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Related Experiment Video

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Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors
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Biotinylated non-ionic amphipols for GPCR ligands screening.

Michaël Bosco1, Marjorie Damian2, Vinay Chauhan1

  • 1Institut des Biomolécules Max Mousseron (UMR 5247 UM-CNRS-ENSCM) & Avignon University, Equipe Chimie Bioorganique et Systèmes amphiphiles, 301 rue Baruch de Spinoza, 84916 Avignon cedex 9, France.

Methods (San Diego, Calif.)
|June 8, 2020
PubMed
Summary

We developed biotin-functionalized polymers (BNAPols) for immobilizing membrane proteins (MPs) onto surfaces. This enables novel sensor devices for ligand screening and studying protein function.

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Area of Science:

  • Polymer Chemistry
  • Biotechnology
  • Surface Science

Background:

  • Membrane proteins (MPs) play crucial roles in cellular functions but are challenging to study due to their hydrophobic nature.
  • Existing methods for MP immobilization often require protein modification or result in loss of function.
  • Developing robust and versatile platforms for MP stabilization and surface attachment is essential for biochemical assays and drug discovery.

Purpose of the Study:

  • To synthesize novel biotin-functionalized polymers (BNAPols) for the efficient immobilization of membrane proteins onto surfaces.
  • To characterize the synthesized BNAPols and confirm their biotinylation.
  • To demonstrate the utility of BNAPols for stabilizing and immobilizing a model G protein-coupled receptor (GPCR) for ligand screening.

Main Methods:

  • Synthesis of BNAPols via free-radical polymerization of an amphiphilic monomer followed by Huisgen-cycloaddition with biotinylated alkyne.
  • Structural confirmation using NMR spectroscopy and biotinylation percentage determination via HABA/avidin assay.
  • Colloidal characterization using dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS).
  • Stabilization and immobilization of a model GPCR (GHSR) and subsequent ligand binding assays.

Main Results:

  • Successful synthesis and characterization of biotin-functionalized polymers (BNAPols).
  • Demonstrated colloidal stability and structural integrity of BNAPols.
  • Effective stabilization of a model GPCR (GHSR) in an non-membrane environment.
  • Successful immobilization of the BNAPols:GHSR complex onto a streptavidin-coated surface, enabling ligand screening.

Conclusions:

  • BNAPols provide a versatile platform for the functional immobilization of unmodified membrane proteins.
  • This approach facilitates the development of novel sensor devices for various applications, including high-throughput ligand screening.
  • The methodology opens new avenues for studying membrane protein structure-function relationships and for drug discovery efforts.