Gast K, Damaschun G, Damaschun H, Misselwitz R, Zirwer D
Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany.
Biochemistry. 1993 Aug 3;32(30):7747-52. doi: 10.1021/bi00081a020.
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.
在轻度去稳定条件下（0.7 M 盐酸胍），当温度从30℃降至1℃时，酵母磷酸甘油酸激酶会经历一个可逆的两步平衡去折叠转变（Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276 - 278）。分别通过动态光散射和远紫外圆二色性研究了紧密性和二级结构变化的动力学。结果表明，在进行适当的温度跃升（T-jump）后，去折叠和重新折叠的方式有很大不同。从30℃跃升至1℃后，观察到多相去折叠行为，反映出N端和C端结构域的独立去折叠，时间常数分别约为7分钟和45分钟。去折叠过程的一个显著特征是紧密性和二级结构的同时变化。从1℃跃升至更高温度后的重新折叠分两个阶段进行。在第一阶段，在温度跃升的死时间内迅速形成了相当数量的二级结构，而多肽链的整体尺寸基本保持不变。因此，在折叠早期形成了一个伸展的折叠中间体。二级结构的进一步形成在几分钟的时间范围内与紧密性增加同时缓慢进行。在30℃时，两个结构域同时重新折叠，而在15℃时，可以观察到独立折叠。结合现有折叠模型的预测对这些发现进行了讨论。
