Kim P S, Baldwin R L
Biochemistry. 1980 Dec 23;19(26):6124-9. doi: 10.1021/bi00567a027.
In the folding reaction of the slow-folding species (US) of ribonuclease A (RNase A), the slow isomerization of wrong proline isomers provides a suitable trap for kinetic folding intermediates at low temperatures (0--10 degrees C). Partly folded intermediates are known to accumulate before proline isomerization takes place, after which native RNase A is formed. We have been able to measure the protection from amide proton exchange which is provided by structure in the intermediates at different times along the folding pathway. Previous work has shown that, by labeling the amide protons of the unfolded protein before initiating refolding, an early folding intermediate can be detected. The new pulse-labeling method presented here can be used to label later folding intermediates. Our results indicate that, in conditions which strongly favor the native protein, intermediates are formed which provide protection against exchange. However, when folding is initiated in 2.5 M Gdn . HCl, 10 degrees C, pH 7.5, conditions in which folding goes to completion but there are no spectroscopically detectable intermediates, then no intermediates are detected by our method. Alternate minimal mechanisms for the folding of US are presented.
在核糖核酸酶A（RNase A）慢折叠物种（US）的折叠反应中，错误脯氨酸异构体的缓慢异构化在低温（0-10摄氏度）下为动力学折叠中间体提供了一个合适的陷阱。已知部分折叠的中间体在脯氨酸异构化发生之前会积累，之后形成天然RNase A。我们已经能够测量在折叠途径中不同时间中间体结构所提供的对酰胺质子交换的保护作用。先前的工作表明，通过在开始重折叠之前标记未折叠蛋白质的酰胺质子，可以检测到早期折叠中间体。这里介绍的新脉冲标记方法可用于标记后期折叠中间体。我们的结果表明，在强烈有利于天然蛋白质的条件下，会形成提供抗交换保护的中间体。然而，当在2.5 M盐酸胍、10摄氏度、pH 7.5的条件下开始折叠时，折叠反应会完成，但没有光谱可检测到的中间体，那么我们的方法就检测不到中间体。文中还提出了US折叠的替代最小机制。
