Isothermal equilibrium unfolding experiments followed by tryptophan fluorescence (A and B) and far-UV CD (C and D).

<p>Unfolding was induced by urea (A, C) or GdmCl (B, D). Protein variants were incubated with denaturant for 16 h. GdmCl and urea stock solution concentration (8.0 M and 10.0 M) was determined using a standard refractometer. The measurements were carried out at 20°C in 20 mM Tris·HCl, 100 mM NaCl, 1 mM EDTA, pH 7.0. The solid lines represent the nonlinear regression fitting (two-state model, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045743#s4" target="_blank">Materials and Methods</a>).