2. Academic Validation
  3. Biotinylated non-ionic amphipols for GPCR ligands screening

Biotinylated non-ionic amphipols for GPCR ligands screening

  • Methods. 2020 Aug 1:180:69-78. doi: 10.1016/j.ymeth.2020.06.001.
Michaël Bosco 1,  Marjorie Damian 2,  Vinay Chauhan 1,  Mélanie Roche 2,  Pierre Guillet 1,  Jean-Alain Fehrentz 2,  Françoise Bonneté 3,  Ange Polidori 1,  Jean-Louis Banères 2,  Grégory Durand 4
Affiliations

Affiliations

  • 1 Institut 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.
  • 2 Institut des Biomolécules Max Mousseron (UMR 5247 UM-CNRS-ENSCM), Faculté de pharmacie, 15 avenue Charles Flahault, 34000 Montpellier, France.
  • 3 Institut 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; Institut de Biologie Physico-Chimique (UMR 7099 CNRS Université de Paris), Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, 13 rue Pierre et Marie Curie, 75005 Paris, France.
  • 4 Institut 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. Electronic address: [email protected].
PMID: 32505829 DOI: 10.1016/j.ymeth.2020.06.001
Abstract

We present herein the synthesis of biotin-functionalized Polymers (BNAPols) that have been developed for the fixation of membrane proteins (MPs) onto surfaces. BNAPols were synthesized by free-radical polymerization of a tris(hydroxymethyl)acrylamidomethane (THAM)-derived amphiphilic monomer in the presence of a thiol-based transfer agent with an azido group. Then a Huisgen-cycloaddition reaction was performed with Biotin-(PEG)8-alkyne that resulted in formation of the biotinylated Polymers. The designed structure of BNAPols was confirmed by NMR spectroscopy, and a HABA/avidin assay was used for estimating the percentage of biotin grafted on the polymer end chain. The colloidal characterization of these biotin-functionalized Polymers was done using both dynamic LIGHT scattering (DLS) and small angle X-ray scattering (SAXS) techniques. BNAPols were used to stabilize a model G protein-coupled receptor (GPCR), the human Growth Hormone Secretagogue Receptor (GHSR), out of its membrane environment. Subsequent immobilization of the BNAPols:GHSR complex onto a streptavidin-coated surface allowed screening of ligands based on their ability to bind the immobilized receptor. This opens the way to the use of biotinylated NAPols to immobilize functional, unmodified, membrane proteins, providing original sensor devices for multiple applications including innovative ligand screening assays.

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