Selective 15N-labeling of the side-chain amide groups of asparagine and glutamine for applications in paramagnetic NMR spectroscopy
Selective 15N-labeling of the side-chain amide groups of asparagine and glutamine for applications in paramagnetic NMR spectroscopy
复制标题
DOI：
10.1007/s10858-014-9844-0
复制
发表时间：
2014-08-01
影响因子：
2.7
通讯作者：
Su, Xun-Cheng
中科院分区：
文献类型：
--
作者：
Cao, Chan;Chen, Jia-Liang;Su, Xun-Cheng
天冬酰胺和谷氨酰胺的侧链酰胺基团在稳定蛋白质的结构折叠、参与氢键网络和蛋白质相互作用中起重要作用。然而，由于蛋白质合成过程中酰胺基团的酶催化交换，侧链酰胺的选择性N-15标记可能是一个挑战。在本研究中，我们开发了一种有效的方法，选择性标记的侧链的天冬酰胺，或天冬酰胺和谷氨酰胺残基与（NH 2）-N-15。利用色氨酸的生物合成途径，还建立了一种方案，用于同时选择性N-15-标记天冬酰胺、谷氨酰胺和色氨酸的侧链NH基团。结合位点特异性标记的靶蛋白与镧系元素离子，我们表明，选择性检测N-15标记的天冬酰胺和谷氨酰胺的侧链允许确定磁化率各向异性张量的基础上，完全基于伪接触位移的酰胺侧链质子。
The side-chain amide groups of asparagine and glutamine play important roles in stabilizing the structural fold of proteins, participating in hydrogen-bonding networks and protein interactions. Selective N-15-labeling of side-chain amides, however, can be a challenge due to enzyme-catalyzed exchange of amide groups during protein synthesis. In the present study, we developed an efficient way of selectively labeling the side chains of asparagine, or asparagine and glutamine residues with (NH2)-N-15. Using the biosynthesis pathway of tryptophan, a protocol was also established for simultaneous selective N-15-labeling of the side-chain NH groups of asparagine, glutamine, and tryptophan. In combination with site-specific tagging of the target protein with a lanthanide ion, we show that selective detection of N-15-labeled side-chains of asparagine and glutamine allows determination of magnetic susceptibility anisotropy tensors based exclusively on pseudocontact shifts of amide side-chain protons.