Bioorthogonal Labeling of Ghrelin Receptor to Facilitate Studies of Ligand-Dependent Conformational Dynamics

Minyoung Park; Bjørn B Sivertsen; Sylvia Els-Heindl; Thomas Huber; Birgitte Holst; Annette G Beck-Sickinger; Thue W Schwartz; Thomas P Sakmar

doi:10.1016/j.chembiol.2015.09.014

Bioorthogonal Labeling of Ghrelin Receptor to Facilitate Studies of Ligand-Dependent Conformational Dynamics

Chem Biol. 2015 Nov 19;22(11):1431-1436. doi: 10.1016/j.chembiol.2015.09.014. Epub 2015 Nov 5.

Authors

Minyoung Park¹, Bjørn B Sivertsen², Sylvia Els-Heindl³, Thomas Huber¹, Birgitte Holst², Annette G Beck-Sickinger³, Thue W Schwartz², Thomas P Sakmar⁴

Affiliations

¹ Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
² Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, University of Copenhagen, The Panum Institute, Blegdamsvej 3b, 2200 Copenhagen, Denmark.
³ Institute of Biochemistry, Faculty of Biosciences, Pharmacy, and Psychology, Universität Leipzig, Brüderstraße 34, 04103 Leipzig, Germany.
⁴ Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA; Department of Neurobiology, Care Sciences and Society, Division for Neurogeriatrics, Center for Alzheimer Research, Karolinska Institutet, 141 57 Huddinge, Sweden. Electronic address: sakmar@rockefeller.edu.

PMID: 26548612
DOI: 10.1016/j.chembiol.2015.09.014

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Antibodies, Monoclonal / chemistry
Antibodies, Monoclonal / immunology
Azides / chemistry
Cycloaddition Reaction
Fluorescence Resonance Energy Transfer
Fluorescent Dyes / chemistry
Humans
Ligands*
Organometallic Compounds / chemistry
Phenylalanine / analogs & derivatives
Phenylalanine / chemistry
Protein Conformation
Receptors, Ghrelin / chemistry*
Receptors, Ghrelin / genetics
Receptors, Ghrelin / metabolism

Substances

Antibodies, Monoclonal
Azides
Fluorescent Dyes
Ligands
Organometallic Compounds
Receptors, Ghrelin
europium(III) trisbipyridine cryptate
4-azidophenylalanine
Phenylalanine