Amino Acid Selective 13C Labeling and 13C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance
Amino Acid Selective 13C Labeling and 13C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance
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DOI：
10.1021/acs.biochem.8b00182
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发表时间：
2018-07-03
期刊：
影响因子：
2.9
通讯作者：
Kojima，Chojiro
中科院分区：
文献类型：
--
作者：
Sugiki，Toshihiko;Furuita，Kyoko;Kojima，Chojiro
氨基酸选择性同位素标记是一项重要的核磁共振技术，特别是对于较大的蛋白质，为明确的共振分配和有关结构、动力学和分子间相互作用的信息提供了强有力的基础。氨基酸选择性15n标记受到氨基酸代谢相互转化引起的同位素稀释的影响，导致靶蛋白内的同位素混乱。羰基13c原子比主链15n原子经历较少的同位素混乱。然而，人们对侧链13c原子知之甚少。本文研究了易发生15n抢序的氨基酸，如Leu、Ile、Tyr、Phe、Thr、Val和Ala的Cα和侧链碳的13c抢序谱。13c α和13c侧链标记的同位素置乱水平明显低于15n标记。利用大肠杆菌-休克表达系统和高密度发酵，我们利用13c的这种减少混乱倾向的特性作为一种简单有效的氨基酸选择性13c标记方法。该方法的13c标记效率分别为Leu和Ile的约80%、Tyr和Phe的约60%、Thr的约50%、Val的约40%和ala的约30-40%。利用15n -{13Cα}自旋回波差异实验，可以很容易地过滤出易发生15n扰频的氨基酸的1h - 15n异核单量子相干信号。该方法适用于55 kDa蛋白的定位。
Amino acid selective isotope labeling is an important nuclear magnetic resonance technique, especially for larger proteins, providing strong bases for the unambiguous resonance assignments and information concerning the structure, dynamics, and intermolecular interactions. Amino acid selective15N labeling suffers from isotope dilution caused by metabolic interconversion of the amino acids, resulting in isotope scrambling within the target protein. Carbonyl13C atoms experience less isotope scrambling than the main-chain15N atoms do. However, little is known about the side-chain13C atoms. Here, the13C scrambling profiles of the Cα and side-chain carbons were investigated for15N scrambling-prone amino acids, such as Leu, Ile, Tyr, Phe, Thr, Val, and Ala. The level of isotope scrambling was substantially lower in13Cα and13C side-chain labeling than in15N labeling. We utilized this reduced scrambling-prone character of13C as a simple and efficient method for amino acid selective13C labeling using anEscherichia colicold-shock expression system and high-cell density fermentation. Using this method, the13C labeling efficiency was >80% for Leu and Ile, ∼60% for Tyr and Phe, ∼50% for Thr, ∼40% for Val, and 30–40% for Ala.1H–15N heteronuclear single-quantum coherence signals of the15N scrambling-prone amino acid were also easily filtered using15N-{13Cα} spin–echo difference experiments. Our method could be applied to the assignment of the 55 kDa protein.