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 unfoldin g transition when the temperature is lowered from 30 to 1-degrees-C (G riko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 24 4, 276-278). The kinetics of the changes in compactness and secondary structure have been studied by means of dynamic light scattering and f ar-UV circular dichroism, respectively. It turned out that unfolding a nd refolding after an appropriate temperature jump (T-jump) was perfor med proceeded in substantially different ways. After a T-jump from 30 to 1-degrees-C, a multiphasic unfolding behavior was observed, reflect ing 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 compac tness and secondary structure. Refolding after a T-jump from 1-degrees -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 polypept ide chain remain essentially unchanged. Thus, an extended folding inte rmediate is formed at an early stage of folding. Further formation of secondary structure proceeds slowly within a time range of minutes in parallel with the increase of compactness. At 30-degrees-C, both domai ns refold simultaneously, while at 15-degrees-C, independent folding c an be observed. These findings are discussed with respect to predictio ns of existing models of folding.