Native adenosine A_2A receptor solubilisation by a library of amphipathic copolymers

Polymer nanodiscs are a research tool that allows membrane proteins (MPs) to be encapsulated by a surrounding amphipathic polymer, isolated and studied to understand their structural and physiological properties. An advantage of using polymer nanodiscs over other membrane mimetics can be found in their ability to natively solubilise membrane proteins (MPs) within an annulus of cellular phospholipids, however, potential polymer interactions with membrane constituents can hinder MP activity making the selection of a suitable polymer critical. This work demonstrates the native solubilisation of G-protein coupled A_2A adenosine receptor (A_2AR) by polymers with alternating units and cationic charge, poly(N-methyl-4-vinyl pyridinium iodide-co-N-alkyl-maleimides) (poly(MVP-co-AlkylMs)), and novel statistical copolymers with pseudozwitterionic charge, poly(potassium 3-sulfopropyl methacrylate-co-2-(trimethyl-amino) ethyl methacrylate-co-n-butyl methacrylate) (poly(KSPMA-co-TMAEMA-co-BMA)), both synthesised using RAFT polymerisation. After surveying a library of polymers within each class, A_2AR extraction was the most efficient using poly(MVP-co-BM) (1 : 1 MVP : BM) and poly(KSPMA-co-TMAEMA-co-BMA) (1 : 1 : 1 KSPMA : TMAEMA : BMA). The optimal pH, temperature, solubilisation time, polymer concentration and ionic strength conditions required for extracting A_2AR were identified and enabled a large-scale A_2AR-nanodisc preparation. The yield of A_2AR-poly(MVP-co-BM) was superior to A_2AR-poly(KSPMA-co-TMAEMA-co-BMA) nanodiscs after affinity purification. Functional assessment of the reconstituted receptors was undertaken using fluorescence correlation spectroscopy (FCS) to determine the ligand binding capacity of A_2AR stabilised within an alternating cationic poly(MVP-co-BM). These native nanodiscs retained their ability to specifically bind A_2AR ligand antagonists.