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 (PG K) have been used in the present study to characterize the urea-induce d unfolding of PGK. A possibility that residual structures might be pr esent in the urea-unfolded state was also investigated. The urea-induc ed unfolding transitions were monitored using circular dichroism (CD) and fluorescence techniques. The presence of stable intermediate(s) du ring urea-induced unfolding is suggested by biphasic transitions detec ted for the mutants containing tryptophans in the N-terminal domain an d by the noncoincidence of transitions detected by various methods for other mutants. The N-terminal tryptophan probes exhibit hyperfluoresc ent properties in the intermediate state and a wavelength of maximum e mission 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 s tate. 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 bin ding in the denaturant concentration range corresponding to the N --> I transition indicates the presence of a partially folded structure wi th exposed hydrophobic surfaces. These results demonstrate that the pa rtially folded intermediates detected during urea-induced denaturation are structurally similar to those detected previously during guanidin e-induced denaturation. No significant differences were detected betwe en the urea- and guanidine-unfolded proteins on the basis of their flu orescence and CD properties. (C) 1996 Academic Press, Inc.