Figure 2.

Urea-induced unfolding of CPR3 studied by fluorescence and CD spectroscopy. (A) Urea-induced unfolding followed by monitoring of the tryptophan fluorescence λ_max for tertiary structure. (B) Urea-induced unfolding followed by monitoring of the tryptophan fluorescence intensity. (C) Urea-induced unfolding followed by CD ellipticity at 226 nm for secondary structure. (D) Normalized transition curves for urea-induced unfolding of CPR3 as followed by CD ellipticity at 226 nm (θ₂₂₆) for secondary structure and by relative tryptophan fluorescence λ_max at different urea concentrations for tertiary structure. A linear extrapolation of the baselines in the pre- and post-transitional regions was used to determine the fraction of unfolded protein within the transition region by assuming a two-state mechanism of unfolding. The curves for CD and fluorescence are red and blue, respectively. (E) The population curve for urea-induced unfolding, in which the difference in the normalized value of λ_max and θ₂₂₆ is plotted against urea concentration. (F) Plot of ANS fluorescence at 470 nm after excitation at 370 nm with increasing urea concentration. To see this figure in color, go online.