13C Spin Dilution for Simplified and Complete Solid-State NMR Resonance Assignment of Insoluble Biological Assemblies
13C Spin Dilution for Simplified and Complete Solid-State NMR Resonance Assignment of Insoluble Biological Assemblies
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DOI：
10.1021/ja200066s
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发表时间：
2011-04-06
影响因子：
15
通讯作者：
Lange, Adam
中科院分区：
文献类型：
--
作者：
Loquet, Antoine;Lv, Guohua;Lange, Adam
提出了一种用固态核磁共振（ssNMR）谱法对不溶性和非晶体蛋白质进行简化和完全共振分配的方法。用[1-C-13]-或[2- c -13]葡萄糖生成的蛋白质被非常稀疏地标记，得到的二维ssNMR光谱显示出更小的线宽（相对于均匀标记的蛋白质，线宽大约为2），并且包含更少的交叉峰。这允许加速和直接的共振分配，而不需要耗时的3D光谱或复杂的脉冲序列。该策略旨在基于双向顺序行走完成主链和侧链共振分配。该方法已成功地通过3型分泌系统PrgI针蛋白的重新定位得到验证。使用一组有限的简单二维实验，我们报告了97%的主链和侧链C-13原子的完整共振分配。
A strategy for simplified and complete resonance assignment of insoluble and noncrystalline proteins by solid-state NMR (ssNMR) spectroscopy is presented. Proteins produced with [1-C-13]- or [2-C-13]glucose are very sparsely labeled, and the resulting 2D ssNMR spectra exhibit smaller line widths (by a factor of similar to 2 relative to uniformly labeled proteins) and contain a reduced number of cross-peaks. This allows for an accelerated and straightforward resonance assignment without the necessity of time-consuming 3D spectroscopy or sophisticated pulse sequences. The strategy aims at complete backbone and side-chain resonance assignments based on bidirectional sequential walks. The approach was successfully demonstrated with the de novo assignment of the Type Three Secretion System PrgI needle protein. Using a limited set of simple 2D experiments, we report a 97% complete resonance assignment of the backbone and side-chain C-13 atoms.