Site specific methyl labeling combined with methyl TROSY offers a powerful NMR approach to study structure and dynamics of proteins and protein complexes of high molecular weight. Robust and cost-effective methods have been developed for site specific protein 1H/13C methyl labeling in an otherwise deuterated background in bacteria. However, bacterial systems are not suitable for expression and isotope labeling of many eukaryotic and membrane proteins. The yeast Pichia pastoris (P. pastoris) is a commonly used host for expression of eukaryotic proteins, and site-specific methyl labeling of perdeuterated eukaryotic proteins has recently been achieved with this system. However, the practical utility of methyl labeling and deuteration in P. pastoris is limited by high costs. Here, we describe an improved method for 1H/13C-labeling of the δ-methyl group of isoleucine residues in a perdeuterated background, which reduces the cost by ≥ 50% without compromising the efficiency of isotope enrichment. We have successfully implemented this method to label actin and a G-protein coupled receptor. Our approach will facilitate studies of the structure and dynamics of eukaryotic proteins by NMR spectroscopy.

中文翻译：

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毕赤酵母中异亮氨酸 1H/13C 甲基标记的改进策略。


位点特异性甲基标记与甲基 TROSY 相结合，提供了一种强大的 NMR 方法来研究蛋白质和高分子量蛋白质复合物的结构和动力学。已经开发出稳健且经济有效的方法，用于在细菌的氘化背景中进行位点特异性蛋白质 1H/13C 甲基标记。然而，细菌系统不适合许多真核蛋白和膜蛋白的表达和同位素标记。酵母毕赤酵母（P. Pastoris）是表达真核蛋白的常用宿主，最近利用该系统实现了全氘化真核蛋白的位点特异性甲基标记。然而，甲基标记和氘化在毕赤酵母中的实际应用受到高成本的限制。在这里，我们描述了一种在全氘化背景下对异亮氨酸残基的 δ-甲基进行 1H/13C 标记的改进方法，该方法可在不影响同位素富集效率的情况下将成本降低 ≥ 50%。我们已经成功地实施了这种方法来标记肌动蛋白和 G 蛋白偶联受体。我们的方法将有助于通过核磁共振波谱研究真核蛋白质的结构和动力学。