NMR Resonance Assignment Methodology: Characterizing Large Sparsely Labeled Glycoproteins
NMR Resonance Assignment Methodology: Characterizing Large Sparsely Labeled Glycoproteins
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DOI：
10.1016/j.jmb.2019.04.029
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发表时间：
2019-05-31
影响因子：
5.6
通讯作者：
Prestegard, James H.
中科院分区：
文献类型：
--
作者：
Chalmers, Gordon R.;Eletsky, Alexander;Prestegard, James H.
使用核磁共振方法对蛋白质进行表征始于指定特定残基的共振峰。这通常是利用核磁共振活性同位素统一标记的蛋白质中核对之间的顺序连接性来实现的。这对于较大的蛋白质来说变得不切实际，特别是对于在哺乳动物细胞中表达最好的蛋白质，包括糖蛋白。这里描述了一种用于指定稀疏标记蛋白质的核磁共振波谱的替代方案，即用N-15或C-13同位素富集的单一氨基酸类型或有限类型子集标记的蛋白质。该协议基于使用简单二维核磁共振实验(相关化学位移、核Overhauser效应、剩余偶极耦合)的扩展收集的数据与从已知蛋白质结构开始的分子动力学轨迹的预测的比较。采用遗传算法ASSIGN_SLP_MD的软件包促进了预测值和实验值的最佳配对。该方法被应用于36 kDa Lumina！唾液酸基转移酶的结构域rST6Ga11，其中所有的苯丙氨酸都用N-15标记，并且通过选择的苯丙氨酸到酪氨酸的单点突变消除共振来验证结果。Assignment允许使用先前发表的顺磁驰豫增强来评估底物类似物在该蛋白质活性部位的放置。该方案将为在哺乳动物细胞中最佳表达的许多糖基化和其他蛋白质的结构表征开辟道路。(C)2019爱思唯尔有限公司。保留所有权利。
Characterization of proteins using NMR methods begins with assignment of resonances to specific residues. This is usually accomplished using sequential connectivities between nuclear pairs in proteins uniformly labeled with NMR active isotopes. This becomes impractical for larger proteins, and especially for proteins that are best expressed in mammalian cells, including glycoproteins. Here an alternate protocol for the assignment of NMR resonances of sparsely labeled proteins, namely, the ones labeled with a single amino acid type, or a limited subset of types, isotopically enriched with N-15 or C-13, is described. The protocol is based on comparison of data collected using extensions of simple two-dimensional NMR experiments (correlated chemical shifts, nuclear Overhauser effects, residual dipolar couplings) to predictions from molecular dynamics trajectories that begin with known protein structures. Optimal pairing of predicted and experimental values is facilitated by a software package that employs a genetic algorithm, ASSIGN_SLP_MD. The approach is applied to the 36-kDa lumina! domain of the sialyltransferase, rST6Ga11, in which all phenylalanines are labeled with N-15, and the results are validated by elimination of resonances via single-point mutations of selected phenylalanines to tyrosines. Assignment allows the use of previously published paramagnetic relaxation enhancements to evaluate placement of a substrate analog in the active site of this protein. The protocol will open the way to structural characterization of the many glycosylated and other proteins that are best expressed in mammalian cells. (C) 2019 Elsevier Ltd. All rights reserved.