Design of zwitterionic fluorescent polymers for membrane protein solubilization into native nanodiscs

Copolymers formed by non-alternating distributions of styrene and maleic acid monomers directly solubilize intact membranes into ∼10 nm discs. However, these copolymers are inherently polydisperse in terms of polymer structure, difficult to detect, prone to precipitation with divalent cations, and have limited working pH ranges due to their charges. The exposed polar sidechain of nanodisc-forming amphipathic copolymers provides a handle for integrating critical chemical features for facile solubilization, purification, detection, and resolution of diverse membrane protein complexes, including 7-transmembrane G-Protein-Coupled Receptor (GPCR) and beta-barrel proteins directly from cellular material. Here, we report that when derivatized with amine oxide (AO) moieties, alternating and intrinsically fluorescent derivatives of poly(styrene-alt-maleic anhydride) (SMAnh) spontaneously convert biological membranes into nanodiscs with diameters of 15-30 nm that can be resolved by dynamic light scattering and electron microscopy. Compared to non-alternating poly(styrene-co-maleic acid) (SMA), their fluorescence signals allow monitoring under diverse solution conditions, whether free or lipid bilayer-bound. These copolymers are useful in a broad pH range, are tolerant of high levels of divalent cations (>200 mM CaCl₂) and are designed to reduce undesirable nonspecific interactions. The resulting nanodiscs can accommodate the PagP palmitoyltransferase expressed in Escherichia coli outer membranes and the human adenosine A_2A receptor expressed into Pichia pastoris membranes, resulting in readily purified proteins that are less likely to be perturbed by polymer charge or hydrophobicity.