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Spirohexene-Tetrazine Ligation Enables Bioorthogonal Labeling of Class B G Protein-Coupled Receptors in Live Cells
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2017-09-15 00:00:00 , DOI: 10.1021/jacs.7b05674 Carlo P. Ramil 1 , Maoqing Dong 2 , Peng An 1 , Tracey M. Lewandowski 1 , Zhipeng Yu 1 , Laurence J. Miller 2 , Qing Lin 1
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2017-09-15 00:00:00 , DOI: 10.1021/jacs.7b05674 Carlo P. Ramil 1 , Maoqing Dong 2 , Peng An 1 , Tracey M. Lewandowski 1 , Zhipeng Yu 1 , Laurence J. Miller 2 , Qing Lin 1
Affiliation
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A new bioorthogonal reactant pair, spiro[2.3]hex-1-ene (Sph) and 3,6-di(2-pyridyl)-s-tetrazine (DpTz), for the strain-promoted inverse electron-demand Diels–Alder cycloaddition, that is, tetrazine ligation, is reported. As compared to the previously reported strained alkenes such as trans-cyclooctene (TCO) and 1,3-disubstituted cyclopropene, Sph exhibits balanced reactivity and stability in tetrazine ligation with the protein substrates. A lysine derivative of Sph, SphK, was site-selectively incorporated into the extracellular loop regions (ECLs) of GCGR and GLP-1R, two members of class B G protein-coupled receptors (GPCRs) in mammalian cells with the incorporation efficiency dependent on the location. Subsequent bioorthogonal reactions with the fluorophore-conjugated DpTz reagents afforded the fluorescently labeled GCGR and GLP-1R ECL mutants with labeling yield as high as 68%. A multitude of functional assays were performed with these GPCR mutants, including ligand binding, ligand-induced receptor internalization, and ligand-stimulated intracellular cAMP accumulation. Several positions in the ECL3s of GCGR and GLP-1R were identified that tolerate SphK mutagenesis and subsequent bioorthogonal labeling. The generation of functional, fluorescently labeled ECL3 mutants of GCGR and GLP-1R should allow biophysical studies of conformation dynamics of this important class of GPCRs in their native environment in live cells.
中文翻译：
Spirohexene-Tetrazine连接使活细胞中BG类蛋白偶联受体的生物正交标记成为可能。
一种新的生物正交反应物对，螺[2.3]己-1-烯（SPH）和3,6-二（2-吡啶基） -小号-tetrazine（DPTZ），用于张力促进逆电子需求狄尔斯-阿尔德环加成报道了四嗪连接。与先前报道的应变烯烃如反式-环辛烯（TCO）和1,3-二取代的环丙烯，Sph在四嗪与蛋白质底物的连接中显示出平衡的反应性和稳定性。将Sph的赖氨酸衍生物SphK选择性插入哺乳动物细胞中BGR类蛋白偶联受体（GPCR）的两个成员GCGR和GLP-1R的细胞外环区（ECL）中，其结合效率取决于地点。随后与荧光团偶联的DpTz试剂的生物正交反应提供了荧光标记的GCGR和GLP-1R ECL突变体，其标记产率高达68％。用这些GPCR突变体进行了许多功能测定，包括配体结合，配体诱导的受体内在化和配体刺激的细胞内cAMP积累。确定了在GCGR和GLP-1R的ECL3中的几个位置可以耐受SphK诱变和随后的生物正交标记。GCGR和GLP-1R的功能性，荧光标记的ECL3突变体的产生，应使这种重要的GPCR类在其天然环境中的活细胞中的构象动力学能够进行生物物理研究。
更新日期：2017-09-15
中文翻译：
Spirohexene-Tetrazine连接使活细胞中BG类蛋白偶联受体的生物正交标记成为可能。
一种新的生物正交反应物对，螺[2.3]己-1-烯（SPH）和3,6-二（2-吡啶基） -小号-tetrazine（DPTZ），用于张力促进逆电子需求狄尔斯-阿尔德环加成报道了四嗪连接。与先前报道的应变烯烃如反式-环辛烯（TCO）和1,3-二取代的环丙烯，Sph在四嗪与蛋白质底物的连接中显示出平衡的反应性和稳定性。将Sph的赖氨酸衍生物SphK选择性插入哺乳动物细胞中BGR类蛋白偶联受体（GPCR）的两个成员GCGR和GLP-1R的细胞外环区（ECL）中，其结合效率取决于地点。随后与荧光团偶联的DpTz试剂的生物正交反应提供了荧光标记的GCGR和GLP-1R ECL突变体，其标记产率高达68％。用这些GPCR突变体进行了许多功能测定，包括配体结合，配体诱导的受体内在化和配体刺激的细胞内cAMP积累。确定了在GCGR和GLP-1R的ECL3中的几个位置可以耐受SphK诱变和随后的生物正交标记。GCGR和GLP-1R的功能性，荧光标记的ECL3突变体的产生，应使这种重要的GPCR类在其天然环境中的活细胞中的构象动力学能够进行生物物理研究。
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