甲基和氨基酸型标记
氘代蛋白质中的质子化碳-13甲基是结构和动力学的极好探针,在研究高分子量蛋白质和蛋白质复合物时至关重要。甲基标记是指将13个CH3或13个CHD2基团引入均匀氘代蛋白质的方法。通过在蛋白质诱导之前将足够量的氨基酸直接添加到细胞培养基中,可以在原核和真核表达系统中实现甲基标记。对于大肠杆菌培养物,通过在诱导前 1 小时将 α-酮酸前体添加到 D2O 基细胞培养基中,可以实现对异亮氨酸、亮氨酸和缬氨酸 (ILV) 甲基的选择性质子化。α-酮异丁酸转化为异亮氨酸,α-酮异戊酸转化为亮氨酸和缬氨酸。当使用甲基TROSY技术时,这种方法显着增加了可通过NMR评估的蛋白质和蛋白质复合物的有用分子量范围。
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Application Notes
➤ Stereospecific Leu/Val Methyl Labeling: An Important Technology for NMR Studies of High-Molecular-Weight Complexes
➤ Production of U-[2H], Thy-γ2[13CH3] Labeled Proteins for methyl-TROSY NMR
➤ Isotope Labeling of Alanine Methyl Groups on a Deuterated Background for NMR Studies of High-Molecular-Weight Proteins
➤ Specific Isotopic Labeling of Methyl Groups Has Extended the Molecular Weight Limits for NMR Studies of Protein Structure and Dynamics
Researcher Perspective
➤ Selective Isotope-Labeling Methods for Protein Structural Studies
Stable Isotopes for Biomolecular NMR
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参考实例
Kano, H.; Toyama, Y.; Imai, S.; et al. 2019. Structural mechanism underlying G protein family-specific regulation of G protein-gated inwardly rectifying potassium channel. Nat Commun, 10(1), 2008. PMID: 31043612
Kurauskas, V.; Schanda, P.; Sounier, R. 2017. Methyl-specific isotope labeling strategies for NMR studies of membrane proteins. Methods Mol Biol, 1635, 109-123. PMID: 28755366