To examine whether ligand binding induces a variation of exposed hydrophobic protein regions, we applied fluorescence spectroscopy to monitor the binding of the hydrophobic probe ANS to SsTroA. ANS is extensively used to detecting the Table S2 . doi:10.1371/journal.pone.0019510.g002 equilibrium and kinetic folding intermediates of proteins, especially for a partially folded protein [43] . Interaction of the probe with a hydrophobic site yields an enhancement of fluorescence intensity, usually accompanied by a blue shift of the maximum. SsTroA significantly enhances the emission intensity of ANS and promotes a blue shift (Fig. 3B) . Meanwhile, the fluorescence intensity of ANS clearly decreases in the metal bound form compared to the apo-SsTroA. As indicated by the diminished fluorescence, ANS displays stronger binding to Mn 2+ -SsTroA than Zn 2+ -SsTroA. The spectra clearly provide evidence that the addition of metal ions induces a large change in the nonpolarity of the ANS-binding site, and the Mn 2+ causes a greater change than Zn 2+ does. These data suggested that the decreased plots observed in the spectra (Fig. 3B) can be attributed to a more compact and ordered structure for SsTroA in the presence of metal.