Cold denaturation of yeast phosphoglycerate kinase: which domain is more stable?

Abstract

Under destabilising conditions both heat and cold denaturation of yeast phosphoglycerate kinase (PGK) can be observed. According to previous interpretation of experimental data and theoretical calculations, the C-terminal domain should be more stable than the N-terminal domain at all temperatures. We report on thermal unfolding experiments with PGK and its isolated domains, which give rise to a revision of this view. While the C-terminal domain is indeed the more stable one on heating, it reveals lower stability in the cold. These findings are of importance, because PGK has been frequently used as a model for protein folding and mutual domain interactions.as separate units when the temperature is lowered. The distinct unfolding of both domains in the temperature range between 20°C and 0°C has been well demonstrated with different physical methods [8,9]. However, we have good evidence that experimentally observed unfolding transitions have been assigned to the wrong domains. Cold denaturation experiments with the isolated domains and a careful re-evaluation of data of the entire protein molecule demonstrate that in contrast to the previous interpretation of data the N-terminal domain is more stable than the C-terminal domain in the cold.

Structure, function, and folding of phosphoglycerate kinase are strongly perturbed by macromolecular crowding

“…Despite numerous studies of PGK's folding stability and dynamics (24)(25)(26) and the motion of its flexible hinge, by experiment (27) as well as by computation (28,29), it remains an open question to what extent the highly crowded environment can affect the PGK folding and structure and, ultimately, its enzymatic activity involving large-scale structural movement inside a cell. Although experimental studies using synthetic crowding agents have shown that the folding properties and enzymatic activity of simple proteins can be perturbed (30), our study shows that crowding leads to major conformational changes of PGK, with a major impact on its enzymatic activity.…”

Tuning the Heterogeneous Early Folding Dynamics of Phosphoglycerate Kinase

“…However, the wild-type yeast PGK used in the previous study contains two tryptophan residues. 43 Tryptophan 333 is buried in the main hydrophobic core. Its fluorescence is quenched strongly under native conditions.…”

“…38 -42 Cold-denatured PGK at low concentrations of denaturant (0.7 M guanidinium hydrochloride (GuHCl)) is nearly a random coil with a radius of gyration of 7.8 nm, and cold denaturation is an apparent two-state process. 42,43 Refolding of yPGK from all denatured states proceeds via a kinetic intermediate. 37 …”

Role of hydrophobic interactions in yeast phosphoglycerate kinase stability

“…yPGK is destabilized by lowering temperature 9,16,18,19 and such phenomenon can be evidenced by performing Gdn-HCl denaturation at various temperatures. By lowering temperature from 22°C to 4°C, a clear shift in the transition midpoint (Cm) value can be observed for GdnHCl induced transitions monitored either by ellipticity at 220 nm or by fluorescence ( Figs.…”

“…This finding is consistent with previously published studies suggesting that the C-terminal domain is more coldsensitive than the N-terminal domain. 19 Further information about the cold denaturation process can be obtained by using various Gdn-HCl concentrations. In particular, for denaturant concentrations lower that 0.6 M, the denaturation is not complete.…”