Structure determination of uniformly 13C, 15N labeled protein using qualitative distance restraints from MAS solid-state 13C-NMR observed paramagnetic relaxation enhancement
Structure determination of uniformly 13C, 15N labeled protein using qualitative distance restraints from MAS solid-state 13C-NMR observed paramagnetic relaxation enhancement
复制标题
使用 MAS 固态 13C-NMR 观察到的顺磁弛豫增强的定性距离限制对均匀 13C、15N 标记的蛋白质进行结构测定
DOI：
10.1007/s10858-015-0010-0
复制
发表时间：
2016
影响因子：
2.7
通讯作者：
M. Demura
中科院分区：
文献类型：
--
作者：
H. Tamaki;A. Egawa;K. Kido;T. Kameda;M. Kamiya;T. Kikukawa;T. Aizawa;T. Fujiwara;M. Demura
魔角旋转（MAS）固体核磁共振（NMR）是测定不溶性生物分子结构的有力手段。然而，通过MAS固态NMR的结构测定仍然具有挑战性，因为由于光谱复杂性，难以获得足够量的距离限制。收集顺磁弛豫增强（PRE）的距离限制是一种很有前途的方法，以减轻这一障碍。然而，由于不完全的平均相互作用和分子间PRE，PRE提供的距离限制的精度是有限的，在固态NMR中。在这份报告中，已成功地确定了链球菌蛋白G（GB 1）的B1结构域的骨架结构相结合的CS-Rosetta协议和定性PRE的限制。相对于X射线结构，衍生结构的Cα RMSD为1.49 μ m。值得注意的是，我们的协议可以确定正确的结构，从只有三个半胱氨酸-EDTA-Mn 2+突变体，因为这个数量的PRE网站是不够的，当使用传统的结构计算方法的基础上限制分子动力学和模拟退火。这项研究表明，定性PRE限制可以有效地用于蛋白质结构测定从有限的构象采样空间使用蛋白质片段库。
Magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) is a powerful method for structure determination of insoluble biomolecules. However, structure determination by MAS solid-state NMR remains challenging because it is difficult to obtain a sufficient amount of distance restraints owing to spectral complexity. Collection of distance restraints from paramagnetic relaxation enhancement (PRE) is a promising approach to alleviate this barrier. However, the precision of distance restraints provided by PRE is limited in solid-state NMR because of incomplete averaged interactions and intermolecular PREs. In this report, the backbone structure of the B1 domain of streptococcal protein G (GB1) has been successfully determined by combining the CS-Rosetta protocol and qualitative PRE restraints. The derived structure has a Cα RMSD of 1.49 Å relative to the X-ray structure. It is noteworthy that our protocol can determine the correct structure from only three cysteine-EDTA-Mn2+mutants because this number of PRE sites is insufficient when using a conventional structure calculation method based on restrained molecular dynamics and simulated annealing. This study shows that qualitative PRE restraints can be employed effectively for protein structure determination from a limited conformational sampling space using a protein fragment library.