Biotinylated RAFT Styrene/Maleic Acid Copolymers for Biosensor Applications of Membrane Proteins

We report the RAFT polymerization of biotinylated styrene/maleic anhydride (SMAnh) copolymers using a biotin-PEGn-modified transfer agent. These polymers exhibit degrees of polymerization ranging from 32 to 45 and narrow molar mass distributions from 4000 to 7000 g mol–1, with a dispersity (D̵) around 1.20. Hydrolysis of SMAnh yielded water-soluble styrene/maleic acid (SMA) copolymers, which formed micelles with diameters of 9–13 nm on average. 1:1 mixtures of biotinylated and nonbiotinylated SMA copolymers were found to solubilize vesicles made from fully saturated zwitterionic phospholipids to form lipid-bilayer nanodiscs. These polymer mixtures were further tested using proteoliposomes composed of unsaturated zwitterionic and anionic phospholipids containing 10 mol % cholesterol as well as melanocortin 2 receptor accessory protein 2 (MRAP2) or human ghrelin receptor (GHSR) as monotopic and polytopic transmembrane proteins, respectively. Mixtures of biotinylated and nonbiotinylated SMA were similarly efficient in solubilizing these proteoliposomes as SMA and superior to di-isobutylene maleic acid (DIBMA) copolymer. After solubilization, the resulting biotinylated nanodiscs were efficiently and specifically immobilized onto streptavidin-coated surfaces, as demonstrated by surface plasmon resonance (SPR) spectroscopy. Crucially, immobilizing nanodiscs using biotinylated SMA did not impair the pharmacology properties of GHSR. From a chemical viewpoint, our approach ensures homogeneous polymer end-chain functionalization, overcoming limitations associated with postpolymerization modification. From an application viewpoint, biotinylated SMA copolymers offer a robust and versatile platform for immobilizing membrane proteins within their native lipid environment for biosensing and ligand screening applications without the need to modify proteins directly.