Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments.
Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments.
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DOI：
10.1007/s10858-010-9447-3
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发表时间：
2010-11
影响因子：
2.7
通讯作者：
Sklenář V
中科院分区：
文献类型：
--
作者：
Motáčková V;Nováček J;Zawadzka-Kazimierczuk A;Kazimierczuk K;Zídek L;Sanderová H;Krásný L;Koźmiński W;Sklenář V
提出了一种对具有高度重复序列的无序蛋白质进行完整主链和侧链共振分配的策略。该协议基于三个分辨率增强的 NMR 实验：5D HN(CA)CONH 提供顺序连接，5D HabCabCONH 用于识别氨基酸类型，5D HC(CC-TOCSY)CONH 用于分配侧链共振。分辨率的提高是通过高维度和长演化时间的结合来实现的，这是通过间接维度的非均匀采样实现的。使用数据点的随机分布和稀疏多维傅立叶变换处理。将分配程序成功应用于特别困难的蛋白质（来自枯草芽孢杆菌的 RNA 聚合酶 δ 亚基），证明了该策略的有效性。研究的蛋白质包含一个由 81 个氨基酸组成的无序 C 端区域，具有高度重复的序列。虽然传统的分配方法由于化学位移的微小差异而完全失败，但所提出的策略提供了完整的主链和侧链分配。本文的在线版本 (doi:10.1007/s10858-010-9447-3) 包含补充材料，可供授权用户使用。
A strategy for complete backbone and side-chain resonance assignment of disordered proteins with highly repetitive sequence is presented. The protocol is based on three resolution-enhanced NMR experiments: 5D HN(CA)CONH provides sequential connectivity, 5D HabCabCONH is utilized to identify amino acid types, and 5D HC(CC-TOCSY)CONH is used to assign the side-chain resonances. The improved resolution was achieved by a combination of high dimensionality and long evolution times, allowed by non-uniform sampling in the indirect dimensions. Random distribution of the data points and Sparse Multidimensional Fourier Transform processing were used. Successful application of the assignment procedure to a particularly difficult protein, δ subunit of RNA polymerase from Bacillus subtilis, is shown to prove the efficiency of the strategy. The studied protein contains a disordered C-terminal region of 81 amino acids with a highly repetitive sequence. While the conventional assignment methods completely failed due to a very small differences in chemical shifts, the presented strategy provided a complete backbone and side-chain assignment. The online version of this article (doi:10.1007/s10858-010-9447-3) contains supplementary material, which is available to authorized users.
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影响因子：
15
作者：
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通讯作者：
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