Thermal denaturation transitions were assessed by monitoring the temperature dependent change in ellipticity at 223 nm to determine apparent thermal denaturation midpoints in the presence or absence of metal ions. As expected, thermal denaturation proceeded, as expected, with a sharp decrease in ellipticity at 223 nm at the melting temperature and a complete loss of secondary structure (Fig. 4B) . The data are fit very well to a twostate model between the folded and unfolded states. Nonetheless, the unfolding profiles are obviously different, the heat denaturation analysis revealed that the apoprotein begins to dramatically lose CD signal at 60-65uC, whereas the Zn-bound or Mn-bound protein starts to significantly lose CD signal at a temperature as high as 65-70uC. The apoprotein exhibits a half-denaturation (T m ) of 71.560.1uC, and addition of Zn 2+ and Mn 2+ significantly stabilizes SsTroA as judged by a substantial enhancement in the Tm value (DTm = 4.660.2uC and 560.1uC, respectively), which agrees with the presence of Zn 2+ or Mn 2+ as seen in CD spectroscopic changes (Fig. 4A) . These results combined with CD spectrum data imply that the ternary protein-metal complex appears to form a compact domain organization, i.e., the metalligand interactions in the SsTroA protein indeed play an important role in imparting extra stability to the metal binding site of the protein. Further, as we proposed that Mn 2+ has a more significant effect on the conformation and thermal stability of SsTroA than Zn 2+ .