Fig. 4.

pH-dependent denaturation of DF2. (a) Stability of DF2 as a function of pH was determined by guanidine denaturation curves measured by CD. Open symbols designate a DF2 concentration of 10.7 μM, and filled symbols are for a DF2 concentration of 33.3 μM. (open diamond) pH 7.0, (closed circle) pH 6.5, (open triangle, point down) pH 6.0, (open square) pH 5.5, (open and closed triangles, point up) pH 5.0, (open circle) pH 4.0. Curves were determined using pH 4.0, pH 5.0, and pH 5.5 data assuming two-state denaturation from dimer to monomer, because trimer concentration at these pH values is small. Dashed line curves are theoretical curves for pH 6.0, pH 6.5, and pH 7.0 predicted from the lower pH data. The calculated curve for pH 6.0 deviates slightly from the experimental data, and the deviation between the curves and the data becomes progressively larger as the pH is raised. (b) DF2 takes up two protons upon folding. The negative of the slope of the plot (Eq. 2 in Results) is the proton uptake, equal to 1.9 ± 0.2. Observed equilibrium constants were determined from curves in a. Higher pH data were not used because of the significant trimer population, and pH 4.0 data were not used because Glu residues would be expected to be protonated in the unfolded state.