As protein aggregation occurs after demetallization, we suspected that apo-SsTroA is unstable compared to the native SsTroA at room temperature, i.e., we assumed that SsTroA displays metal-dependent conformational stability of SsTroA. To obtain further information on the metal sensitivity of the SsTroA and details on possible structural changes occurring upon its interaction with metal ions, we applied CD spectroscopy to monitor these processes. Proteins were prepared by incubation with Zn 2+ and Mn 2+ as previously described [31] . In the far-UV regions (Fig. 4A) , the CD spectra are generally characterized by distinct minima at 208 and 223 nm respectively, which are the features of proteins that contain a-helix conformational elements, consistent with the crystal structure data. The metal bound SsTroA samples both displayed spectra similar to apo-SsTroA, only with minor differences, though both has deeper 208 nm and 223 nm minima relative to apo-SsTroA. The intensity of the 223-nm band of SsTroA is in the order: apo-SsTroA.Zn 2+ -SsTroA.Mn 2+ -SsTroA, suggesting a decrease of the content of secondary structure in the apo form compared to the metal-bound forms. While previous work demonstrates that TpTroA does not manifest changes discernible by CD upon metal binding, in contrast, T. pallidum ZnuA does undergo conformational changes when Zn 2+ is loaded [31] . These data indicated that TpTroA and SsTroA present differential binding behaviors upon metal binding.