Rapidly Forming Early Intermediate Structures Dictate the Pathway of Capsid Assembly
Rapidly Forming Early Intermediate Structures Dictate the Pathway of Capsid Assembly
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DOI：
10.1021/jacs.0c01092
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发表时间：
2020-04-29
影响因子：
15
通讯作者：
Raviv, Uri
中科院分区：
文献类型：
--
作者：
Asor, Roi;Schlicksup, Christopher John;Raviv, Uri
在从B型肝炎病毒衣壳蛋白二聚体到120-二聚体衣壳的组装路径上，存在类似于10（30）种可能的中间体。如果每个中间体都被测试，组装通常会陷入熵阱，基本上每个衣壳都会遵循一条独特的组装路径。然而，衣壳快速组装时捕获的中间体最少，这是Levinthal悖论的实现。为了理解衣壳组装的基本机制，解决反应的早期阶段至关重要。我们已经使用时间分辨的小角度X射线散射，这是敏感的溶质的大小和形状，并具有毫秒的时间分辨率。使用最大熵原理，将散射曲线拟合到组装中间体的化学策划库。最大熵也为物种的选择提供了一个物理学原理。我们发现，衣壳组装途径是精致的初始组装条件敏感。在测试的最温和条件下，反应似乎是从二聚体到120-二聚体衣壳的两种状态，具有一些二聚体的二聚体和二聚体的三聚体。在稍微更具侵略性的条件下，我们观察到瞬时积累的十聚体的二聚体和90-二聚体衣壳的外观。在有可测量的动力学捕获的条件下，我们发现高度多样化的早期中间体在几分之一秒内积累，并传播到长寿命的动力学捕获状态（>= 90聚体）。在所有情况下，中间体之间的35和90个亚基没有积累。这些结果与连接早期和晚期中间体的低屏障路径的存在一致，并将最终组装路径引导至可以暂停组装的晚期中间体。
There are similar to 10(30) possible intermediates on the assembly path from hepatitis B capsid protein dimers to the 120-dimer capsid. If every intermediate was tested, assembly would often get stuck in an entropic trap and essentially every capsid would follow a unique assembly path. Yet, capsids assemble rapidly with minimal trapped intermediates, a realization of the Levinthal paradox. To understand the fundamental mechanisms of capsid assembly, it is critical to resolve the early stages of the reaction. We have used time-resolved small angle X-ray scattering, which is sensitive to solute size and shape and has millisecond temporal resolution. Scattering curves were fit to a thermodynamically curated library of assembly intermediates, using the principle of maximum entropy. Maximum entropy also provides a physical rationale for the selection of species. We found that the capsid assembly pathway was exquisitely sensitive to initial assembly conditions. With the mildest conditions tested, the reaction appeared to be two-state from dimer to 120-dimer capsid with some dimers-of-dimers and trimers-of-dimers. In slightly more aggressive conditions, we observed transient accumulation of a decamer-of-dimers and the appearance of 90-dimer capsids. In conditions where there is measurable kinetic trapping, we found that highly diverse early intermediates accumulated within a fraction of a second and propagated into long-lived kinetically trapped states (>= 90-mer). In all cases, intermediates between 35 and 90 subunits did not accumulate. These results are consistent with the presence of low barrier paths that connect early and late intermediates and direct the ultimate assembly path to late intermediates where assembly can be paused.