Computational and Structural Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
J Am Chem Soc. 2010 Mar 24;132(11):3842-6. doi: 10.1021/ja909723f.
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 (15)N nuclei of the protein backbone and the (31)P 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.
由于 RNA 的灵活性以及蛋白质-RNA 界面的灵活性，通过 X 射线晶体学对大型 RNP 复合物进行结构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，尽管典型的 RNP 复合物的尺寸较大可能会限制溶液 NMR 的适用性，但 NMR 光谱学是晶体学的一种有吸引力的替代方法。然而，固态 NMR 光谱学不受研究对象大小的任何内在限制的影响，仅受到仪器灵敏度的限制。此外，它不需要大而有序的晶体，因此可用于柔性、部分无序的复合物。在这里，我们首次表明固态 NMR 光谱学可用于探测 RNP 复合物中蛋白质-RNA 界面处的分子间相互作用。在 TEDOR 实验中可以测量蛋白质骨架的 (15)N 核与 RNA 骨架的 (31)P 核之间的距离，并将其用作结构计算中的约束条件。如具有晶体结构的 L7Ae-box C/D RNA 复合物的测试案例所示，距离测量是准确的。这里呈现的结果揭示了固态 NMR 光谱学在研究大型 RNP 复合物方面尚未开发的潜力。
