Equilibrium unfolding of yeast phosphoglycerate kinase and its mutants lacking one or both native tryptophans: a circular dichroism and steady-state and time-resolved fluorescence study.
Yeast 3-phosphoglycerate kinase contains two tryptophans, both situated in the carboxy-terminal domain, and seven tyrosines, five in the amino-terminal domain, one in the domain-domain interface, and one in the carboxy-terminal domain. Site-specific mutagenesis has been used to construct two single-tryptophan mutants and one no-tryptophan mutant by replacing one or both native tryptophans, W308 and W333, with phenylalanines. The mutations have been shown to have a relatively small effect on the overall structure and enzymatic properties of the mutants. Both tryptophans are quenched in the folded state. The steady-state emission spectra and tryptophan quantum yields are the same in the single-tryptophan mutants and in the wild-type protein. Large changes in the tryptophan emission maxima and steady-state emission intensities are observed upon unfolding. Far-UV circular dichroism and steady-state as well as time-resolved fluorescence spectroscopy have been used to monitor the equilibrium unfolding transitions of these mutants and wild-type PGK. For each protein, the transitions followed by CD and steady-state fluorescence are nearly coincident, suggesting that the structural changes monitored by local fluorescence probes and ellipticity changes, which are sensitive to the changes in the overall structure, report a single cooperative transition, consistent with a two-state unfolding mechanism. Both tryptophans have three lifetimes, which follow a similar pattern as a function of denaturant concentration. The amplitude terms associated with the two longer lifetimes increase with unfolding while the short lifetime amplitude decreases. It thus appears that these population amplitudes represent markers for the unfolded and folded states, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Szpikowska
B K
BK
Division of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Beechem
J M
JM
Sherman
M A
MA
Mas
M T
MT
D002942:Circular Dichroism; D009154:Mutation; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry*
D014364:Tryptophan; D010735:Phosphoglycerate Kinase