期刊
CHEMBIOCHEM
卷 20, 期 5, 页码 683-692出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201800582
关键词
crosslinking; molecular modeling; peptide-protein interactions; peptides; sulfur
资金
- Emmy Noether program of the German Research Foundation (DFG) [CO822/2-1]
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.
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