Spherical nanoparticle-supported lipid bilayers (SSLBs) combine precision nanoparticle engineering with biocompatible interfaces for various applications, ranging from drug delivery platforms to structural probes for membrane proteins. Although the bulk, spontaneous assembly of vesicles and larger silica nanoparticles (>100 nm) robustly yields SSLBs, it will only occur with low charge density vesicles for smaller nanoparticles (<100 nm), a fundamental barrier in increasing SSLB utility and efficacy. Here, through whole mount and cryogenic transmission electron microscopy, we demonstrate that mixing osmotically loaded vesicles with smaller nanoparticles robustly drives the formation of SSLBs with high membrane charge density (up to 60% anionic lipid or 50% cationic lipid). We show that the osmolyte load necessary for SSLB formation is primarily a function of absolute membrane charge density and is not lipid headgroup-dependent, providing a generalizable, tunable approach toward bulk production of highly curved and charged SSLBs with various membrane compositions.

While it is generally accepted that neuronal protein α-synuclein binds to highly curved and highly charged lipid membranes, its biological function beyond binding remains unknown despite its fundamental link to Parkinson’s disease. Herein, we utilize spherical nanoparticle lipid bilayers (SSLBs) to recapitulate the charge and curvature limit of membrane organelles with which α-synuclein associates and probe how α-synuclein affects SSLB structure and dynamics as a proxy for interorganelle interactions. Small-angle X-ray scattering and X-ray photon correlation spectroscopy reveal our SSLBs form aggregates that are clearly broken up by the addition of α-synuclein, a clear indication that α-synuclein confers steric stabilization to membrane surfaces.

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While -Synuclein, an intrinsically disordered protein linked to Parkinson's disease, has been shown to associate with membrane organelles, its overall cellular function remains nebulous. -Synuclein binds to membranes through its amino-terminal domain (first ~ 100 residues), but there is no This article is protected by copyright. All rights reserved. 2 consensus on the biophysical function of the carboxyl-terminal domain (last ~ 40 residues) due, in part, to its lack of strong interaction partners and persisting intrinsic disorder even when membranebound. Here, by directly applying force on -Synuclein bound to spherical nanoparticle-supported lipid bilayers (SSLBs) and tracking higher-order structural changes through small-angle X-ray scattering, we present strong evidence that -Synuclein sterically stabilizes membrane surfaces through its carboxyl-terminal domain. Full-length -Synuclein dramatically increases the critical osmotic pressure at which SSLBs cluster (P C ~ 1.3 x 10 5 Pa) compared to -Synuclein without the carboxyl-terminal domain (P C ~ 1.9 x 10 4 Pa) at physiological salt and temperature conditions. We show this clustering of -Synuclein-bound SSLBs to be reversible and sensitive to monovalent/divalent salt, both features of grafted polyelectrolyte-mediated steric stabilization. In elucidating the biophysical function of -Synuclein in the framework of polymer science, we demonstrate that the carboxyl-terminal domain can potentially utilize its persisting intrinsic disorder to functionalize membrane surfaces.

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