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.
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.
Membrane transport of small, non-polar gas molecules, such as O 2 and CO 2 , is among fundamental processes of living cells. Because of their size and chemical properties, these molecules may unimpededly transit through the lipid phase of the membrane. However, varying degrees of gas permeability in membranes with different lipid compositions have been reported experimentally, suggesting involvement of protein channels, particularly aquaporins (AQPs), in gas transport across membranes. To account for membrane heterogeneity and to probe potential roles of AQPs in gas transport, we performed extensive MD simulations of gas diffusion through lipid membranes. The systems simulated included POPC bilayers embedded with an AQP tetramer (i.e., AQP1, AQP5 or AQP7), and protein-free lipid bilayer mixtures of POPC, cholesterol (CHL) and sphingomyelin (SM) or DPPC. Each of them contained an excess amount (125 molecules) of a gas species and was simulated for several hundred nanoseconds. Since the gas molecules were allowed to diffuse freely, their permeability coefficients were directly estimated by measuring their flow through the lipid and protein phases. The results showed that gas molecules diffused through the membrane via monomeric water and hydrophobic central pores of AQPs, and via the lipid phase. For fluid-like membranes (e.g., 100% POPC) which exhibited high gas permeability, AQPs would not facilitate the permeation. Highly-ordered or gel-like membranes (e.g., 100% SM and 50:50 SM:CHL or DPPC:CHL), on the other hand, exhibited reduced gas permeability as low as or even lower than through AQPs, suggesting that in such conditions, AQPs would become imminent in facilitating gas transport. Consistently with experimentally observed low gas permeability in high SM-CHL containing membranes, such as those of erythrocytes and ocular lens, our study suggests biological significance of protein-facilitated gas permeation across membranes.
The discovery that alpha-synuclein, an intrinsically disordered protein, was linked to a multitude of neurodegenerative diseases (''synucleinopathies'') prompted intense efforts to understand the function of alpha-synuclein. While many hypotheses centrally involve the ability of alpha-synuclein to bind to highly-curved lipid membranes, it is difficult to reconcile the diversity of sub-neuronal organelles (and corresponding lipid membranes) with which alpha-synuclein is purported to interact. To that end, we have pioneered a generalizable technique to encapsulate uniform spherical-nanoparticles with a single membrane (with lipid composition of our choosing), creating an in vitro probe with precise membrane curvature in a previously un-realizable biological limit (35-50 nm). Using isothermal titration calorimetry we report the binding affinities of alpha-synuclein variants as a function of increasing membrane curvature and lipid composition. While previous measurements required an un-physiological high mol% of anionic lipids to recapitulate strong alpha-synuclein binding, we demonstrate the complex interplay between lipid charge, lipid intrinsic curvature, and membrane curvature to tease out the biochemical and biophysical parameters that drive alpha-synuclein binding. These parameters will be correlated to states in the dynamic lipid composition of sub-neuronal organelles, providing the data necessary to develop hypotheses on the molecular action and dysfunction of alpha-synuclein within the neuron.
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