PERTURBATION OF A TERTIARY HYDROGEN-BOND IN BARSTAR BY MUTAGENESIS OFTHE 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-cha in hydroxyl group of Tyr30 and the side-chain N delta 1 atom of His17 in the small, monomeric, single-domain protein, barstar, has been pert urbed by site-directed mutagenesis of the sole histidine residue (His1 7) to a glutamine residue. The effect of the perturbation has been stu died in the resultant mutant protein, H17Q, by equilibrium unfolding m ethods. Both guanidine hydrochloride (GdnHCl)-induced denaturation and thermal denaturation studies have been performed, with unfolding moni tored by UV absorption, intrinsic tryptophan fluorescence, near-UV and far-UV circular dichroism (CD), and size exclusion chromatography. Wh ile 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-indu ced denaturation, the midpoint of the fluorescence-monitored denaturat ion curve is 1.4 +/- 0.1 M, that of the near-UV CD-monitored denaturat ion curve is 1.6 +/- 0.1 M, and that of the far-UV CD-monitored denatu ration curve is 1.8 +/- 0.1 M. The accumulation of I is also evident i n gel filtration experiments which indicate that I forms slowly from t he fully-folded form, F, and that once formed, I rapidly equilibrates with the unfolded form, U. The gel filtration data for H17Q suggest th at in 1.5 M GdnHCl, there is no F present and that I is the predominan t form. I does not appear to possess hydrated hydrophobic surfaces, wh ich is reflected in its inability to bind 8-anilino-1-naphthalenesulfo nic acid (ANS). At least one equilibrium-unfolding intermediate is als o observed upon thermal denaturation. The midpoints of the thermal den aturation 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; an d 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 cha racterized by a greater disruption of tertiary structure than of secon dary structure.