We applied isothermal titration calorimetry (ITC) to monitor the energetics of Zn 2+ and Mn 2+ binding to the apo-SsTroA. ITC measures the heat directly released or absorbed upon an interaction triggered by mixing two components, and is capable of calculating both the extent of the ligand binding affinity and the free energy values (DG) and enthalpy(DH) changes, from which entropy (DS) is determined [38] . The isotherms for loading with Zn 2+ and Mn 2+ appeared in normal sigmoidal titration curves, in agreement with our ICP-MS data. Again the binding stoichiometry (n) was determined to be ,1 in both cases (Fig. 2) , confirming that a specific 1:1 complex is formed in each case. The association constants (Ka) for each metal ion binding to SsTroA were around 10 7 M 21 (Fig. 2) . SsTroA affinities for Zn 2+ and Mn 2+ were calculated from duplicate measurements with K D s ( = 1/Ka) of 43469 nM and 254613 nM, respectively, indicating the formation of tight complexes. It should be noted that SsTroA binds Mn 2+ with a higher affinity than that of Zn 2+ . These values are of a similar magnitude to those described for binding of Zn 2+ and Mn 2+ to N. gonorrhoeae MntC [33] . In each metal ion titration with both metal ions, we observed negative enthalpy and entropy values, demonstrating that binding of each ion to SsTroA is an exothermic event. The thermodynamic profiles for metal binding by SsTroA are not identical to those previously reported for TpTroA [31] . Intrinsic metal binding to proteins is usually entropically driven (DH.0) due to the very high dehydration energies of divalent cations. However, the binding enthalpy can become exothermic (DH,0) if metal binding is coupled to a protein conformational change [39, 40] . Our ITC data are consistent with a significant conformational change observed in The four metal-coordinating residues are highlighted in green. The LXXC lipoprotein motif (type II signal peptides for Sec-dependent transport) [34] is highlighted in red. The two conserved residues predicted to form the salt bridge are marked in yellow. Secondary structure elements for SsTroA are shown above the sequence. TpTroA, TroA from Treponema pallidum; SpPsaA, PsaA from Streptococcus pneumonia; SyMntC, MntC from Synechocytis 6803. doi:10.1371/journal.pone.0019510.g001 our nuclear magnetic resonance (NMR) and CD spectra. Thermodynamic parameters for the binding of Zn 2+ /Mn 2+ to SsTroA are summarized in Table S2 .