Far-UV CD spectra were recorded at a protein concentration of 10 mM in 20 mM sodium acetate (pH 6.5) buffer on a Jasco 810 spectropolarimeter (Jasco Inc.). CD spectra were measured for apo-SsTroA alone and in the presence of 100 mM Zn 2+ or Mn 2+ at room temperature using a 0.1 cm quartz cell, 1 nm band width, 50 nm min 21 scan speed, and 1 nm step. Each curve represents the average of three accumulations and a blank containing the same buffer and detergent, buffer spectra were always subtracted, and ellipticity results were expressed in millidegree. Calculations of the fractional percentage of secondary structures were performed using the computer programs Spectra-Manager (Jasco) and K2d2 [65] . The thermal reactions were monitored on the Jasco 810 spectropolarimeter (Jasco Inc.) connected to a digitally controlled water bath (Julabo). The data were monitored for apo-SsTroA alone and loaded with Zn 2+ or Mn 2+ at 223 nm with a constant heating rate of 0.5uC/min,at temperatures between 25uC and 90uC. The results analysis yielded the transition temperature (Tm) as described previously [66] , and the data were fit using SigmaPlot software.