A novel way of amino acid-specific assignment in 1H-15N HSQC spectra with a wheat germ cell-free protein synthesis system
A novel way of amino acid-specific assignment in 1H-15N HSQC spectra with a wheat germ cell-free protein synthesis system
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DOI：
10.1023/b:jnmr.0000042956.65678.b8
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发表时间：
2004-09-01
影响因子：
2.7
通讯作者：
Kohno, T
中科院分区：
文献类型：
--
作者：
Morita, EH;Shimizu, M;Kohno, T
为了进行高通量的蛋白质结构分析，开发可靠的蛋白质过表达系统是必不可少的。虽然已经开发了许多蛋白质过表达系统，例如利用大肠杆菌细胞的蛋白质过表达系统，但许多在溶液中起作用的蛋白质仍然是以不可溶的形式合成的。最近，人们开发了一种新型的小麦胚芽无细胞蛋白质合成系统，其中许多蛋白质是以可溶性形式合成的。这意味着这种蛋白质合成方法在确定可溶性蛋白质的功能结构方面的适用性。在我们之前的工作中，我们利用这个小麦胚芽无细胞系统合成了N-15标记的蛋白质，并基于天然蛋白质的H-1-N-15 HSQC谱与合成蛋白质的H-1-N-15 HSQC谱具有很强的相似性，证实了这种适用性。在本研究中，我们利用蛋白质合成过程中转氨酶和谷氨酰胺合成酶的几种抑制剂，建立了一种简便可靠的蛋白质H-1-N-15 HSQC谱中氨基酸选择性归属的方法。H-1-N-15HSQC谱的氨基酸选择性指认是监测蛋白质折叠、分子间相互作用等特征的有力手段。这也是小麦胚芽提取物中存在活性转氨酶和谷氨酰胺合成酶的第一个直接实验证据。
For high-throughput protein structural analyses, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones utilizing E. coli cells, have been developed, a lot of proteins functioning in solution still were synthesized as insoluble forms. Recently, a novel wheat germ cell-free protein synthesis system was developed, and many of such proteins were synthesized as soluble forms. This means that the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we synthesized N-15-labeled proteins with this wheat germ cell-free system, and confirmed this applicability on the basis of the strong similarity between the H-1-N-15 HSQC spectra for native proteins and the corresponding ones for synthesized ones.In this study, we developed a convenient and reliable method for amino acid selective assignment in H-1-N-15 HSQC spectra of proteins, using several inhibitors for transaminases and glutamine synthase in the process of protein synthesis. Amino acid selective assignment in H-1-N-15 HSQC spectra is a powerful means to monitor the features of proteins, such as folding, intermolecular interactions and so on. This is also the first direct experimental evidence of the presence of active transaminases and glutamine synthase in wheat germ extracts.