+ Titration into apo-SsTroA by ITC Binding of Zn 2+ and Mn 2+ to apo-SsTroA was determined by Isothermal Titration Calorimetry (ITC) at 25uC using a NANO ITC 2G MicroCalorimeter (TA Instruments). The metal and protein solutions were prepared in 20 mM sodium acetate buffer, pH 6.5. Prior to experiments, the reference cell was filled with deionized water, and both metal solutions and protein solutions were clarified for 10 minutes at 16,000 g and degassed for 20 minutes to eliminate air bubbles, using the ThermoVac accessory of the microcalorimeter. Upon experimental setup, the protein solution present in the sample cell was stirred at 200 rpm. After a stable baseline was achieved the titration was initiated with 25 consecutive 10 ml injections of 500 mM Zn 2+ into the sample cell (volume = 1.5 ml) containing 90 mM apo-SsTroA,and then 200 mM Mn 2+ was injected into 30 mM apo-SsTroA. The background heat of dilution is collected by titration with buffer alone, and the integrated data were subtracted from data obtained with apo-SsTroA present. Integrated heat data obtained for Zn 2+ and Mn 2+ titrations were analyzed using the NanoAnalyze software (TA Instruments) fitting them to an independent binding model. The free energy (DG) and entropy (DS) of the binding reaction can be determined with Equations 1 and 2, respectively: