FOLDING OF RIBONUCLEASE-T1 .1. EXISTENCE OF MULTIPLE UNFOLDED STATES CREATED BY PROLINE ISOMERIZATION
FOLDING OF RIBONUCLEASE-T1 .1. EXISTENCE OF MULTIPLE UNFOLDED STATES CREATED BY PROLINE ISOMERIZATION
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DOI：
10.1021/bi00464a023
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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
我们的目标是阐明蛋白质折叠机制的分子方面。我们使用核糖核酸酶T1作为模型蛋白，因为它是一个小的单结构域蛋白，具有明确的二级和三级结构，在存在和不存在二硫键的情况下都是稳定的。此外，有效的诱变系统可用于产生具有确定的序列变异的蛋白质分子。在这里，我们提出了核糖核酸酶T1的折叠动力学的初步表征。它的去折叠和再折叠反应是可逆的，这表现在去折叠/再折叠循环后催化活性的定量恢复。重折叠是一个复杂的过程，其中天然蛋白质在三个可区分的途径上形成。有3.5%的快速折叠分子，在毫秒的时间范围内重新折叠，和96.5%的缓慢折叠物种，在几分钟到几小时的时间范围内恢复到天然状态。这些缓慢折叠的分子引起两个主要的平行重折叠反应。快速和缓慢折叠分子的混合物是在解折叠后通过链平衡反应缓慢产生的，该反应显示脯氨酸异构化的性质。我们的结论是，部分的RNase T1折叠的动力学复杂性可以解释的基础上的脯氨酸模型的蛋白质折叠。这是支持的发现，这种蛋白质的缓慢重折叠反应的酶脯氨酰异构酶的存在下加速。然而，核糖核酸酶T1重折叠的几个属性，如探针的相对振幅的依赖性，用于以下折叠，不容易解释一个简单的脯氨酸模型。
It is our aim to elucidate molecular aspects of the mechanism of protein folding. We use ribonuclease T1 as a model protein, because it is a small single-domain protein with a well-defined secondary and tertiary structure, which is stable in the presence and absence of disulfide bonds. Also, an efficient mutagenesis system is available to produce protein molecules with defined sequence variations. Here we present a preliminary characterization of the folding kinetics of ribonuclease T1. Its unfolding and refolding reactions are reversible, which is shown by the quantitative recovery of the catalytic activity after an unfolding/refolding cycle. Refolding is a complex process, where native protein is formed on three distinguishable pathways. There are 3.5% fast-folding molecules, which refold within the millisecond time range, and 96.5% slow-folding species, which regain the native state in the time range of minutes to hours. These slow-folding molecules give rise to two major, parallel refolding reactions. The mixture of fast- and slow-folding molecules is produced slowly after unfolding by chain equilibration reactions that show properties of proline isomerization. We conclude that part of the kinetic complexity of RNase T1 folding can be explained on the basis of the proline model for protein folding. This is supported by the finding that the slow refolding reactions of this protein are accelerated in the presence of the enzyme prolyl isomerase. However, several properties of ribonuclease T1 refolding, such as the dependence of the relative amplitudes on the probes, used to follow folding, are not readily explained by a simple proline model.