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 situate d in the carboxy-terminal domain, and seven tyrosines, five in the ami no-terminal domain, one in the domain-domain interface, and one in the carboxy-terminal domain. Site-specific mutagenesis has been used to c onstruct two single-tryptophan mutants and one no-tryptophan mutant by replacing one or both native tryptophans, W308 and W333, with phenyla lanines. The mutations have been shown to have a relatively small effe ct on the overall structure and enzymatic properties of the mutants. B oth tryptophans are quenched in the folded state. The steady-state emi ssion 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 mon itor the equilibrium unfolding transitions of these mutants and wild-t ype PGK. For each protein, the transitions followed by CD and steady-s tate fluorescence are nearly coincident, suggesting that the structura l changes monitored by local fluorescence probes and ellipticity chang es, which are sensitive to the changes in the overall structure, repor t a single cooperative transition, consistent with a two-state unfoldi ng mechanism. Both tryptophans have three lifetimes, which follow a si milar pattern as a function of denaturant concentration. The amplitude terms associated with the two longer lifetimes increase with unfoldin g while the short lifetime amplitude decreases. It thus appears that t hese population amplitudes represent markers for the unfolded and fold ed states, respectively. The transition midpoints calculated from the analysis of each amplitude term are identical with those determined fr om the steady-state total intensity changes. In contrast, those determ ined from the lifetime changes and from the preexponential associated with the long correlation time appear to precede the transitions follo wed by steady-state intensity changes and CD. The time-resolved anisot ropy decays associated with WT, W308, and W333 are distinct in the fol ded proteins and become essentially identical in the unfolded state.