Determining the topology of virus assembly intermediates using ion mobility spectrometry-mass spectrometry.
Determining the topology of virus assembly intermediates using ion mobility spectrometry-mass spectrometry.
复制标题
DOI：
10.1002/rcm.4732
复制
发表时间：
2010-10-30
期刊：
影响因子：
--
通讯作者：
Ashcroft AE
中科院分区：
文献类型：
--
作者：
Knapman TW;Morton VL;Stonehouse NJ;Stockley PG;Ashcroft AE
我们将离子迁移谱-质谱与串联质谱相结合，在一个实验中就质量、形状（横截面积）和稳定性（解离）方面对大的非共价结合的大分子复合物进行了定性。结果表明，一个复杂的四级结构影响其残留的形状后，通过碰撞诱导解离串联质谱去除一个单一的亚基。其亚基与几个相邻亚基结合以产生环状三维（3D）结构的复合物在解离时经历显著的塌陷。相比之下，在复合物内仅具有单个相邻亚基的亚基在复合物解离后保留其大部分原始形状。具体来说，我们已经确定了两个短暂的，在体外病毒衣壳组装过程中观察到的途径中间体的架构。每个病毒组装中间体的质量、化学计量和横截面积的知识使我们能够基于外壳蛋白构建块的已知X射线结构来模拟一系列潜在结构。比较解离前后这些潜在结构的横截面积为复合物的拓扑结构的分配提供了切实的证据，已经发现复合物的拓扑结构包括最终二十面体病毒壳的3倍和5倍对称轴。这些见解提供了关于病毒组装途径的独特信息，可以允许针对组装步骤设计抗病毒治疗剂。这种方法可以很容易地应用到许多其他非共价键结合的大分子复合物及其组装途径的结构表征。
We have combined ion mobility spectrometry–mass spectrometry with tandem mass spectrometry to characterise large, non-covalently bound macromolecular complexes in terms of mass, shape (cross-sectional area) and stability (dissociation) in a single experiment. The results indicate that the quaternary architecture of a complex influences its residual shape following removal of a single subunit by collision-induced dissociation tandem mass spectrometry. Complexes whose subunits are bound to several neighbouring subunits to create a ring-like three-dimensional (3D) architecture undergo significant collapse upon dissociation. In contrast, subunits which have only a single neighbouring subunit within a complex retain much of their original shape upon complex dissociation. Specifically, we have determined the architecture of two transient, on-pathway intermediates observed during in vitro viral capsid assembly. Knowledge of the mass, stoichiometry and cross-sectional area of each viral assembly intermediate allowed us to model a range of potential structures based on the known X-ray structure of the coat protein building blocks. Comparing the cross-sectional areas of these potential architectures before and after dissociation provided tangible evidence for the assignment of the topologies of the complexes, which have been found to encompass both the 3-fold and the 5-fold symmetry axes of the final icosahedral viral shell. Such insights provide unique information about virus assembly pathways that could allow the design of anti-viral therapeutics directed at the assembly step. This methodology can be readily applied to the structural characterisation of many other non-covalently bound macromolecular complexes and their assembly pathways.