Chemistry & Biology
Volume 22, Issue 11, 19 November 2015, Pages 1431-1436
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Brief Communication
Bioorthogonal Labeling of Ghrelin Receptor to Facilitate Studies of Ligand-Dependent Conformational Dynamics

https://doi.org/10.1016/j.chembiol.2015.09.014Get rights and content
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Highlights

  • Functional GhrR was site-specifically tagged with the unnatural amino acid, azF

  • GhrR azF mutants were labeled bioorthogonally with a fluorophore using SpAAC

  • Novel assays were developed to probe binding of fluorescent ligands to labeled GhrR

  • Ligand-induced conformational changes could be detected in fluorescent GhrR

Summary

Ghrelin receptor (GhrR) is a promising drug target because of its central role in energy homeostasis. GhrR, known for high constitutive activity, is thought to display multi-state conformations during activation and signaling. We used genetically encoded unnatural amino acids and bioorthogonal labeling reactions to engineer multiple fluorescent donor-acceptor pairs to probe ligand-directed structural changes in GhrR. We demonstrate how conformational dynamics of a G-protein-coupled receptor can be measured in reconstituted systems.

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