Specific isotopic labelling and reverse labelling for protein NMR spectroscopy: using metabolic precursors in sample preparation.
Specific isotopic labelling and reverse labelling for protein NMR spectroscopy: using metabolic precursors in sample preparation.
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蛋白质NMR光谱的特定同位素标记和反向标记：在样品制备中使用代谢前体。
DOI：
10.1042/bst20210586
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发表时间：
2022-12-16
影响因子：
3.9
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中科院分区：
文献类型：
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通过NMR光谱学研究蛋白质结构、动力学和功能通常需要用稳定同位素13 C和/或15 N富集（“标记”）的样品。标准方法是用这些核中的一个或两个均匀地标记蛋白质，使得所有C和/或N位点原则上是“NMR可见的”。均匀标记的蛋白质的NMR光谱可能非常复杂，并且存在信号重叠。此外，随着分子尺寸的增加，NMR信号的线宽变宽，这降低了灵敏度并导致进一步的光谱拥塞。这两种效应都可能限制NMR数据中可用信息的类型和质量。与信号重叠和信号增宽相关的问题通常可以通过使用替代的、不均匀的同位素标记模式来缓解。特定的同位素标记在选定的位点“打开”信号，而蛋白质的其余部分是NMR不可见的。相反，特定的同位素去标记（也称为“反向”标记）“关闭”选定的信号，而蛋白质的其余部分仍然是NMR可见的。这两种方法可以简化NMR光谱，提高灵敏度，促进共振分配，并允许一系列不同的NMR策略时，与其他标记工具和NMR实验相结合。在这里，我们审查的方法生产蛋白质与稳定的NMR可见同位素富集，特别侧重于残基特异性标记和反向标记，使用大肠杆菌表达系统。我们还探讨了这些方法如何帮助蛋白质的NMR研究。
The study of protein structure, dynamics and function by NMR spectroscopy commonly requires samples that have been enriched (‘labelled') with the stable isotopes 13C and/or 15N. The standard approach is to uniformly label a protein with one or both of these nuclei such that all C and/or N sites are in principle ‘NMR-visible'. NMR spectra of uniformly labelled proteins can be highly complicated and suffer from signal overlap. Moreover, as molecular size increases the linewidths of NMR signals broaden, which decreases sensitivity and causes further spectral congestion. Both effects can limit the type and quality of information available from NMR data. Problems associated with signal overlap and signal broadening can often be alleviated though the use of alternative, non-uniform isotopic labelling patterns. Specific isotopic labelling ‘turns on' signals at selected sites while the rest of the protein is NMR-invisible. Conversely, specific isotopic unlabelling (also called ‘reverse' labelling) ‘turns off' selected signals while the rest of the protein remains NMR-visible. Both approaches can simplify NMR spectra, improve sensitivity, facilitate resonance assignment and permit a range of different NMR strategies when combined with other labelling tools and NMR experiments. Here, we review methods for producing proteins with enrichment of stable NMR-visible isotopes, with particular focus on residue-specific labelling and reverse labelling using Escherichia coli expression systems. We also explore how these approaches can aid NMR studies of proteins.