STABILIZATION OF BARSTAR BY CHEMICAL MODIFICATION OF THE BURIED CYSTEINES

The internal packing of residues in the small monomeric protein barsta r was severely perturbed by chemical modification of the two buried cy steine residues with the thiol reagent 5,5'-dithiobis(2-nitrobenzoic a cid) (DTNB) after prior unfolding of the protein using guanidine hydro chloride (GdnHCl). The modification produces mixed disulfides between 5-thio(2-nitrobenzoic acid) and the two Cys residues. To understand th e effects of the modification of the individual cysteine residues, Cys 40 and Cys82, the modification was also carried out on the two single Cys-->Ala mutant forms of barstar, C40A and C82A, whose structures, ac tivities, and stabilities were first shown to be similar to those of w t barstar. Equilibrium GdnHCl-induced denaturation studies an wt barst ar show that the modification causes the midpoint of the denaturation curve to increase by 0.6 M and tile 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 stru ctures of the modified and unmodified proteins have been compared usin g circular dichroism (CD) spectroscopy, UV difference absorption spect roscopy, 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 prote ins, as well as in modified wt barstar. The far-UV CD spectra of the m odified and unmodified proteins are similar, but the mean residue elli pticity at 220 nm of wt barstar is reduced by 30% upon modification, S uch a decrease is not seen for either C40A or C82A. The barnase-inhibi ting activities of the three modified proteins are shown to be similar to those of the corresponding unmodified proteins. Thus, the severe p erturbations of the internal packing, which result in a significant in crease in stability, do not appear to affect the overall fold of barst ar.