Mapping a ligand binding site using genetically encoded photoactivatable crosslinkers
Amy Grunbeck1, Thomas Huber, Thomas P Sakmar
1Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, USA.
Abstract:
G protein-coupled receptor (GPCR) signaling complexes are important for mediating many different biological processes. Uncovering the mechanism for how a ligand triggers a GPCR to elicit a specific response is an active area of research. One step toward understanding this mechanism is through identifying a ligand's binding site on a GPCR. We have optimized a targeted photocrosslinking technology to detect the residues in a receptor that are within a precise distance from a bound ligand in the receptor-ligand complex. Here, we describe the method for introducing photoactivable crosslinkers into a GPCR using the amber stop codon suppression technology. In addition, we review the steps to identify the binding site of a fluorescein-tagged peptide ligand and a tritium-labeled small molecule ligand.
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