Exploring Pairwise Chemical Crosslinking To Study Peptide–Receptor Interactions

Lisa Seidel, Barbara Zarzycka, Vsevolod Katritch, Irene Coin*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


Pairwise crosslinking is a powerful technique to characterize interactions between G protein 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 α-chloro- or α-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, GTPγS, and Gαs-depleted HEK293 cells to modulate Gs coupling, it is shown that yields of crosslinking increase in the presence of elevated levels of Gs.

Original languageEnglish
Pages (from-to)683-692
Number of pages10
Issue number5
Publication statusPublished - 1 Mar 2019
Externally publishedYes


  • crosslinking
  • molecular modeling
  • peptides
  • peptide–protein interactions
  • sulfur


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