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.
Division of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
B K
BK
Szpikowska
M T
MT
Mas
D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D010735:Phosphoglycerate Kinase / Q000737:chemistry*; D017354:Point Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan; D014508:Urea / Q000494:pharmacology*
D006146:Guanidines; D014364:Tryptophan; D014508:Urea; D010735:Phosphoglycerate Kinase; D019791:Guanidine