High-Resolution Studies of Uniformly 13C,15N-Labeled RNA by Solid-State NMR Spectroscopy
High-Resolution Studies of Uniformly 13C,15N-Labeled RNA by Solid-State NMR Spectroscopy
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DOI：
10.1002/anie.200906885
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发表时间：
2010-01-01
影响因子：
16.6
通讯作者：
Schwalbe, Harald
中科院分区：
文献类型：
--
作者：
Cherepanov, Alexey V.;Glaubitz, Clemens;Schwalbe, Harald
魔角旋转固体核磁共振波谱（MAS NMR）是研究生物大分子结构和动力学的一种先进的无创技术。MAS NMR实验可以在冷冻溶液、膜、微晶或冷冻干燥蛋白质中进行。这些研究提供了有关核间距离、扭转角、分子取向和功能动力学的信息。MAS核磁共振波谱提供了一个独特的机会来研究大分子在其自身的自然环境中，在体外和体内，无论是单一纯化蛋白，[1]大的多蛋白复合物，[2,3]分子原纤维，[4]细胞器，[5]整个细胞或组织，[6,7]或整个生物体此外，固态核磁共振波谱成为大分子折叠和催化的时间分辨研究的有力工具。[9-11]通过改变冷冻样品的温度，可以选择性地捕获或实时监测结构和化学转变。[11-13]本文以cUUCGg四环发夹为模型，应用固态核磁共振波谱法对RNA进行原子研究。我们最近在溶液中对这种发夹进行了表征，并获得了精细的高分辨率结构（RMSD= 0.3）。[14,15]在这里，我们使用13C MAS NMR将我们的研究扩展到冷冻溶液，将结果与溶液NMR数据进行比较，并将差异与发夹结构联系起来。据我们所知，这是首次对均匀13C， 15n标记的RNA进行高分辨率MAS NMR研究。固体2H核磁共振光谱用于描述HIV-1蛋白识别过程中TAR RNA中选定残基的运动采用15N MAS NMR谱法对（CUG） 97 RNA中的NH···N氢键进行了检测。NHHN、CHHC和NHHC实验中1H相关性与高分辨率光谱维度相结合尽管信号强度令人印象深刻，但低色散阻碍了单个自旋的分配。RNA 14-mer的13C、13C射频驱动的偶极重耦合（RFDR） MAS NMR谱如图1所示。光谱中的所有交叉峰都源于残留内的相关性。在168个可能的短程直接相干转移交叉峰中，有158个存在于固态光谱中。对于胞苷，C4C5相关性无法检测到。对于尿苷类化合物，在混合时间为5.74 ms时出现了弱的C4C5交叉峰。总共鉴定了132个碳原子中的116个。歧义共振来自碱基的C2和C8原子。其中9个被分配，因为峰没有重叠，溶液位移的差异小于0.3 ppm（图1，标记为对角峰）。除了相邻碳原子之间的交叉峰外，我们还观察到距离约为2.4的中范围相关性（C1'C3 ', C3‘C5 ’和C4C6）。杂原子桥接碳（例如，C1 ‘ c4 ’和C2C4）之间没有发现交叉峰，这意味着存在中继相干传递机制，例如，C1 ' ！C2”!C3”。在112个传递的交叉峰中，观察到30个，分配到18个（图1，红色标签）。图1显示12个继电器组成4个复合交叉峰（图1，峰1 - 4）。对于嘌呤，观察到的接力来源于C4C6转移。在嘧啶中没有发现类似的相关性。13C化学位移数据、溶液核磁共振数据、[14]和计算差值汇总在支持信息中的表1中。图1和表1表明，固态中的位移与溶液中的位移密切对应：89%的位移相差小于0.3 ppm。只有6个碳核的差异超过1ppm。
Solid-state NMR spectroscopy with magic angle spinning (MAS NMR) is an advanced noninvasive technique to study the structure and dynamics of biologic macromolecules. MAS NMR experiments can be performed in frozen solution, in membranes, and in microcrystalline or freeze-dried proteins. These studies yield information on internuclear distances, torsion angles, molecular orientation, and functional dynamics. MAS NMR spectroscopy provides a unique opportunity to study macromolecules in their own natural environment, in vitro and in vivo, be it a single purified protein,[1] large multiprotein complexes,[2, 3] molecular fibrils,[4] cell organelles,[5] the entire cell or tissue,[6, 7] or the whole organism.[8] In addition, solid-state NMR spectroscopy emerges as a powerful tool for the time-resolved study of macromolecular folding and catalysis.[9–11] By varying the temperature of the frozen sample, structural and chemical transitions can be selectively trapped or monitored in real time.[11–13] Herein we apply solid-state NMR spectroscopy for atomic studies on RNA using a cUUCGg tetraloop hairpin as a model. We have recently characterized this hairpin in solution and obtained a refined high-resolution structure (RMSD= 0.3).[14, 15] Here, we use 13C MAS NMR spectroscopy to extend our studies to frozen solution, compare the results with solution NMR data, and relate the differences to the hairpin structure. To our knowledge, this is the first high-resolution MAS NMR study on uniformly 13C, 15N-labeled RNA. Solidstate 2H NMR spectroscopy was used to describe motion of selected residues in TAR RNA from HIV-1 during protein recognition.[16] NH··· N hydrogen bonds in (CUG) 97 RNA were detected by 15N MAS NMR spectroscopy.[17] 1H correlation was combined with high-resolution spectral dimensions in NHHN, CHHC, and NHHC experiments.[18] Despite the impressive signal intensity, low dispersion precluded assignment of individual spins.The 13C, 13C radio-frequency-driven dipolar recoupling (RFDR) MAS NMR spectrum of the RNA 14-mer is shown in Figure 1. All cross peaks in the spectrum originate from intraresidue correlations. Out of 168 possible short-range direct coherence transfer cross peaks, 158 are found in the solid-state spectrum. For cytidines, the C4C5 correlations could not be detected. For uridines, weak C4C5 cross peaks appeared with a mixing time of 5.74 ms. In total, 116 out of 132 carbon atoms were identified. Ambiguous resonances originate from the C2 and C8 atoms of the bases. Nine of them were assigned because the peaks did not overlap and the solution shifts differed by less than 0.3 ppm (Figure 1, labeled diagonal peaks). In addition to the cross peaks between adjacent carbon atoms, we observed medium-range correlations over a distance of approximately 2.4 (C1’C3’, C3’C5’, and C4C6). Cross peaks between the heteroatom-bridged carbons (for example, C1’C4’and C2C4) were not found, implying a relayed coherence transfer mechanism, for example, C1’! C2’! C3’. Of 112 relayed cross peaks, 30 were observed and 18 assigned (Figure 1, red labels). Figure 1 shows that 12 relays form four composite cross peaks (Figure 1, peaks 1–4). For purines, the observed relays derived from C4C6 transfer. Similar correlations in pyrimidines were not found. The 13C chemical shift data, the solution NMR data,[14] and calculated differences are summarized in TableS1 in the Supporting Information. Figure1 and TableS1 indicate that the shifts in the solid state closely correspond to those in solution: 89% of the shifts differ by less than 0.3 ppm. Only six carbon nuclei show differences exceeding 1 ppm.