NMR “Crystallography” for Uniformly ( 13 C, 15 N)‐Labeled Oriented Membrane Proteins
NMR “Crystallography” for Uniformly ( 13 C, 15 N)‐Labeled Oriented Membrane Proteins
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NMR – 晶体学 – 用于均匀 ( 13 C, 15 N) – 标记的定向膜蛋白
DOI：
10.1002/anie.201915110
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Nevzorov, Alexander A.
中科院分区：
文献类型：
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作者：
Awosanya, Emmanuel O.;Lapin, Joel;Nevzorov, Alexander A.
在膜蛋白的定向样品（OS）固态核磁共振中，角度依赖性偶极耦合和化学位移为结构计算提供了直接输入。然而，到目前为止，仅对定向 15 N 标记样品中的 1 H–15 N 偶极耦合和 15 N 化学位移进行了常规评估。将这种技术扩展到任意拓扑的膜蛋白的主要障碍仍然是缺乏额外的实验限制。我们开发了一种新的实验性三重共振 NMR 技术，该技术应用于磁性排列的 DMPC/DHPC bicelles 中均匀双 (15N,13C) 标记的 Pf1 外壳蛋白。以前无法实现的 1Hα-13Cα 偶极耦合已经被测量，这使得在不先验假设 α 螺旋结构的情况下确定肽平面之间的扭转角成为可能。每个肽平面的三个角度约束的拟合和通过 Rosetta 评分函数的过滤已经产生了 Pf1 蛋白的一致 α 螺旋跨膜结构。
In oriented‐sample (OS) solid‐state NMR of membrane proteins, the angular‐dependent dipolar couplings and chemical shifts provide a direct input for structure calculations. However, so far only1H–15N dipolar couplings and15N chemical shifts have been routinely assessed in oriented15N‐labeled samples. The main obstacle for extending this technique to membrane proteins of arbitrary topology has remained in the lack of additional experimental restraints. We have developed a new experimental triple‐resonance NMR technique, which was applied to uniformly doubly (15N,13C)‐labeled Pf1 coat protein in magnetically aligned DMPC/DHPC bicelles. The previously inaccessible1Hα–13Cαdipolar couplings have been measured, which make it possible to determine the torsion angles between the peptide planes without assuming α‐helical structure a priori. The fitting of three angular restraints per peptide plane and filtering by Rosetta scoring functions has yielded a consensus α‐helical transmembrane structure for Pf1 protein.