Application of reductive ¹³C-methylation of lysines to enhance the sensitivity of conventional NMR methods.
Application of reductive ¹³C-methylation of lysines to enhance the sensitivity of conventional NMR methods.
复制标题
赖氨酸的还原性αC-甲基化的应用以增强常规NMR方法的敏感性。
DOI：
10.3390/molecules18067103
复制
发表时间：
2013-06-18
期刊：
影响因子：
--
通讯作者：
Gaponenko V
中科院分区：
文献类型：
--
作者：
Chavan TS;Abraham S;Gaponenko V
NMR通常用于研究大分子相互作用。然而，敏感性问题阻碍了其用于研究在低生理相关浓度下的这种相互作用。在高浓度下，蛋白质或肽倾向于聚集。为了克服这个问题，我们利用还原性13 C甲基化来研究低微摩尔浓度下的蛋白质相互作用。二甲基赖氨酸中的甲基是简并的，与脂肪族氨基酸中的一个碳和三个质子相比，具有由两个碳和六个质子产生的一个13 CH 3信号。提高的灵敏度使我们能够在非常低的微摩尔浓度下研究蛋白质-蛋白质或蛋白质-肽的相互作用。我们证明了这种方法的效用，通过研究的人原癌基因GT3-K-Ras和钙传感器蛋白钙调素的后-acetylidated高变区之间的相互作用。钙调素特异性结合K-Ras并调节其下游信号传导。这种结合特异性归因于K-Ras独特的脂化高变区。在低微摩尔浓度下，K-Ras的后修饰高变区以非特异性方式聚集并结合钙调蛋白，因此常规NMR技术不能用于研究这种相互作用，然而，在还原甲基化钙调蛋白的赖氨酸后，我们检测到K-Ras的脂化高变区在生理相关纳摩尔浓度下的信号。因此，我们利用赖氨酸的13 C-还原甲基化来提高常规NMR方法在低浓度下研究蛋白质相互作用的灵敏度。
NMR is commonly used to investigate macromolecular interactions. However, sensitivity problems hamper its use for studying such interactions at low physiologically relevant concentrations. At high concentrations, proteins or peptides tend to aggregate. In order to overcome this problem, we make use of reductive 13C-methylation to study protein interactions at low micromolar concentrations. Methyl groups in dimethyl lysines are degenerate with one 13CH3 signal arising from two carbons and six protons, as compared to one carbon and three protons in aliphatic amino acids. The improved sensitivity allows us to study protein-protein or protein-peptide interactions at very low micromolar concentrations. We demonstrate the utility of this method by studying the interaction between the post-translationally lipidated hypervariable region of a human proto-oncogenic GTPase K-Ras and a calcium sensor protein calmodulin. Calmodulin specifically binds K-Ras and modulates its downstream signaling. This binding specificity is attributed to the unique lipidated hypervariable region of K-Ras. At low micromolar concentrations, the post-translationally modified hypervariable region of K-Ras aggregates and binds calmodulin in a non-specific manner, hence conventional NMR techniques cannot be used for studying this interaction, however, upon reductively methylating the lysines of calmodulin, we detected signals of the lipidated hypervariable region of K-Ras at physiologically relevant nanomolar concentrations. Thus, we utilize 13C-reductive methylation of lysines to enhance the sensitivity of conventional NMR methods for studying protein interactions at low concentrations.
登录
查看更多内容
影响因子：
14.8
作者：
Ismail, Shehab A.;Chen, Yong-Xiang;Wittinghofer, Alfred
通讯作者：
Wittinghofer, Alfred
影响因子：
4.8
作者：
Liao, Jinhui;Planchon, Sarah M.;Wolfman, Alan
通讯作者：
Wolfman, Alan
影响因子：
2.7
作者：
Larda, Sacha Thierry;Bokoch, Michael P.;Prosser, R. Scott
通讯作者：
Prosser, R. Scott
影响因子：
2.9
作者：
Abraham, Sherwin J.;Nolet, Ryan P.;Calvert, Richard J.;Anderson, Lucy M.;Gaponenko, Vadim
通讯作者：
Gaponenko, Vadim
影响因子：
5.3
作者：
Esteban, LM;Vicario-Abejón, C;Santos, E
通讯作者：
Santos, E