Exploring Pairwise Chemical Crosslinking To Study Peptide–Receptor Interactions. (25th January 2019) - Seidel, Lisa
Abstract: 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. Abstract : S(−S)nap ! Intermolecular pairs of vicinal amino acids in ligand–receptor complexes can be conveniently identified by using the "thiol trapping" method, which exploits the proximity‐enhanced reaction between haloacetamides and cysteine. The method provides outcomes comparable to those of classical disulfide trapping and is compatible with the presence of reducing reagents during electrophoretic analysis.