Amino-acid type identification in 15N-HSQC spectra by combinatorial selective 15N-labelling
Amino-acid type identification in 15N-HSQC spectra by combinatorial selective 15N-labelling
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DOI：
10.1007/s10858-005-5021-9
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发表时间：
2006-01-01
影响因子：
2.7
通讯作者：
Otting, G
中科院分区：
文献类型：
--
作者：
Wu, PSC;Ozawa, K;Otting, G
无细胞蛋白质合成与组合选择性N-15-标记相结合的效率提供了一种用于将N-15-HSQC交叉峰快速分配给来自5个N-15-HSQC光谱的19种不同非脯氨酸氨基酸类型的方法。用大肠杆菌DNA聚合酶III全酶的tau亚基的C-末端结构域V的两种不同构建体tau（C）16和tau（C）14探索了该策略。由于五个N-15-HSQC光谱中的每一个仅含有均匀标记样品中存在的交叉峰的约三分之一，因此光谱重叠大大减少。骨架酰胺的所有N-15-HSQC交叉峰可归属于正确的氨基酸类型。残基类型信息的可用性极大地帮助了对tau（C）16与tau（C）14中相应残基观察到的化学位移变化的评价，以及对tau（C）16中存在但tau（C）14中不存在的C-末端残基的结构和迁移率的分析。
The efficiency of cell-free protein synthesis combined with combinatorial selective N-15-labelling provides a method for the rapid assignment of N-15-HSQC cross-peaks to the 19 different non-proline amino-acid types from five N-15-HSQC spectra. This strategy was explored with two different constructs of the C-terminal domain V of the tau subunit of the Escherichia coli DNA polymerase III holoenzyme, tau(C)16 and tau(C)14. Since each of the five N-15-HSQC spectra contained only about one third of the cross-peaks present in uniformly labelled samples, spectral overlap was much reduced. All N-15-HSQC cross-peaks of the backbone amides could be assigned to the correct amino-acid type. Availability of the residue-type information greatly assisted the evaluation of the changes in chemical shifts observed for corresponding residues in tau(C)16 vs. those in tau(C)14, and the analysis of the structure and mobility of the C-terminal residues present in tau(C)16 but not in tau(C)14.