Cao Chan, Chen Jia-Liang, Yang Yin, Huang Feng, Otting Gottfried, Su Xun-Cheng
State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
J Biomol NMR. 2014 Aug;59(4):251-61. doi: 10.1007/s10858-014-9844-0. Epub 2014 Jul 8.
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 (15)N-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 (15)NH2. Using the biosynthesis pathway of tryptophan, a protocol was also established for simultaneous selective (15)N-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 (15)N-labeled side-chains of asparagine and glutamine allows determination of magnetic susceptibility anisotropy tensors based exclusively on pseudocontact shifts of amide side-chain protons.
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方面发挥着重要作用。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（15）N标记可能具有挑战性。在本研究中，我们开发了一种用（15）NH2选择性标记天冬酰胺或天冬酰胺和谷氨酰胺残基侧链的有效方法。利用色氨酸的生物合成途径，还建立了同时选择性（15）N标记天冬酰胺、谷氨酰胺和色氨酸侧链NH基团的方案。结合用镧系离子对目标蛋白质进行位点特异性标记，我们表明，对天冬酰胺和谷氨酰胺的（15）N标记侧链进行选择性检测，可以仅基于酰胺侧链质子的赝接触位移来确定磁化率各向异性张量。
