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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 PrP^Sc The high-resolution structure of the infectious PrP^Sc 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 PrP^Sc 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.

SUBMITTER: Esmaili M

PROVIDER: S-EPMC7307199 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The membrane-embedded protein rhodopsin is widely produced in organisms as a photoreceptor showing a variety of light-dependent biological functions. To investigate its molecular features, rhodopsin is often extracted from cellular membrane lipids by a suitable detergent as "micelles." The extracted protein is purified by column chromatography and then is often reconstituted into "liposomes" by removal of the detergent. The styrene-maleic acid ("SMA") copolymer spontaneously forms nanostructures containing lipids without detergent. In this study, we applied SMA to characterize two microbial rhodopsins, a thermally stable rhodopsin, Rubrobacter xylanophilus rhodopsin (RxR), and an unstable one, Halobacterium salinarum sensory rhodopsin I (HsSRI), and evaluated their physicochemical properties in SMA lipid particles compared with rhodopsins in micelles and in liposomes. Those two rhodopsins were produced in Escherichia coli cells and were successfully extracted from the membrane by the addition of SMA (5 w/v %) without losing their visible color. Analysis by dynamic light scattering revealed that RxR in SMA lipid particles (RxR-SMA) formed a discoidal structure with a diameter of 54 nm, which was 10 times smaller than RxR in phosphatidylcholine liposomes. The small particle size of RxR-SMA allowed us to obtain scattering-less visible spectra with a high signal-to-noise ratio similar to RxR in detergent micelles composed of n-dodecyl-β-D-maltoside. High-speed atomic force microscopy revealed that a single particle contained an average of 4.1 trimers of RxR (12.3 monomers). In addition, RxR-SMA showed a fast cyclic photoreaction (k = 13 s-1) comparable with RxR in phosphatidylcholine liposomes (17 s-1) but not to RxR in detergent micelles composed of n-dodecyl-β-D-maltoside (0.59 s-1). By taking advantage of SMA, we determined the dissociation constant (Kd) of chloride for HsSRI as 34 mM. From these results, we conclude that SMA is a useful molecule forming a membrane-mimicking assembly for microbial rhodopsins having the advantages of detergents and liposomes.

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

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