Evidence for Initial Non-specific Polypeptide Chain Collapse During the Refolding of the SH3 Domain of PI3 Kinase
Evidence for Initial Non-specific Polypeptide Chain Collapse During the Refolding of the SH3 Domain of PI3 Kinase
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DOI：
10.1016/j.jmb.2010.08.046
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发表时间：
2010-10-29
影响因子：
5.6
通讯作者：
Udgaonkar, Jayant B.
中科院分区：
文献类型：
--
作者：
Dasgupta, Amrita;Udgaonkar, Jayant B.
从盐酸胍（GdnHCl）-未折叠状态的PI3激酶的SH3结构域的重折叠已被探测与毫秒（停止流动）和亚毫秒（连续流动）的荧光，圆二色性，ANS荧光和三个网站的荧光共振能量转移（FRET）效率的变化的测量。荧光测量无法检测到结构变化之前的限速步骤的折叠，而在ANS荧光和FRET效率的变化的测量表明，多肽链崩溃之前的主要结构转变。初始链崩溃反应在150 μ s内完成。此时的折叠形式具有ANS结合的疏水簇。每三个测量的分子内距离收缩的程度预测的FRET信号在完全展开的蛋白质变性剂浓度的依赖性，表明收缩是非特异性的。在亚毫秒折叠的塌陷产物中的每个分子内距离的收缩程度随着[GdnHCl]的减少而连续增加。逐渐收缩是连续的，在完全展开的蛋白质中看到的逐渐收缩，并且其对[GdnHCl]的依赖性并不表明全或无塌陷反应。收缩的程度对[盐酸钆]的依赖性是相似的三个距离，表明链崩溃发生在一个同步的方式跨越不同的多肽链段。亚毫秒测量的GdnHCl中的折叠无法确定是否疏水簇的形成，探测ANS荧光测量，之前的链收缩探测FRET。为了确定氢键是否在初始链崩溃中起作用，通过稀释尿素未折叠状态来引发折叠。在亚毫秒折叠在尿素中的塌陷产物中的至少一个分子内距离的收缩程度类似于在GdnHCl中看到的收缩程度，并且在尿素中的初始收缩也似乎是渐进的。(C)2010爱思唯尔有限公司保留所有权利。
Refolding of the SH3 domain of PI3 kinase from the guanidine hydrochloride (GdnHCl)-unfolded state has been probed with millisecond (stopped flow) and sub-millisecond (continuous flow) measurements of the change in fluorescence, circular dichroism, ANS fluorescence and three-site fluorescence resonance energy transfer (FRET) efficiency. Fluorescence measurements are unable to detect structural changes preceding the rate-limiting step of folding, whereas measurements of changes in ANS fluorescence and FRET efficiency indicate that polypeptide chain collapse precedes the major structural transition. The initial chain collapse reaction is complete within 150 mu s. The collapsed form at this time possesses hydrophobic clusters to which ANS binds. Each of the three measured intra-molecular distances has contracted to an extent predicted by the dependence of the FRET signal in completely unfolded protein on denaturant concentration, indicating that contraction is non-specific. The extent of contraction of each intra-molecular distance in the collapsed product of sub-millisecond folding increases continuously with a decrease in [GdnHCl]. The gradual contraction is continuous with the gradual contraction seen in completely unfolded protein, and its dependence on [GdnHCl] is not indicative of an all-or-none collapse reaction. The dependence of the extent of contraction on [GdnHCl] was similar for the three distances, indicating that chain collapse occurs in a synchronous manner across different segments of the polypeptide chain. The sub-millisecond measurements of folding in GdnHCl were unable to determine whether hydrophobic cluster formation, probed by ANS fluorescence measurement, precedes chain contraction probed by FRET. To determine whether hydrogen bonding plays a role in initial chain collapse, folding was initiated by dilution of the urea-unfolded state. The extent of contraction of at least one intra-molecular distance in the collapsed product of sub-millisecond folding in urea is similar to that seen in GdnHCl, and the initial contraction in urea too appears to be gradual. (C) 2010 Elsevier Ltd. All rights reserved.