Native nanodiscs formed by styrene maleic acid copolymer derivatives help recover infectious prion multimers bound to brain-derived lipids

Abstract

Prions are lipidated proteins that interact with endogenous lipids and metal ions. They also assemble into multimers and propagate into the infectious scrapie form known as PrPSc. The high-resolution structure of the infectious PrPSc state remains unknown, and its analysis largely relies on detergent-based preparations devoid of endogenous ligands. Here we designed polymers that allow isolation of endogenous membrane:protein assemblies in native nanodiscs without exposure to conventional detergents that destabilize protein structures and induce fibrillization. A set of styrene–maleic acid (SMA) polymers including a methylamine derivative facilitated gentle release of the infectious complexes for resolution of multimers, and a thiol-containing version promoted crystallization. Polymer extraction from brain homogenates from Syrian hamsters infected with Hyper prions and WT mice infected with Rocky Mountain Laboratories prions yielded infectious prion nanoparticles including oligomers and microfilaments bound to lipid vesicles. Lipid analysis revealed the brain phospholipids that associate with prion protofilaments, as well as those that are specifically enriched in prion assemblies captured by the methylamine-modified copolymer. A comparison of the infectivity of PrPSc attached to SMA lipid particles in mice and hamsters indicated that these amphipathic polymers offer a valuable tool for high-yield production of intact, detergent-free prions that retain in vivo activity. This native prion isolation method provides an avenue for producing relevant prion:lipid targets and potentially other proteins that form multimeric assemblies and fibrils on membranes.

Extraction and reconstitution of membrane proteins into lipid nanodiscs encased by zwitterionic styrene-maleic amide copolymers

“…Buffer incompatibilities of SMAs and SMALPs constitute a major problem when polyvalent cations at mM concentration or low pH are needed 14,[17][18][19] . Newer copolymers that offer distinct advantages over the traditional SMA copolymers have been studied recently 19,[47][48][49][50] . Diisobutylene/maleic acid copolymer (DIBMA), poly(styrene-co-maleimide) (SMI), styrene maleimide quaternary ammonium (SMA-QA) and our zSMAs are compatible with high concentrations of divalent cations 19,[47][48][49] .…”

Screening potential P-glycoprotein inhibitors by combination of a detergent-free membrane protein extraction with surface plasmon resonance biosensor

“…In recent years, styrene maleic acid (SMA) polymer is more and more widely used in the extraction and purification of MPs. SMA can realize self-assembly in cell membrane: SMA ring surrounds lipids, forms a disk-shaped structure of layered membrane, and produces very uniform particles with a diameter of 9 to 20 nm 13 , 14 . The advantage of SMA over detergents and artificial membranes is that it extracts the target protein into SMA lipid particles (SMALPs) by recognizing the spontaneous assembly of endogenous lipids around the target protein, and the protein is thus retained in the environment of natural lipid bilayer.…”

Membrane protein extraction and purification using partially-esterified SMA polymers

“…This agrees with a recent study showing that increasing levels of esterification of SMA with butoxyethanol causes increased sensitivity to Mg 2+ [46] . In contrast, the modification of a 1:1 styrene:maleic acid polymer by reaction of methylamine with the maleic anhydride, forming an amide derivative ( SMAma ) rather than an ester, does indeed show increased tolerance to divalent cations [48] . Overall, it seems that the divalent cation sensitivity is more complex than previously thought.…”