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 (MA) 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 wild-type mice infected with Rocky Mountain Laboratories (RML) 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 MA-modified copolymer. A comparison of the infectivity of PrPSc attached to SMA lipid particles (SMALPs) 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.
Keywords: SMALP; lipid; lipid binding protein; lipid-protein interaction; membrane protein; nanotechnology; native nanodisc; neurodegenerative disease; prion; protein misfolding; styrene maleic acid copolymer; styrene-maleic acid lipid particle (SMALP).
Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
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