Canady M A, Tsuruta H, Johnson J E
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
J Mol Biol. 2001 Aug 24;311(4):803-14. doi: 10.1006/jmbi.2001.4896.
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 (NomegaV) 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.
时间分辨小角X射线散射（TR-SAXS）被用于研究二十面体病毒成熟过程中发生的大尺度构象变化的动力学。研究表明，T=4的无包膜RNA病毒——卡普斯角无尾病毒（NomegaV）的病毒样颗粒（VLP）会发生大尺度的pH依赖性构象变化。电子冷冻显微镜（cryoEM）和X射线溶液散射结果表明，前体VLP（原衣壳）的直径比最终的衣壳大16%，cryoEM研究显示最终衣壳与感染性成熟病毒粒子极为相似。在pH 7.5时观察到VLP的原衣壳形式，在pH 5.0时则转变为衣壳形式。对介于这两个pH值之间的溶液中的VLP进行静态SAXS测量，确定转变的半滴定点为pH 6.0。通过使用停流装置将pH从7.5变为5.0，对VLP溶液进行时间分辨SAXS实验，结果表明构象变化的时间尺度在亚秒范围内。使用较温和的pH变化（将pH降至5.8或5.5），构象变化发生得更慢，在亚分钟或分钟时间尺度上，并且在转变过程中检测到一种快速形成的中间体。使用静态SAXS测量进行的进一步表征表明，构象变化最初是可逆的，但在自催化成熟完成约15%后变为不可逆。除了表征这种大尺度的四级构象变化外，我们首次证明它发生在亚秒时间尺度上，这一情况与在其他多亚基组装体中观察到的情况相当。
