The deposition of coassemblies made of the small presynaptic protein, α-synuclein, and lipids in the brains of patients is the hallmark of Parkinson’s disease. In this study, we used natural abundance 13C and 31P magic-angle spinning nuclear magnetic resonance spectroscopy together with cryo-electron microscopy and differential scanning calorimetry to characterize the fibrils formed by α-synuclein in the presence of vesicles made of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine or 1,2-dilauroyl-sn-glycero-3-phospho-L-serine. Our results show that these lipids coassemble with α-synuclein molecules to give thin and curly amyloid fibrils. The coassembly leads to slower and more isotropic reorientation of lipid molecular segments and a decrease in both the temperature and enthalpy of the lipid chain-melting compared with those in the protein-free lipid lamellar phase. These findings provide new insights into the properties of lipids within protein–lipid assemblies that can be associated with Parkinson’s disease.
Cooperative binding is a key feature of metabolic pathways, signaling, and transport processes. It provides tight regulation over a narrow concentration interval of a ligand, thus enabling switching to be triggered by small concentration variations. The data presented in this work reveal strong positive cooperativity of α-synuclein binding to phospholipid membranes. Fluorescence cross-correlation spectroscopy, confocal microscopy, and cryo -TEM results show that in excess of vesicles α-synuclein does not distribute randomly but binds only to a fraction of all available vesicles. Furthermore, α-synuclein binding to a supported lipid bilayer observed with total internal reflection fluorescence microscopy displays a much steeper dependence of bound protein on total protein concentration than expected for independent binding. The same phenomenon was observed in the case of α-synuclein binding to unilamellar vesicles of sizes in the nm and μm range as well as to flat supported lipid bilayers, ruling out that nonuniform binding of the protein is governed by differences in membrane curvature. Positive cooperativity of α-synuclein binding to lipid membranes means that the affinity of the protein to a membrane is higher where there is already protein bound compared to a bare membrane. The phenomenon described in this work may have implications for α-synuclein function in synaptic transmission and other membrane remodeling events.
α-synuclein is a small neuronal protein that reversibly associates with lipid membranes. The membrane interactions are believed to be central to the healthy function of this protein involved in synaptic plasticity and neurotransmitter release. α-synuclein has been speculated to induce vesicle fusion as well as fission, processes which are analogous to each other but proceed in different directions and involve different driving forces. In the current work, we analyze α-synuclein-induced small unilamellar vesicle deformation from a thermodynamics point of view. We show that the structures interpreted in the literature as fusion intermediates are in fact a stable deformed state and no fusion nor vesicle clustering occurs. We speculate on the driving force for the observed deformation and put forward a hypothesis that α-synuclein self-assembly on the lipid membrane precedes and induces membrane remodeling.
The presence of amyloid fibrils is a hallmark of several neurodegenerative diseases. Some amyloidogenic proteins, such as α-synuclein and amyloid β, can interact with lipids, and this interaction can strongly favor the formation of amyloid fibrils. In particular the primary nucleation step, i.e. the de novo formation of amyloid fibrils, has been shown to be accelerated by lipids. However, the exact mechanism of this acceleration is still mostly unclear. Here we use a range of scattering methods, such as dynamic light scattering (DLS) and small angle X-ray and neutron scattering (SAXS and SANS) to obtain structural information on the binding of α-synuclein to vesicles formed from negatively charged lipids and their co-assembly into amyloid fibrils. We find that the lipid vesicles do not simply act as a surface that catalyses the nucleation reaction, but that lipid molecules take an active role in the reaction. The binding of α-synuclein to the lipid vesicles immediately induces a major structural change in the lipid assembly, which leads to a break-up into small, cylindrical and disc-like lipid-protein particles. This transition can be largely reversed by temperature changes or proteolytic protein removal. Incubation of these small, cylindrical and disc-like lipid-α-synuclein particles for several hours, however, yields amyloid fibril formation, whereby the lipids are incorporated into the fibrils.
Interactions of lipid vesicles play important roles in a large variety of functions and dysfunctions in the human body. Vital for several biochemical functions is the interaction between monomeric proteins and lipid membranes, and the induced phenomena such as fusion between vesicles and cell membranes, lipid exchange between the membranes, or vesicle fission. Identification of single events and their frequency of occurrence would provide valuable information about protein-lipid interactions in both healthy and degenerative pathways. In this work, we present a single-vesicle intensity and colocalization fluorescence microscopy assay with a custom-written MATLAB analysis program. The assay can be used to study lipid exchange as well as vesicle fusion and fission between two vesicle populations labeled with different fluorescent dyes. Vesicles from the two populations are first mixed and docked to a glass surface. The sample is then simultaneously imaged using two separate wavelength channels monitoring intensity changes and colocalization of vesicles from the two populations. The monomeric pre-synaptic protein α-synuclein (α-syn) and small unilamellar vesicles consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, (DOPS), and monosialotetrahexosylganglioside (GM1) were used as a model system to evaluate the method. From our analysis, neither α-syn induced fusion nor lipid exchange was observed for vesicles consisting of DOPC:DOPS (7:3). However, including 10% GM1 in the vesicles resulted in a 91% increase of the number of vesicles within 10 min, combined with a 57% decrease in the average fluorescence intensity per vesicle, indicating that approximately half of the vesicles underwent fission. The method facilitates the study of lipid vesicle fusion, fission, and lipid exchange under controlled conditions. It also allows these events to be studied for systems with more complex composition including exosomes and lipid-based drug carriers, to enable a better understanding of their physicochemical properties.
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