pH Dependence of the stability of barstar to chemical and thermal denaturation

Abstract

Equilibrium unfolding of barstar with guanidine hydrochloride (GdnHCl) and urea as denaturants as well as thermal unfolding have been carried out as a function of pH using fluorescence, far-UV and near-UV CD, and absorbance as probes. Both GdnHCl-induced and urea-induced denaturation studies at pH 7 show that barstar unfolds through a two-state F⇌U mechanism and yields identical values for δG_U the free energy difference between the fully folded (F) and unfolded (U) forms, of 5.0±0.5 kcal·mol⁻¹ at 25°C. Thermal denaturation of barstar also follows a two-state F⇌U unfolding transition at pH 7, and the value of δG_U at 25°C is similar to that obtained from chemical denaturation. The pH dependence of denaturation by GdnHCl is complex. The C_m value (midpoint of the unfolding transition) has been used as an index for stability in the pH range 2-10, because barstar does not unfold through a two-state transition on denaturation by GdnHCl at all pH values studied. Stability is maximum at pH 2-3, where barstar exists in a molten globule-like form that forms a large soluble oligomer. The stability decreases with an increase in pH to 5, the isoelectric pH of the protein. Above pH 5, the stability increases as the pH is raised to 7. Above pH 8, it again decreases as the pH is raised to 10. The decrease in stability from pH 7 to 5 in wild-type (wt) barstar, which is shown to be characterized by an apparent pK_a of 6.2±0.2, is not observed in H17Q, a His 17±Gln 17 mutant form of barstar. This decrease in stability has therefore been correlated with the protonation of His 17 in barstar. The decrease in stability beyond pH 8 in wt barstar, which is characterized by an apparent pK_a of 9.2±0.2, is not detected in BSCCAA, the Cys 40 Cys 82→Ala 40 Ala 82 double mutant form of barstar. Thus, this decrease in stability has been correlated with the deprotonation of at least one of the two cysteines present in wt barstar. The increase in stability from pH 5 to 3 is characterized by an apparent pK_a of 4.6±0.2 for wt barstar and BSCCAA, which is similar to the apparent pK_a that characterizes the structural transition leading to the formation of the A form. The use of C_m as an index of stability has been supported by thermal denaturation studies. In the pH range where both chemical denaturation and thermal denaturation studies were possible, both C_m and T_m, the midpoint of a thermal denaturation curve displays similar trends. Very high pH (pH 12) is shown to completely unfold the protein in a fully reversible manner.