J Biomol NMR. 2013 Nov;57(3):251-62. doi: 10.1007/s10858-013-9785-z.
The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D2O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d10. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.
在 M9/D2O 培养基中过表达蛋白质，并添加标记的α-酮异戊酸（用于标记四个前手性甲基）或 2-乙酰乳酸（用于立体特异性标记缬氨酸和亮氨酸侧链），通常可以实现蛋白质中天冬氨酸和谷氨酸侧链的特定质子化。然而，当这些标记方案应用于大型蛋白质组装体时，缬氨酸和亮氨酸甲基之间的相关性会发生显著重叠，从而阻碍了二维甲基-TROSY 谱的分析。亮氨酸和缬氨酸生物合成途径的分析表明，通过添加外源性 l-亮氨酸-d10，可以抑制亮氨酸途径中标记前体的掺入。我们利用这一特性，通过最小程度的亮氨酸残基交换，立体特异性地标记缬氨酸的 pro-R 和 pro-S 甲基。该新标记方案应用于 468 kDa 同源十二聚体肽酶 TET2，以降低其 NMR 谱的复杂性。通过结合突变和这种创新的标记方法，分配了 TET2 所有的 pro-S 缬氨酸甲基共振峰。通过使用最佳标记样品和一组三重共振实验，将这些分配转移到 pro-R 基团。该改进的标记方案使我们能够克服前手性甲基 NMR 谱中拥挤的主要限制，这是在非常大的蛋白质组装体中进行结构和动态参数的位点特异性测量或研究相互作用的前提。
