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 selective ¹⁵N labeling suffers from isotope dilution caused by metabolic interconversion of the amino acids, resulting in isotope scrambling within the target protein. Carbonyl ¹³C atoms experience less isotope scrambling than the main-chain ¹⁵N atoms do. However, little is known about the side-chain ¹³C atoms. Here, the ¹³C scrambling profiles of the Cα and side-chain carbons were investigated for ¹⁵N scrambling-prone amino acids, such as Leu, Ile, Tyr, Phe, Thr, Val, and Ala. The level of isotope scrambling was substantially lower in ¹³Cα and ¹³C side-chain labeling than in ¹⁵N labeling. We utilized this reduced scrambling-prone character of ¹³C as a simple and efficient method for amino acid selective ¹³C labeling using an Escherichia coli cold-shock expression system and high-cell density fermentation. Using this method, the ¹³C 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 the ¹⁵N scrambling-prone amino acid were also easily filtered using ¹⁵N-{¹³Cα} spin–echo difference experiments. Our method could be applied to the assignment of the 55 kDa protein.

中文翻译：

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用于蛋白质核磁共振的大肠杆菌中蛋白质α和侧链碳的氨基酸选择性¹³ C标记和¹³ C加扰曲线分析

氨基酸选择性同位素标记是一种重要的核磁共振技术，特别是对于较大的蛋白质，为明确的共振分配以及有关结构，动力学和分子间相互作用的信息提供了坚实的基础。氨基酸选择性¹⁵ N标记遭受氨基酸代谢互变引起的同位素稀释，导致目标蛋白内的同位素加扰。羰基¹³ C原子比主链¹⁵ N原子经历更少的同位素加扰。然而，关于侧链¹³ C原子知之甚少。在这里，研究了¹⁵个Cα和侧链碳的¹³ C加扰曲线Ñ扰频发的氨基酸，如亮氨酸，异亮氨酸，酪氨酸，苯丙氨酸，苏氨酸，缬氨酸，和Ala。同位素的水平加扰中被显着降低¹³ Cα和¹³ C侧链标记比在¹⁵ Ñ标记。我们利用这种降低的¹³ C易扰性特征作为使用大肠杆菌冷休克表达系统和高细胞密度发酵进行氨基酸选择性¹³ C标记的简单有效方法。使用这种方法，Leu和Ile的¹³ C标记效率> 80％，Tyr和Phe的〜60％，Thr的〜50％，Val的〜40％，Ala的30–40％，¹ H– ¹⁵所述N个异核单量子相干信号¹⁵ Ñ扰频发氨基酸也容易使用过滤¹⁵ N- { ¹³ Cα}自旋回波差实验。我们的方法可以应用于55 kDa蛋白的分配。