Urea-induced equilibrium unfolding of single tryptophan mutants of yeast phosphoglycerate kinase: evidence for a stable intermediate.

Several single-tryptophan mutants of yeast phosphoglycerate kinase (PGK) have been used in the present study to characterize the urea-induced unfolding of PGK. A possibility that residual structures might be present in the urea-unfolded state was also investigated. The urea-induced unfolding transitions were monitored using circular dichroism (CD) and fluorescence techniques. The presence of stable intermediate(s) during urea-induced unfolding is suggested by biphasic transitions detected for the mutants containing tryptophans in the N-terminal domain and by the noncoincidence of transitions detected by various methods for other mutants. The N-terminal tryptophan probes exhibit hyperfluorescent properties in the intermediate state and a wavelength of maximum emission that lies between that of the native and unfolded state. This unfolding intermediate exhibits a major decrease in the ellipticity at 220 nm, but only a minor decrease at 278 nm, relative to the native state. These results suggest a significant loss of secondary structure content and a relatively small change in the asymmetric environment of tyrosine residues. Increased 1-anilinonaphthalene-8-sulfonic acid binding in the denaturant concentration range corresponding to the N --> I transition indicates the presence of a partially folded structure with exposed hydrophobic surfaces. These results demonstrate that the partially folded intermediates detected during urea-induced denaturation are structurally similar to those detected previously during guanidine-induced denaturation. No significant differences were detected between the urea- and guanidine-unfolded proteins on the basis of their fluorescence and CD properties.