Biotinylated non-ionic amphipols for GPCR ligands screening

Michaël Bosco (1) , Marjorie Damian (1) , Vinay Chauhan (1) , Mélanie Roche (1) , Pierre Guillet (1) , Jean-Alain Fehrentz (1) , Françoise Bonnete (1, 2) , Ange Polidori (1) , Jean-Louis Banères (1) , Grégory Durand (1)

1 IBMM - Institut des Biomolécules Max Mousseron [Pôle Chimie Balard]
2 LBPC-PM (UMR_7099) - Laboratoire de biologie physico-chimique des protéines membranaires

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. These 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 both on their ability to bind the immobilized receptor and to trigger GHSR conformational changes using the fluorescence energy transfer (FRET)-based assay. 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.

Domaines: Polymères

Michaël Bosco, Marjorie Damian, Vinay Chauhan, Mélanie Roche, Pierre Guillet, et al.. Biotinylated non-ionic amphipols for GPCR ligands screening. Methods, 2020, 180, pp.69 - 78. ⟨10.1016/j.ymeth.2020.06.001⟩. ⟨hal-03009212v2⟩