Stabilization of barstar by chemical modification of the buried cysteines

Abstract

The internal packing of residues in the small monomeric protein barstar was severely perturbed by chemical modification of the two buried cysteine residues with the thiol reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) after prior unfolding of the protein using guanidine hydrochloride (GdnHCl). The modification produces mixed disulfides between 5-thio(2-nitrobenzoic acid) and the two Cys residues. To understand the effects of the modification of the individual cysteine residues, Cys40 and Cys82, the modification was also carried out on the two single Cys→Ala mutant forms of barstar, C40A and C82A, whose structures, activities, and stabilities were first shown to be similar to those of wt barstar. Equilibrium GdnHCl-induced denaturation studies on wt barstar show that the modification causes the midpoint of the denaturation curve to increase by 0.6 M and the stability to increase by 1.3 kcal mol⁻¹. Both C40A and C82A also denature at higher concentrations of GdnHCl after modification. Modification of Cys40 has approximately the same stabilizing contribution as does modification of Cys82. The structures of the modified and unmodified proteins have been compared using circular dichroism (CD) spectroscopy, UV difference absorption spectroscopy, and fluorescence spectroscopy. It is shown that the 5-thio(2-nitrobenzoic acid) groups introduced by reaction with DTNB are buried in hydrophobic environments in the modified C40A and C82A mutant proteins, as well as in modified wt barstar. The far-UV CD spectra of the modified and unmodified proteins are similar, but the mean residue ellipticity at 220 nm of wt barstar is reduced by 30% upon modification. Such a decrease is not seen for either C40A or C82A. The barnase-inhibiting activities of the three modified proteins are shown to be similar to those of the corresponding unmodified proteins. Thus, the severe perturbations of the internal packing, which result in a significant increase in stability, do not appear to affect the overall fold of barstar.