Equilibrium folding of CKs.

<p>(A) Mean residue molar ellipticity at 222 nm during unfolding. (B) Parameter A (<i>I</i>₃₆₅/<i>I</i>₃₂₀) of intrinsic fluorescence during unfolding. The data were fitted by the three-state model shown in Scheme 1, and the thermodynamic parameters were listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024681#pone-0024681-t002" target="_blank">Table 2</a>. The fitted curves are presented as solid lines. (C) ANS fluorescence during unfolding. (D) ANS fluorescence during refolding. For clarity, the transition curves of refolding monitored by CD and Trp fluorescence are not shown. Protein unfolding were carried out by incubating 0.2 mg/ml proteins in 20 mM Tris-HCl buffer (pH 8.0) containing various concentrations of GdnHCl at 25°C overnight. The fully-unfolded proteins were prepared by denaturing 24 mg/ml proteins in 6 M GdnHCl for 1 h, and the refolding was conducted by a manual dilution (1∶120) of the denatured proteins in 20 mM Tris-HCl buffer (pH 8.0) containing various concentration of GdnHCl and incubated at 25°C overnight.