European Biophysics Journal

, Volume 35, Issue 8, pp 709–712 | Cite as

Direct analysis of a GPCR-agonist interaction by surface plasmon resonance

  • Peter J. Harding
  • Timothy C. Hadingham
  • James M. McDonnell
  • Anthony Watts
Biophysics Letter

Abstract

Despite their clinical importance, detailed analysis of ligand binding at G-protein coupled receptors (GPCRs) has proved difficult. Here we successfully measure the binding of a GPCR, neurotensin receptor-1 (NTS-1), to its ligand, neurotensin (NT), using surface plasmon resonance (SPR). Specific responses were observed between NT and purified, detergent-solublised, recombinant NTS-1, using a novel configuration where the biotinylated NT ligand was immobilised on the biosensor surface. This SPR approach shows promise as a generic approach for the study of ligand interactions with other suitable GPCRs.

Keywords

GPCR Neurotensin receptor Surface plasmon resonance Ligand-binding High-throughput screening 

Abbreviations

CHAPS

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate

CHS

Cholesteryl hemisuccinate

DCM

Dichloromethane

DDM

Dodecyl-β-d-maltoside

EDTA

Ethylenediaminetetraacetate

GPCR

GTP-binding protein-coupled receptor

HPLC

High pressure liquid chromatography

MBP

Escherichia coli maltose binding protein

NT

Neurotensin

NTR/NTS

Neurotensin receptor

NTS-1A

MBP-rT43NTR-TrxA-H10

SPR

Surface plasmon resonance

TES

Triethylsilane

TFA

Trifluoroacetic acid

Notes

Acknowledgements

We acknowledge the Medical Research Council (MRC), Biotechnology and Biological Sciences Research Council (BBSRC) and the UK Bionanotechnology IRC for funding this work. We thank GlaxoSmithKine (GSK), specifically Claus Spitzfaden, Martin Hibbs, Emma Jones, and Mark Hessey for allowing access to large scale fermentation, purification and SPR facilities. We would like to thank Dr. Simon Davis (Nuffield Department of Medicine, Oxford) for the use of his SPR machine.

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Copyright information

© EBSA 2006

Authors and Affiliations

  • Peter J. Harding
    • 1
  • Timothy C. Hadingham
    • 1
  • James M. McDonnell
    • 2
  • Anthony Watts
    • 1
  1. 1.Biomembrane Structure Unit, Department of BiochemistryUniversity of OxfordOxfordUK
  2. 2.The Laboratory of Molecular Biophysics, Department of BiochemistryUniversity of OxfordOxfordUK

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