Analysis of rapid, large-scale protein quaternary structural changes:: Time-resolved X-ray solution scattering of Nudaurelia capensis ω virus (NωV) maturation
Analysis of rapid, large-scale protein quaternary structural changes:: Time-resolved X-ray solution scattering of Nudaurelia capensis ω virus (NωV) maturation
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DOI：
10.1006/jmbi.2001.4896
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发表时间：
2001-08-24
影响因子：
5.6
通讯作者：
Johnson, JE
中科院分区：
文献类型：
--
作者：
Canady, MA;Tsuruta, H;Johnson, JE
时间分辨小角X射线散射（TR-SAXS）用于研究二十面体病毒成熟过程中发生的大构象变化的动力学。T = 4无包膜RNA病毒角裸壳虫omega病毒（N omegaV）的病毒样颗粒（VLP）显示经历大的pH依赖性构象变化。使用电子冷冻显微镜（cryoEM）和X射线溶液散射显示前体VLP（原衣壳）的直径比所得衣壳大16%，cryoEM研究显示所得衣壳与感染性成熟病毒体非常相似。原壳体。在pH 7.5下观察到VLP的形式，并在pH 5.0下转化为衣壳形式。在这些pH值之间的溶液中的VLP的静态SAXS测量确定了过渡的半滴定点为pH 6.0。时间分辨SAXS实验进行VLP解决方案，通过启动pH值从7.5到5.0的变化，使用停流装置，和时间尺度的构象变化发生在亚秒范围内。使用不太剧烈的pH值变化（将pH值降低至5.8或5.5），构象变化发生得更慢，在亚分钟或分钟的时间尺度上，检测到过渡中的快速形成的中间体。使用静态SAXS测量的进一步表征表明，构象变化最初是可逆的，但在自蛋白水解成熟完成约15%后变得不可逆。除了表征大的四级构象变化，我们已经能够第一次证明，它发生在亚秒级的时间尺度上，一个政权在其他多亚基组件中观察到的。(C)北京：科学出版社.
Time-resolved small-angle X-ray scattering (TR-SAXS) was used to study the kinetics of a large conformational change that occurs during the maturation of an icosahedral virus. Virus-like particles (VLPs) of the T = 4 non-enveloped RNA virus Nudaurelia capensis omega virus (N omegaV) were shown to undergo a large pH-dependent conformational change. Electron cryo-microscopy (cryoEM) and X-ray solution scattering were used to show that the precursor VLP (procapsid) was 16% larger in diameter, than the resulting capsid, which was shown by the cryoEM study to, closely resemble the infectious mature virion. The procapsid. form of the VLPs was observed at pH 7.5 and was converted to the capsid form at pH 5.0. Static SAXS measurements of the VLPs in solutions ranging between these pH values determined that the half-titration point of the transition was pH 6.0. Time-resolved SAXS experiments were performed on VLP solutions by initiating a pH change from 7.5 to 5.0 using a stopped-flow device, and the time-scale of the conformational change occurred in the subsecond range. Using a less drastic pH change (lowering the pH to 5.8 or 5.5), the conformational change occurred more slowly, on the subminute or minute time-scale, with the detection of a fast-forming intermediate in the transition. Further characterization using static SAXS measurements showed that the conformational change was initially reversible but became irreversible after autoproteolytic maturation was about 15% complete. In addition to characterizing the large quaternary conformational change, we have been able for the first time to demonstrate that it takes place on the subsecond time-scale, a regime comparable to that observed in other multisubunit assemblies. (C) 2001 Academic Press.