An NMR experiment for measuring methyl-methyl NOEs in 13C-labeled proteins with high resolution
An NMR experiment for measuring methyl-methyl NOEs in 13C-labeled proteins with high resolution
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DOI：
10.1021/ja981205z
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发表时间：
1998-08-05
影响因子：
15
通讯作者：
Kay, LE
中科院分区：
文献类型：
--
作者：
Zwahlen, C;Gardner, KH;Kay, LE
提出了一个三维核磁共振实验来关联质子化、N-15、c -13标记或甲基质子化、高度氘化的N-15、c -13标记蛋白质中甲基之间的noe。该实验的高分辨率有助于在甲基之间分配在其他方法中很难分辨的noe；三维和四维实验，并将基于溶液的核磁共振结构研究扩展到分子量为40 kDa的蛋白质。介绍了对370个麦芽糖结合蛋白（122个甲基）的甲基质子化、高度氘化样品和人类STAT-4的124个氨基末端结构域（59个甲基）的二聚体的应用。该方法也非常适合于研究完全质子化的蛋白质，如应用涉及160残基磷酸酪氨酸结合域从果蝇麻。从本实验中获得的距离限制在生成全局蛋白质折叠中特别有用，因为许多甲基位于疏水性蛋白质核心中，因此甲基间限制连接二级结构的元件，这些元件通常位于初级序列中较远的位置。
A three-dimensional NMR experiment is presented for correlating NOEs between methyl groups in protonated, N-15,C-13-labeled or methyl protonated, highly deuterated N-15,C-13-labeled proteins. The high resolution of this experiment facilitates the assignment of NOEs between methyls that can be poorly resolved in other; three- and four-dimensional experiments and extends the utility of solution-based NMR structural studies to proteins in the 40 kDa molecular weight regime. Applications to methyl protonated, highly deuterated samples of the 370 residue maltose binding protein (122 methyl groups) and the dimer of the 124 residue amino terminal domain of human STAT-4 (59 methyls) are presented. The method is also well suited for studies of fully protonated proteins, as demonstrated with an application involving the 160 residue phosphotyrosine binding domain from Drosophila Numb. Distance restraints obtained from the present experiment are particularly useful in the generation of global protein folds since many methyls are located in hydrophobic protein cores and inter-methyl restraints therefore link elements of secondary structure that are often distant in the primary sequence.