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首页> 美国卫生研究院文献>Proceedings of the National Academy of Sciences of the United States of America >Ultrarapid mixing experiments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease A
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Ultrarapid mixing experiments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease A

机译：超快速混合实验为核糖核酸酶A折叠过程中初始构象集合的特征提供了新的思路

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摘要

The earliest folding events in single-tryptophan mutants of RNase A were investigated by fluorescence measurements by using a combination of stopped-flow and continuous-flow mixing experiments covering the time range from 70 μs to 10 s. An ultrarapid double-jump mixing protocol was used to study refolding from an unfolded ensemble containing only native proline isomers. The continuous-flow measurements revealed a series of kinetic events on the submillisecond time scale that account for the burst-phase signal observed in previous stopped-flow experiments. An initial increase in fluorescence within the 70-μs dead time of the continuous-flow experiment is consistent with a relatively nonspecific collapse of the polypeptide chain whereas a subsequent decrease in fluorescence with a time constant of ≈80 μs is indicative of a more specific structural event. These rapid conformational changes are not observed if RNase A is allowed to equilibrate under denaturing conditions, resulting in formation of nonnative proline isomers. Thus, contrary to previous expectations, the isomerization state of proline peptide bonds can have a major impact on the structural events during early stages of folding.
机译：RNase A的单个色氨酸突变体中最早的折叠事件是通过荧光测量来研究的，方法是将停流和连续流混合实验相结合，覆盖的时间范围为70μs至10 s。超快速双跳跃混合方案用于研究仅包含天然脯氨酸异构体的未折叠集合的重折叠。连续流测量揭示了在毫秒级时间尺度上的一系列动力学事件，这些动力学事件解释了先前停止流实验中观察到的猝发相信号。在连续流实验的70-s死区时间内荧光的最初增加与多肽链的相对非特异性折叠相一致，而随后以时间常数≈80μs的荧光减少表明结构更特异性事件。如果在变性条件下使RNase A平衡，则不会观察到这些快速的构象变化，导致形成非天然脯氨酸异构体。因此，与先前的预期相反，脯氨酸肽键的异构化状态可对折叠早期的结构事件产生重大影响。

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