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Methods Enzymol. 2013;520:307-22. doi: 10.1016/B978-0-12-391861-1.00014-9.

Mapping a ligand binding site using genetically encoded photoactivatable crosslinkers.

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1
Laboratory 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.

[Indexed for MEDLINE]

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