Spectral and Hydrodynamic Analysis of West Nile Virus RNA-Protein Interactions by Multiwavelength Sedimentation Velocity in the Analytical Ultracentrifuge.
Spectral and Hydrodynamic Analysis of West Nile Virus RNA-Protein Interactions by Multiwavelength Sedimentation Velocity in the Analytical Ultracentrifuge.
复制标题
通过分析超速离心机中的多波长沉降速度对西尼罗河病毒 RNA-蛋白质相互作用进行光谱和流体动力学分析。
DOI：
10.1021/acs.analchem.6b03926
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发表时间：
2017
影响因子：
7.4
通讯作者：
Demeler,Borries
中科院分区：
文献类型：
--
作者：
Zhang,Jin;Pearson,JosephZ;Gorbet,GaryE;Cölfen,Helmut;Germann,MarkusW;Brinton,MargoA;Demeler,Borries
核酸和蛋白质之间的相互作用对许多细胞过程都是至关重要的，它们的研究对生物化学、细胞生物学和病毒学的许多领域都是至关重要的。在这里，我们介绍了一种新的基于沉降速度(SV)分析超速离心法的分析方法，并结合一种新的多波长检测器来表征这种相互作用。我们鉴定了西尼罗河病毒RNA干环结构与人类T细胞限制性细胞内抗原-1相关蛋白相互作用过程中形成的复合体的化学计量比和摩尔质量。长期以来，SV一直被证明是研究溶液中生理条件下动态组装过程的一种强有力的技术。在这里，我们首次展示了如何利用新的多波长技术来研究蛋白质-RNA相互作用，并展示了来自新检测器的光谱信息如何补充来自分析超速离心法的传统流体力学信息。我们的方法允许通过光谱分解来分离蛋白质和核酸信号，以便可以基于它们的光谱特征来清楚地识别来自每个单独物种的沉积信息，包括任何复合体。这里提出的方法扩展到任何交互系统，其中交互伙伴在频谱上是可分离的。
Interactions between nucleic acids and proteins are critical for many cellular processes, and their study is of utmost importance to many areas of biochemistry, cellular biology, and virology. Here, we introduce a new analytical method based on sedimentation velocity (SV) analytical ultracentrifugation, in combination with a novel multiwavelength detector to characterize such interactions. We identified the stoichiometry and molar mass of a complex formed during the interaction of a West Nile virus RNA stem loop structure with the human T cell-restricted intracellular antigen-1 related protein. SV has long been proven as a powerful technique for studying dynamic assembly processes under physiological conditions in solution. Here, we demonstrate, for the first time, how the new multiwavelength technology can be exploited to study protein–RNA interactions, and show how the spectral information derived from the new detector complements the traditional hydrodynamic information from analytical ultracentrifugation. Our method allows the protein and nucleic acid signals to be separated by spectral decomposition such that sedimentation information from each individual species, including any complexes, can be clearly identified based on their spectral signatures. The method presented here extends to any interacting system where the interaction partners are spectrally separable.