Specifically collapsed intermediate in the early stage of the folding of ribonuclease A
Specifically collapsed intermediate in the early stage of the folding of ribonuclease A
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DOI：
10.1016/j.jmb.2005.04.074
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发表时间：
2005-07-08
影响因子：
5.6
通讯作者：
Takahashi, S
中科院分区：
文献类型：
--
作者：
Kimura, T;Akiyama, S;Takahashi, S
蛋白质折叠的爆发相信号的本质一直是一个备受争议的话题，即这些信号代表的是早期中间体的形成，还是未折叠多肽的非特异性塌缩。为了区分这两种可能性，对核糖核酸酶A（RNase A）的亚毫秒级折叠动力学进行了研究，并与核糖核酸酶A的二硫键断裂类似物（r - RNase A）进行了比较。对RNase A的圆二色性测量显示，在折叠反应开始后的320微秒内出现爆发相信号，这与在r - RNase A中观察到的相同。相比之下，在1 - 苯胺基 - 8 - 萘磺酸（ANS）的外在荧光中观察到RNase A有爆发相增加，而r - RNase A则没有。ANS荧光强度的动力学滴定实验表明，在RNase A的快速重折叠组分中存在ANS的特异性结合位点。在折叠反应开始约22毫秒时进行的小角X射线散射测量表明，RNase A的中速和慢速重折叠组分的爆发相构象明显小于r - RNase A。这些结果表明了RNase A和r - RNase A在爆发相构象上的差异。由于r - RNase A在生理溶液条件下是变性的，因此RNase A的爆发相信号被解释为具有特定构象的折叠中间体的形成。（c）2005爱思唯尔有限公司。保留所有权利。
Nature of the burst-phase signals of protein folding has been the subject of much debate as to whether the signals represent the formation of early intermediates or the non-specific collapse of unfolded polypeptides. To distinguish the two possibilities, the submillisecond folding dynamics of ribonuclease A (RNase A) was examined, and compared with those of the disulfide bond-ruptured analog of RNase A (r-RNase A). The circular dichroism measurements on RNase A showed the burst-phase signal within 320 mu s after the initiation of the folding reaction, which was identical to that observed for r-RNase A. In contrast, the burst phase increase in the extrinsic fluorescence from 1-anilino-8-naphthalene sulfonate (ANS) was observed for RNase A but not for r-RNase A. The kinetic titration experiment of the ANS fluorescence intensity showed the presence of a specific binding site for ANS in the fast-refolding component of RNase A. The small-angle X-ray scattering measurements at similar to 22 ms after initiating the folding reaction demonstrated that the burst phase conformations of the medium and slow-refolding components of RNase A were distinctly smaller than that of r-RNase A. These results indicated the difference in the burst phase conformations of RNase A and r-RNase A. Since r-RNase A is denatured in the physiological solution condition, the burst-phase signal of RNase A was interpreted as the formation of the folding intermediate with specific conformations. (c) 2005 Elsevier Ltd. All rights reserved.