PERTURBATION OF A TERTIARY HYDROGEN-BOND IN BARSTAR BY MUTAGENESIS OF THE SOLE HIS RESIDUE TO GLN LEADS TO ACCUMULATION OF AT LEAST ONE EQUILIBRIUM FOLDING INTERMEDIATE

A specific tertiary hydrogen bond that is present between the side-chain hydroxyl group of Tyr30 and the side-chain N delta 1 atom of His17 in the small, monomeric, single-domain protein, barstar, has been perturbed by site-directed mutagenesis of the sole histidine residue (His17) to a glutamine residue. The effect of the perturbation has been studied in the resultant mutant protein, H17Q, by equilibrium unfolding methods. Both guanidine hydrochloride (GdnHCl)-induced denaturation and thermal denaturation studies have been performed, with unfolding monitored by UV absorption, intrinsic tryptophan fluorescence, near-UV and far-UV circular dichroism (CD), and size exclusion chromatography. While wild-type (wt) barstar shows a two-state unfolding transition when denatured by either GdnHCl or heat, the mutant protein H17Q undergoes unfolding through a transition that involves at least one equilibrium intermediate I, which is populated at intermediate concentrations of denaturant or at intermediate temperatures. In the case of GdnHCl-induced denaturation, the midpoint of the fluorescence-monitored denaturation curve is 1.4 +/- 0.1 M, that of the near-UV CD-monitored denaturation curve is 1.6 +/- 0.1 M, and that of the far-UV CD-monitored denaturation curve is 1.8 +/- 0.1 M. The accumulation of I is also evident in gel filtration experiments which indicate that I forms slowly from the fully-folded form, F, and that once formed, I rapidly equilibrates with the unfolded form, U. The gel filtration data for H17Q suggest that in 1.5 M GdnHCl, there is no F present and that I is the predominant form. I does not appear to possess hydrated hydrophobic surfaces, which is reflected in its inability to bind 8-anilino-1-naphthalenesulfonic acid (ANS). At least one equilibrium-unfolding intermediate is also observed upon thermal denaturation. The midpoints of the thermal denaturation curves of H17Q are 63.0 +/- 0.5 degrees C when monitored by absorbance at 287 nm or by intrinsic fluorescence at 332 nm; 65.0 +/- 0.5 degrees C when monitored by mean residue ellipticity at 275 nm; and 68.3 +/- 0.5 degrees C when monitored by mean residue ellipticity at 220 nm. In contrast, all four optical probes yield the same midpoint, 71.5 +/- 0.5 degrees C, for the wt protein. The results indicate that perturbation of the tertiary hydrogen bond leads to the accumulation of at least one intermediate (I) in both thermal denaturation studies and GdnHCl-induced denaturation studies. The intermediate(s) I are characterized by a greater disruption of tertiary structure than of secondary structure.