Cold denaturation of yeast phosphoglycerate kinase: kinetics of changes in secondary structure and compactness on unfolding and refolding.
Under mildly destabilizing conditions (0.7 M GuHCl), phosphoglycerate kinase from yeast undergoes a reversible two-step equilibrium unfolding transition when the temperature is lowered from 30 to 1 degree C (Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278). The kinetics of the changes in compactness and secondary structure have been studied by means of dynamic light scattering and far-UV circular dichroism, respectively. It turned out that unfolding and refolding after an appropriate temperature jump (T-jump) was performed proceeded in substantially different ways. After a T-jump from 30 to 1 degree C, a multiphasic unfolding behavior was observed, reflecting the independent unfolding of the N-terminal and C-terminal domains with time constants of about 7 and 45 min, respectively. A remarkable feature of the unfolding process is the simultaneous change of compactness and secondary structure. Refolding after a T-jump from 1 degree C to higher temperatures occurs in two stages. At the first stage an appreciable amount of secondary structure is formed rapidly within the dead time of the T-jump, while the overall dimensions of the polypeptide chain remain essentially unchanged. Thus, an extended folding intermediate is formed at an early stage of folding. Further information of secondary structure proceeds slowly within a time range of minutes in parallel with the increase of compactness. At 30 degrees C, both domains refold simultaneously, while at 15 degrees C, independent folding can be observed. These findings are discussed with respect to predictions of existing models of folding.
Gast
K
K
Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany.
Damaschun
G
G
Damaschun
H
H
Misselwitz
R
R
Zirwer
D
D
D003080:Cold Temperature; D007700:Kinetics; D010735:Phosphoglycerate Kinase / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D010735:Phosphoglycerate Kinase