High-resolution measurement of long-range distances in RNA: pulse EPR spectroscopy with TEMPO-labeled nucleotides.
High-resolution measurement of long-range distances in RNA: pulse EPR spectroscopy with TEMPO-labeled nucleotides.
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DOI：
10.1039/c5sc04631a
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发表时间：
2016-05-01
期刊：
影响因子：
8.4
通讯作者：
Bennati M
中科院分区：
文献类型：
--
作者：
Halbmair K;Seikowski J;Tkach I;Höbartner C;Sezer D;Bennati M
通过脉冲EPR与TEMPO标记的核苷酸在RNA中的距离测量允许将距离无模型转换为碱基对分离。生物分子组装体（如RNA和RNA蛋白质复合物）的原子分辨率结构信息是理解生物功能的基础。现代光谱方法在这方面提供了特殊的机会。在这里，我们提出了脉冲EPR报告的能力，高分辨率的远程距离RNA的装置，最近开发的自旋标记的核苷酸，它携带的克里思组直接连接到核碱基，并保留沃森克里克碱基配对。在一个具有代表性的RNA双链体中，自旋-标记分离高达28个碱基对（1.88 nm），我们证明，如果在Q波段频率（34 GHz）的宽带脉冲激发，该标记允许自旋间距离无模型转换为碱基对分离（Δbp）。观察到的距离分布从Δbp = 10时的±0.2 nm增加到Δbp = 28时的±0.5 nm，与“理想”A型RNA结构只有很小的偏差一致。在20 °C下进行的分子动力学（MD）模拟显示了标记的受限构象自由度。MD产生的结构偏差从一个“理想的”A-RNA几何形状有助于解开的标签和其相邻的核碱基和RNA双螺旋的实验距离分布的全局变形的局部灵活性的贡献。该研究表明，我们简单但具有战略意义的自旋标记程序可以在与大分子RNA复合物大小相匹配的距离上以原子分辨率访问RNA的详细结构信息。
Distance measurements in RNAs by pulse EPR with TEMPO-labeled nucleotides allow for model free conversion of distances into base-pair separation. Structural information at atomic resolution of biomolecular assemblies, such as RNA and RNA protein complexes, is fundamental to comprehend biological function. Modern spectroscopic methods offer exceptional opportunities in this direction. Here we present the capability of pulse EPR to report high-resolution long-range distances in RNAs by means of a recently developed spin labeled nucleotide, which carries the TEMPO group directly attached to the nucleobase and preserves Watson–Crick base-pairing. In a representative RNA duplex with spin-label separations up to 28 base pairs (≈8 nm) we demonstrate that the label allows for a model-free conversion of inter-spin distances into base-pair separation (Δbp) if broad-band pulse excitation at Q band frequencies (34 GHz) is applied. The observed distance distribution increases from ±0.2 nm for Δbp = 10 to only ±0.5 nm for Δbp = 28, consistent with only small deviations from the “ideal” A-form RNA structure. Molecular dynamics (MD) simulations conducted at 20 °C show restricted conformational freedom of the label. MD-generated structural deviations from an “ideal” A-RNA geometry help disentangle the contributions of local flexibility of the label and its neighboring nucleobases and global deformations of the RNA double helix to the experimental distance distributions. The study demonstrates that our simple but strategic spin labeling procedure can access detailed structural information on RNAs at atomic resolution over distances that match the size of macromolecular RNA complexes.
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影响因子：
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