Exploring Pairwise Chemical Crosslinking To Study Peptide-Receptor Interactions
Exploring Pairwise Chemical Crosslinking To Study Peptide-Receptor Interactions
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DOI：
10.1002/cbic.201800582
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发表时间：
2019-03-01
期刊：
影响因子：
3.2
通讯作者：
Coin, Irene
中科院分区：
文献类型：
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作者：
Seidel, Lisa;Zarzycka, Barbara;Coin, Irene
成对交联是研究G蛋白偶联受体与其配体在活细胞中相互作用的有效方法。在这项工作中，巯基捕获方法，利用卤代乙酰胺和半胱氨酸之间的邻近增强反应，检查，以确定分子间的邻位位置对。通过将半胱氨酸纳入促肾上腺皮质激素释放因子受体和-氯-或-溴乙酰胺基团到其配体中，它表明，硫醇捕获提供了高度可重复的信号和低背景，并代表了一个有效的替代经典的二硫化物捕获。如果在样品分析期间需要还原剂，则该方法是有利的。此外，它可以提供部分不同的空间约束，从而为分子建模提供更广泛的数据集。最后，通过应用重组mini-Gs、GTPS和Gs耗尽的HEK 293细胞来调节Gs偶联，表明在升高水平的Gs存在下交联的产率增加。
Pairwise crosslinking is a powerful technique to characterize interactions between Gprotein coupled receptors and their ligands in the live cell. In this work, the thiol trapping method, which exploits the proximity-enhanced reaction between haloacetamides and cysteine, is examined to identify intermolecular pairs of vicinal positions. By incorporating cysteine into the corticotropin-releasing factor receptor and either -chloro- or -bromoacetamide groups into its ligands, it is shown that thiol trapping provides highly reproducible signals and a low background, and represents a valid alternative to classical disulfide trapping. The method is advantageous if reducing agents are required during sample analysis. Moreover, it can provide partially distinct spatial constraints, thus giving access to a wider dataset for molecular modeling. Finally, by applying recombinant mini-Gs, GTPS, and Gs-depleted HEK293 cells to modulate Gs coupling, it is shown that yields of crosslinking increase in the presence of elevated levels of Gs.