Magic Angle Spinning NMR Structure Determination of Proteins from Pseudocontact Shifts
Magic Angle Spinning NMR Structure Determination of Proteins from Pseudocontact Shifts
复制标题
通过赝接触位移对蛋白质进行魔角旋转 NMR 结构测定
DOI：
10.1021/ja4021149
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发表时间：
2013-06-05
影响因子：
15
通讯作者：
Yang, Jun
中科院分区：
文献类型：
--
作者：
Li, Jianping;Pilla, Kala Bharath;Yang, Jun
魔角旋转固体核磁共振是一种独特的技术，用于研究抗结晶或太大而无法用溶液核磁共振技术研究的生物大分子的原子分辨率结构。然而，在获得足够数量的长程距离限制使用偶极耦合为基础的光谱的困难阻碍了在固态NMR蛋白质的结构测定的过程。本研究将伪接触位移（pseudocontactshifts，PCS）的测量与Rosetta计算相结合，在不使用传统偶极-偶极耦合距离约束的情况下，实现了蛋白质固相结构的高分辨率测定。通过将外源顺磁性金属离子螯合到标记4-巯基甲基-吡啶二羧酸，其共价连接到蛋白G（GB 1）的56个残基的免疫球蛋白结合结构域中的不同残基位点来产生PCS。从实验观测到的光子晶体中定量地提取了金属-原子核距离为20埃的长程结构约束，这些约束与使用X射线结构模型反算的距离吻合得很好。此外，我们证明，使用几个顺磁性离子与不同的顺磁性suspectibilities，以及在不同的网站引入顺磁性标签可以显着增加远程限制的数量，并覆盖不同区域的蛋白质。从固态NMR PCS约束结合Rosetta计算产生的结构相对于X射线结构具有0.7埃的均方根偏差。
Magic angle spinning solid-state NMR is a unique technique to study atomic-resolution structure of biomacromoleles which resist crystallization or are too large to study by solution NMR techniques. However, difficulties in obtaining sufficient number of long-range distance restraints using dipolar coupling based spectra hamper the process of structure determination of proteins in solid-state NMR. In this study it is sown that high-resolution structure of proteins in solid phase can e determined without the use of traditional dipolar-dipolar coupling based distance restraints by combining the measurements of pseudocontact shifts (PCSs) with Rosetta calculations. The PCSs were generated by chelating exogenous paramagnetic metal ions to a tag 4-mercaptomcthyl-dipicolinic acid, which is covalently attached to different residue sites in a 56-residue immunogobulin-binding domain of protein G(GB1). The long-range structural restraints with metal-nucleus distance of up similar to 20 angstrom are quantitatively extracted from experimentally observed PCSs, and these are in good agreement with the distances back-calculated using an X-ray structure model. Moreover, we demonstrate that using several paramagnetic ions with varied paramagnetic suspectibilities as well as the introduction of paramagnetic labels at different sites can dramatically increase the number of long-range restraints and cover different regions of the protein. The structure generated from solid-state NMR PCSs restraints combined with Rosetta calculations has 0.7 angstrom root-mean-square deviation relative to X-ray structure.