Paramagnetic shifts in solid-state NMR of proteins to elicit structural information
Paramagnetic shifts in solid-state NMR of proteins to elicit structural information
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DOI：
10.1073/pnas.0708460105
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发表时间：
2008-11-11
影响因子：
11.1
通讯作者：
Luchinat, Claudio
中科院分区：
文献类型：
--
作者：
Balayssac, Stephane;Bertini, Ivano;Luchinat, Claudio
最近在顺磁性蛋白质的 C-13 高分辨率固态 NMR 中观察到的赝接触位移 (pcs) 为其作为结构限制的应用开辟了道路。在此，通过研究钴 (II) 取代的基质金属蛋白酶 12 [CoMMP-12 (159 AA，17.5 kDa)] 的微晶样品，结果表明，蛋白质标记和顺磁性物质稀释的组合策略（即，在未标记的 ZnMMP-12 中稀释 C-13-、N-15 标记的 CoMMP-12，以及C-13-,N-15-标记的 ZnMMP-12 稀释在未标记的 CoMMP-12 中）允许人们轻松地将源自蛋白质内部金属（分子内 pcs）的 pcs 贡献与晶格中相邻蛋白质中的金属（分子间 pcs）的贡献分开，并且两者都可用于结构目的。事实证明，分子内 PC 是重要的结构限制，有助于提高结构的精度和准确性，这是当今固态结构生物学的需要。此外，分子间颗粒提供了有关固相中相邻蛋白质分子的位置和方向的独特信息。
The recent observation of pseudocontact shifts (pcs) in C-13 high-resolution solid-state NMR of paramagnetic proteins opens the way to their application as structural restraints. Here, by investigating a microcrystalline sample of cobalt(II)-substituted matrix metalloproteinase 12 [CoMMP-12 (159 AA, 17.5 kDa)], it is shown that a combined strategy of protein labeling and dilution of the paramagnetic species (i.e., C-13-,N-15-labeled CoMMP-12 diluted in unlabeled ZnMMP-12, and C-13-,N-15-labeled ZnMMP-12 diluted in unlabeled CoMMP-12) allows one to easily separate the pcs contributions originated from the protein internal metal (intramolecular pcs) from those due to the metals in neighboring proteins in the crystal lattice (intermolecular pcs) and that both can be used for structural purposes. It is demonstrated that intramolecular pcs are significant structural restraints helpful in increasing both precision and accuracy of the structure, which is a need in solid-state structural biology nowadays. Furthermore, intermolecular pcs provide unique information on positions and orientations of neighboring protein molecules in the solid phase.