A Double TROSY hNCAnH Experiment for Efficient Assignment of Large and Challenging Proteins
A Double TROSY hNCAnH Experiment for Efficient Assignment of Large and Challenging Proteins
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DOI：
10.1021/ja9046685
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发表时间：
2009-09-16
影响因子：
15
通讯作者：
Wagner, Gerhard
中科院分区：
文献类型：
--
作者：
Frueh, Dominique P.;Arthanari, Haribabu;Wagner, Gerhard
我们提出了一个实验，允许一个简单的分配NMR共振，即使在大型和/或具有挑战性的蛋白质。一个单一的3D双TROSY实验提供了两个α碳，两个酰胺质子和两个氮原子核之间的三对顺序相关性。因此，所有相关的核可以很容易地可视化内的所有维度的所得光谱，和连续的氨基酸的链延长可以与这个单一的数据集的影响。这解决了传统方法中出现的模糊性，传统方法涉及耗时和繁琐的三重共振光谱系列获得的条带比较。该实验利用双TROSY技术来解释在转移和进化期间的信号强度损失，并在33 kDa非核糖体肽合成酶蛋白EntB的500 μ M样品上进行测试。
We present an experiment that allows for a straightforward assignment of NMR resonances, even in large and/or challenging proteins. A single 3D double-TROSY experiment provides three pairs of sequential correlations between two alpha carbons, two amide protons, and two nitrogen nuclei. Thus, all correlated nuclei can readily be visualized within all dimensions of the resulting spectrum, and chain elongation of sequential amino acids can be effected with this single data set. This resolves ambiguities occurring in traditional methods which involve time-consuming and cumbersome strip comparisons obtained with series of triple-resonance spectra. The experiment makes use of the double TROSY technique to account for signal intensity losses during transfer and evolution periods and was tested on a 500 mu M sample of the 33 kDa nonribosomal peptide synthetase protein EntB.