Intracellular bioorthogonal labeling of glucagon receptor via tetrazine ligation.
Intracellular bioorthogonal labeling of glucagon receptor via tetrazine ligation.
复制标题
DOI：
10.1016/j.bmc.2021.116256
复制
发表时间：
2021-08-01
影响因子：
3.5
通讯作者：
Lin Q
中科院分区：
文献类型：
--
作者：
Tian Y;Fang M;Lin Q
胰高血糖素受体 (GCGR) 胞质面的第三个细胞内环 (ICL3) 在受体激活过程中经历显着的构象转变。因此，在我们构建基于荧光的 GPCR 生物传感器的过程中，它为引入有机荧光团提供了一个有吸引力的位点。在此，我们报告了使用生物正交化学策略对 ICL3 细胞内荧光标记进行的共聚焦显微镜研究。我们的方法包括通过遗传密码扩展将应变烯烃氨基酸位点特异性引入 ICL3，然后与荧光四嗪探针进行高度特异性的逆电子需求狄尔斯-阿尔德反应。在检查的三种应变烯烃氨基酸中，SphK 和 2’-aTCOK 均使用适当的四嗪探针成功地荧光标记了 GCGR ICL3。同时，4'-TCOK 由于其细胞内滞留而产生高背景荧光。荧光四嗪探针是根据无背景细胞内荧光标记的计算模型设计的；然而，它们在活细胞成像中的性能差异很大，因为强烈的非特异性信号会干扰特异性信号。在所有带有应变烯烃的 GCGR ICL3 突变体中，H339SphK/2'-aTCOK 突变体为 BODIPY-Tz1/4 试剂在生物正交标记反应中提供了最佳反应伙伴。这项研究的结果强调了在活细胞成像研究中识别适合低丰度受体细胞内标记的生物正交反应物对的挑战。
The third intracellular loop (ICL3) in the cytosolic face of glucagon receptor (GCGR) experiences significant conformational transition during receptor activation. It thus offers an attractive site for the introduction of organic fluorophores in our efforts to construct fluorescence-based GPCR biosensors. Herein, we report our confocal microscopic study of intracellular fluorescent labeling of ICL3 using a bioorthogonal chemistry strategy. Our approach involves the site-specific introduction of a strained alkene amino acid into the ICL3 through genetic code expansion, followed by a highly specific inverse electron-demand Diels-Alder reaction with the fluorescent tetrazine probes. Among the three strained alkene amino acids examined, both SphK and 2’-aTCOK offered successful fluorescent labeling of GCGR ICL3 with the appropriate tetrazine probes. At the same time, 4’-TCOK gave high background fluorescence due to its intracellular retention. The fluorescent tetrazine probes were designed following a computational model for background-free intracellular fluorescent labeling; however, their performance varied significantly in live-cell imaging as the strong non-specific signals interfered with the specific ones. Among all GCGR ICL3 mutants bearing a strained alkene, the H339SphK/2’-aTCOK mutants provided the best reaction partners for the BODIPY-Tz1/4 reagents in the bioorthogonal labeling reactions. The results from this study highlight the challenges in identifying bioorthogonal reactant pairs suitable for intracellular labeling of low-abundance receptors in live-cell imaging studies.
登录
查看更多内容
影响因子：
64.8
作者：
Jazayeri, Ali;Rappas, Mathieu;Marshall, Fiona H.
通讯作者：
Marshall, Fiona H.
影响因子：
16.6
作者：
Carlson, Jonathan C. T.;Meimetis, Labros G.;Hilderbrand, Scott A.;Weissleder, Ralph
通讯作者：
Weissleder, Ralph
影响因子：
48
作者：
Grimm JB;Muthusamy AK;Liang Y;Brown TA;Lemon WC;Patel R;Lu R;Macklin JJ;Keller PJ;Ji N;Lavis LD
通讯作者：
Lavis LD
影响因子：
18.2
作者：
Devaraj NK
通讯作者：
Devaraj NK
影响因子：
5.6
作者：
Culhane KJ;Liu Y;Cai Y;Yan EC
通讯作者：
Yan EC