Figure 2. Urea denaturation of scFV-3D6h WT and its variants. The maximum of the Trp-fluorescence emission-spectra is plotted as a function of urea concentration. (A-D) Fitting to the three-state model. (A) WT, (B) C1, (C) C2, (D) C3. (E) Comparison of the fitted data. Two transitions are observed during the red-shift. The plateau in the 4–6M urea region is indicative of the occurrence of an intermediate state. (F) Magnification of the first transition shows incremented thermodynamic stability upon mutation. WT, black; C1, dark gray; C2, mild gray; C3, light gray.