FOLDING OF RIBONUCLEASE-T1 .2. KINETIC-MODELS FOR THE FOLDING AND UNFOLDING REACTIONS
FOLDING OF RIBONUCLEASE-T1 .2. KINETIC-MODELS FOR THE FOLDING AND UNFOLDING REACTIONS
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DOI：
10.1021/bi00464a024
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发表时间：
1990-03-27
期刊：
影响因子：
2.9
通讯作者：
SCHMID, FX
中科院分区：
文献类型：
--
作者：
KIEFHABER, T;QUAAS, R;SCHMID, FX
用不同的探针研究了核糖核酸酶T1的缓慢复性。具有二级结构的结构中间体在折叠过程中很早就形成了，这表明酰胺区迅速恢复了类似天然的圆二色谱。这种广泛的结构形成比缓慢的复性步骤要快得多，后者的速度受到错误的脯氨酸异构体的重新异构化的限制。利用折叠中间体相对于天然蛋白质稳定性降低的特点，通过展开实验检测到了瞬时折叠中间体。这篇论文和前一篇论文的结果[Kiefhaber等人。(1990)生物化学(本期前文)]被用来提出核糖核酸酶T1的去折叠和重折叠的动力学模型。去折叠机制是基于这样的假设，即在结构去折叠步骤之后，变性蛋白质中两个X-Pro多肽键的缓慢异构化相互独立地发生。在平衡状态下，少量的快折叠物种与三个慢折叠物种共存：两个物种各有一个错误的脯氨酸异构体，另一个是优势物种，它们都处于错误的异构体状态。在重折叠机制中，我们假设所有缓慢折叠的分子都可以在折叠早期迅速重新获得大部分二级结构和部分三级结构。错误的脯氨酸肽键的再异构化构成了缓慢的、限速的复性步骤。折叠动力学模型的一个特殊特征是，具有两个错误的Pro异构体的主要未折叠物种可以位于两条不同的折叠路径的中心，这取决于两条重新异构体中的哪一条最先发生异构化。在快速形成的中间体阶段，各个脯氨酸肽键的重新异构化的相对速率决定了途径的选择。在Pro异构酶的存在下，它会发生变化，因为这种酶以不同的效率催化这两种异构化反应，从而导致从非常慢的复性途径转移到中间复性途径。
The slow refolding of ribonuclease T1 was investigated by different probes. Structural intermediates with secondary structure are formed early during refolding, as indicated by the rapid regain of a native-like circular dichroism spectrum in the amide region. This extensive structure formation is much faster than the slow steps of refolding, which are limited in rate by the reisomerization of incorrect proline isomers. The transient folding intermediates were also detected by unfolding assays, which make use of the reduced stability of folding intermediates relative to that of the native protein. The results of this and the preceding paper [Kiefhaber et al. (1990) Biochemistry (preceding paper in this issue)] were used to propose kinetic models for the unfolding and refolding of ribonuclease T1. The unfolding mechanism is based on the assumption that, after the structural unfolding step, the slow isomerizations of two X-Pro peptide bonds occur independently of each other in the denatured protein. At equilibrium a small amount of fast-folding species coexists with three slow-folding species: two with one incorrect proline isomer each and another, dominant species with both these prolines in the incorrect isomeric state. In the mechanism for refolding we assume that all slow-folding molecules can rapidly regain most of the secondary and part of the tertiary structure early in folding. Reisomerizations of incorrect proline peptide bonds constitute the slow, rate-limiting steps of refolding. A peculiar feature of the kinetic model for refolding is that the major unfolded species with two incorrect proline isomers can center two alternative folding pathways, depending on which of the two reisomerizes first. The relative rates of reisomerization of the respective proline peptide bonds at the stage of the rapidly formed intermediate determine the choice of pathway. It is changed in the presence of prolyl isomerase, because this enzyme catalyzes these two isomerizations with different efficiency and consequently leads to a shift from the very slow to the intermediate refolding pathway.