Figure 4.

Equilibrium unfolding (closed symbols) and refolding (open symbols) of wild-type HγD-Crys in GuHCl probed by fluorescence emission. (A) The solid black line represents a two-state fit of equilibrium unfolding data. Residuals of the fit are shown. (B) Equilibrium unfolding data fit to a three-state model (solid black line), including residuals of the fit. Fluorescence spectra were recorded for each sample using an excitation wavelength of 295 nm. Fluorescence intensity at 360/320 nm was used in order to simultaneously monitor changes in the unfolding and native maxima. Samples were allowed to equilibrate in GuHCl at 37°C for 24 h prior to recording fluorescence emission spectra. By inspection, both fits appear sui ; however, residuals of the three-state fit are of lower magnitude and more random than those of the two-state fit. This observation along with other factors described in the text suggests that the three-state fit is a better description of the data.