An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein
An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein
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DOI：
10.1007/s10858-008-9294-7
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发表时间：
2009-02-01
影响因子：
2.7
通讯作者：
Boisbouvier, Jerome
中科院分区：
文献类型：
--
作者：
Ayala, Isabel;Sounier, Remy;Boisbouvier, Jerome
介绍了一种将（H-1， c -13-甲基）-丙氨酸引入过氘化蛋白的策略。在添加了800 mg/l的2-[H-2]， 3-[C-13] l-丙氨酸的M9/D2O细菌培养基中过表达蛋白，可使丙氨酸甲基的特异性质子化达到95%。然而，这种方法虽然简单，但由于同位素通过不同的氨基酸代谢途径进行混乱，导致不希望的非特异性背景标记。经过对已知代谢途径的仔细分析，我们发现，将α -酮异戊酸-d(7)、琥珀酸-d(4)和l-异亮氨酸-d（10）与标记的l-丙氨酸共添加，可将不需要的背景标记减少到< 1%。当与最近开发的甲基TROSY实验相结合时，这种甲基特异性标记方案允许获得高分子量蛋白质中丙氨酸甲基的高质量相关光谱。与之前报道的方法相比，我们的成本效益策略显著提高了甲基标记丙氨酸在过表达蛋白中的掺入水平。
A strategy for the introduction of (H-1,C-13-methyl)-alanine into perdeuterated proteins is described. Specific protonation of alanine methyl groups to a level of 95% can be achieved by overexpressing proteins in M9/D2O based bacterial growth medium supplemented with 800 mg/l of 2-[H-2], 3-[C-13] l-alanine. However, though simple, this approach results in undesired, non-specific background labeling due to isotope scrambling via different amino acid metabolic pathways. Following a careful analysis of known metabolic pathways we found that co-addition of perdeuterated forms of alpha-ketoisovalerate-d(7), succinate-d(4) and l-isoleucine-d(10) with labeled l-alanine, reduces undesired background labeling to < 1%. When combined with recently developed methyl TROSY experiments, this methyl-specific labeling protocol permits the acquisition of excellent quality correlation spectra of alanine methyl groups in high molecular weight proteins. Our cost effective strategy offers a significant enhancement in the level of incorporation of methyl-labeled alanine in overexpressed proteins over previously reported methods.