Methyl groups as probes for proteins and complexes in in-cell NMR experiments
Methyl groups as probes for proteins and complexes in in-cell NMR experiments
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DOI：
10.1021/ja049977k
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发表时间：
2004-06-09
影响因子：
15
通讯作者：
Dötsch, V
中科院分区：
文献类型：
--
作者：
Serber, Z;Straub, W;Dötsch, V
通过细胞内NMR研究大的细胞内复合物的蛋白质组分迄今为止是不可能的，因为骨架共振由于其缓慢的翻滚速率而不可观察。我们描述了一种方法，克服了这一困难，通过选择性标记的甲基，具有更有利的松弛行为。对三种不同蛋白质（钙调蛋白、NmerA和FKBP）的不同细胞内标记方案的比较表明，用甲硫氨酸和丙氨酸上的[（13）C]甲基进行选择性标记可以以极低的成本提供出色的灵敏度和低背景水平。
Studying protein components of large intracellular complexes by in-cell NMR has so far been impossible because the backbone resonances are unobservable due to their slow tumbling rates. We describe a methodology that overcomes this difficulty through selective labeling of methyl groups, which possess more favorable relaxation behavior. Comparison of different in-cell labeling schemes with three different proteins, calmodulin, NmerA, and FKBP, shows that selective labeling with [(13)C]methyl groups on methionine and alanine provides excellent sensitivity with low background levels at very low costs.