Assignment of backbone resonances is a necessary initial step in every protein NMR investigation. Standard assignment procedure is based on the set of 3D triple-resonance (¹H–¹³C–¹⁵N) spectra and requires at least several days of experimental measurements. This limits its application to the proteins with low stability. To speed up the assignment procedure, combinatorial selective labeling (CSL) can be used. In this case, sequence-specific information is extracted from 2D spectra measured for several selectively ¹³C,¹⁵N-labeled samples, produced in accordance with a special CSL scheme. Here we review previous applications of the CSL approach and present novel deterministic ‘CombLabel’ algorithm, which generates CSL schemes minimizing the number of labeled samples and their price and maximizing assignment information that can be obtained for a given protein sequence. Theoretical calculations revealed that CombLabel software outperformed previously proposed stochastic algorithms. Current implementation of CombLabel robustly calculates CSL schemes containing up to six samples, which is sufficient for moderately sized (up to 200 residues) proteins. As a proof of concept, we calculated CSL scheme for the first voltage-sensing domain of human Nav1.4 channel, a 134 residue four helical transmembrane protein having extremely low stability in micellar solution (half-life ~ 24 h at 45 °C). Application of CSL doubled the extent of backbone resonance assignment, initially obtained by conventional approach. The obtained assignment coverage (~ 50%) is sufficient for ligand screening and mapping of binding interfaces.

中文翻译：

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CombLabel：优化的特定于序列的组合标记方案的合理设计。在低稳定性膜蛋白的骨架分配中的应用

骨架共振的分配是每个蛋白质NMR研究中必不可少的初始步骤。标准分配程序基于3D三重共振（¹ H– ¹³ C– ¹⁵ N）光谱集，需要至少几天的实验测量。这限制了其对具有低稳定性的蛋白质的应用。为了加快分配过程，可以使用组合选择性标记（CSL）。在这种情况下，将从2D光谱中提取的序列特异性信息提取出来，该2D光谱是根据特殊CSL方案生产的几个选择性¹³ C，¹⁵ N标记的样品测得的。在这里，我们回顾了CSL方法的先前应用，并提出了新颖的确定性' CombLabel”算法，该算法生成CSL方案，以最小化标记样品的数量及其价格，并最大化可从给定蛋白质序列获得的分配信息。理论计算表明，CombLabel软件的性能优于先前提出的随机算法。CombLabel的当前实现稳健地计算包含多达六个样本的CSL方案，这足以用于中等大小（多达200个残基）的蛋白质。作为概念验证，我们为人Nav1.4通道的第一个电压感测域计算了CSL方案，该胶束溶液是134个残基的四个螺旋跨膜蛋白，在胶束溶液中具有极低的稳定性（在45°C下半衰期为〜24 h） 。CSL的应用使最初通过常规方法获得的主干共振分配范围加倍。获得的分配覆盖率（〜50％）足以进行配体筛选和结合界面的定位。