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GPCR Solubilization and Quality Control

  • Tamara Miljus
  • David A. Sykes
  • Clare R. Harwood
  • Ziva Vuckovic
  • Dmitry B. VeprintsevEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2127)

Abstract

G protein-coupled receptors (GPCRs) are versatile membrane proteins involved in the regulation of many physiological processes and pathological conditions, making them interesting pharmacological targets. In order to study their structure and function, GPCRs are traditionally extracted from membranes using detergents. However, due to their hydrophobic nature, intrinsic instability in aqueous solutions, and their denaturing effects, the isolation of properly folded and functional GPCRs is not trivial. Therefore, it is of crucial importance to solubilize receptors under mild conditions and control the sample quality subsequently. Here we describe widely used methods for small-scale GPCR solubilization, followed by quality control based on fluorescence size-exclusion chromatography, SDS-PAGE, temperature-induced protein unfolding (CPM dye binding) and fluorescent ligand binding assay. These methods can easily be used to assess the thermostability and functionality of a GPCR sample exposed to different conditions, such as the use of various detergents, addition of lipids and ligands, making them valuable for obtaining an optimal sample quality for structural and functional studies.

Key words

GPCR solubilization FSEC In-gel fluorescence Thermal shift assay Fluorescent ligand binding assay 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Tamara Miljus
    • 1
    • 2
    • 3
    • 4
  • David A. Sykes
    • 3
    • 5
  • Clare R. Harwood
    • 3
    • 5
  • Ziva Vuckovic
    • 1
    • 2
    • 6
  • Dmitry B. Veprintsev
    • 1
    • 2
    • 3
    • 5
    Email author
  1. 1.Laboratory of Biomolecular ResearchPaul Scherrer InstituteVilligen PSISwitzerland
  2. 2.Department of BiologyETH ZürichZürichSwitzerland
  3. 3.Centre of Membrane Proteins and Receptors (COMPARE)University of Birmingham and University of NottinghamMidlandsUK
  4. 4.Institute of Metabolism and Systems Research, College of Medical and Dental SciencesUniversity of BirminghamBirminghamUK
  5. 5.Division of Physiology, Pharmacology & Neuroscience, School of Life SciencesUniversity of NottinghamNottinghamUK
  6. 6.Drug Discovery BiologyMonash Institute of Pharmaceutical Sciences, Monash UniversityParkvilleAustralia

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