Lysine methylation strategies for characterizing protein conformations by NMR
Lysine methylation strategies for characterizing protein conformations by NMR
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DOI：
10.1007/s10858-012-9664-z
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发表时间：
2012-10-01
影响因子：
2.7
通讯作者：
Prosser, R. Scott
中科院分区：
文献类型：
--
作者：
Larda, Sacha Thierry;Bokoch, Michael P.;Prosser, R. Scott
在甲醛和温和的还原剂存在下，已知还原甲基化可以在非变性条件下在水介质中实现蛋白质氨基的近乎完全的二甲基化。蛋白质的氨甲基化用于质谱学、结晶学和核磁共振研究。在生物合成标记被禁止的地方，氨基C-13甲基化为用核磁共振监测折叠、动力学、蛋白质-蛋白质和蛋白质-DNA相互作用提供了一个有吸引力的选择。在这里，我们展示了相对于传统的C-13还原甲基化方案的两个改进：(1)通过明智地选择化学计量比和pH，可以优先将表位氨基单甲基化。与二甲基赖氨酸相比，单甲基标签的干扰更少，通常表现出更高的分辨率，以及(2)通过使用氚还原剂和C-13-甲醛，可以用(CH2D)-C-13标签标记氨基。使用氢-C-13-甲醛可以得到(CHD2)-C-13或(CD3)-C-13探针，这取决于所选择的还原剂。利用C-13-H-2标量耦合，我们证明了一种消除自然丰度C-13信号的滤波方案。
In the presence of formaldehyde and a mild reducing agent, reductive methylation is known to achieve near complete dimethylation of protein amino groups under non-denaturing conditions, in aqueous media. Amino methylation of proteins is employed in mass spectrometric, crystallographic, and NMR studies. Where biosynthetic labeling is prohibitive, amino C-13-methylation provides an attractive option for monitoring folding, kinetics, protein-protein and protein-DNA interactions by NMR. Here, we demonstrate two improvements over traditional C-13-reductive methylation schemes: (1) By judicious choice of stoichiometry and pH, epsilon-aminos can be preferentially monomethylated. Monomethyl tags are less perturbing and generally exhibit improved resolution over dimethyllysines, and (2) By use of deuterated reducing agents and C-13-formaldehyde, amino groups can be labeled with (CH2D)-C-13 tags. Use of deutero-C-13-formaldehyde affords either (CHD2)-C-13, or (CD3)-C-13 probes depending on choice of reducing agent. Making use of C-13-H-2 scalar couplings, we demonstrate a filtering scheme that eliminates natural abundance C-13 signal.