Intermolecular Protein-RNA Interactions Revealed by 2D 31P-15N Magic Angle Spinning Solid-State NMR Spectroscopy
Intermolecular Protein-RNA Interactions Revealed by 2D 31P-15N Magic Angle Spinning Solid-State NMR Spectroscopy
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DOI：
10.1021/ja909723f
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发表时间：
2010-03-24
影响因子：
15
通讯作者：
Carlomagno, Teresa
中科院分区：
文献类型：
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作者：
Jehle, Stefan;Falb, Melanie;Carlomagno, Teresa
由于RNA和蛋白质-RNA界面的灵活性，通过X射线晶体学对大RNP复合物的结构进行研究可能是一项艰巨的任务，这可能会阻碍结晶。在这些情况下，NMR光谱是晶体学的一个有吸引力的替代，虽然典型的RNP复合物的大尺寸可能限制溶液NMR的适用性。然而，固态NMR光谱学不受任何关于被研究对象的尺寸的固有限制，仅受仪器灵敏度的限制。此外，它不需要大的、有序的晶体，因此可以应用于柔性的、部分无序的复合物。在这里，我们首次表明，固态NMR光谱可用于探测RNP复合物中蛋白质-RNA界面的分子间相互作用。蛋白质骨架的N-15核和RNA骨架的P-31核之间的距离可以在TEDOR实验中测量，并用作结构计算中的限制。距离测量是准确的，如L7 Ae盒C/D RNA复合物的测试情况所证明的，其晶体结构是可用的。这里提出的结果揭示了尚未开发的潜力，固态NMR光谱在大型RNP配合物的调查。
The structural investigation of large RNP complexes by X-ray crystallography can be a difficult task due to the flexibility of the RNA and of the protein-RNA interfaces, which may hinder crystallization. In these cases, NMR spectroscopy is an attractive alternative to crystallography, although the large size of typical RNP complexes may limit the applicability of solution NMR. Solid-state NMR spectroscopy, however, is not subject to any intrinsic limitations with respect to the size of the object under investigation, with restrictions imposed solely by the sensitivity of the instrumentation. In addition, it does not require large, well-ordered crystals and can therefore be applied to flexible, partially disordered complexes. Here we show for the first time that solid-state NMR spectroscopy can be used to probe intermolecular interactions at the protein-RNA interface in RNP complexes. Distances between the N-15 nuclei of the protein backbone and the P-31 nuclei of the RNA backbone can be measured in TEDOR experiments and used as restraints in structure calculations. The distance measurement is accurate, as proven for the test case of the L7Ae-box C/D RNA complex, for which a crystal structure is available. The results presented here reveal the as yet unexplored potential of solid-state NMR spectroscopy in the investigation of large RNP complexes.