alpha-synuclein and 1-palmitoyl-2-oleoylglycero-3-phosphoglycerol

The neuronal protein α-synuclein, linked to Parkinson's disease, binds to negatively charged vesicles adopting a partial α-helix structure, but helix arrangement at the vesicle surface is not fully understood. Using linear dichroism spectroscopy (LD), we study the interaction of monomeric α-synuclein with large unilamellar vesicles of 1,2-dioleoyl-

Topics: alpha-Synuclein; Amino Acid Sequence; Phosphatidylglycerols; Phosphatidylserines; Protein Binding; Protein Conformation, alpha-Helical; Unilamellar Liposomes

Both normal and pathological functions of α-synuclein (αSN), an abundant protein in the central and peripheral nervous system, have been linked to its interaction with membrane lipid bilayers. The ability to characterize structural transitions of αSN upon membrane complexation will clarify molecular mechanisms associated with αSN-linked pathologies, including Parkinson's disease (PD), multiple systems atrophy, and other synucleinopathies. In this work, time-resolved electrospray ionization hydrogen/deuterium exchange mass spectrometry (TRESI-HDX-MS) was employed to acquire a detailed picture of αSN's conformational transitions as it undergoes complexation with nanodisc membrane mimics with different headgroup charges (zwitterionic DMPC and negative POPG). Using this approach, αSN interactions with DMPC nanodiscs were shown to be rapid exchanging and to have little impact on the αSN conformational ensemble. Interactions with nanodiscs containing lipids known to promote amyloidogenesis (e.g., POPG), on the other hand, were observed to induce substantial and specific changes in the αSN conformational ensemble. Ultimately, we identify a region corresponding residues 19-28 and 45-57 of the αSN sequence that is uniquely impacted by interactions with "amyloidogenic" lipid membranes, supporting the existing "broken-helix" model for α-synuclein/membrane interactions, but do not detect a "helical extension" that is also thought to play a role in αSN aggregation.

Topics: alpha-Synuclein; Chromatography, Gel; Dimyristoylphosphatidylcholine; Humans; Hydrogen Deuterium Exchange-Mass Spectrometry; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Lipids; Models, Chemical; Nanostructures; Phosphatidylglycerols; Phospholipids; Protein Conformation; Spectrometry, Mass, Electrospray Ionization

Topics: alpha-Synuclein; Gold; Immobilized Proteins; Membrane Lipids; Phosphatidylglycerols; Polyethylene Glycols; Protein Binding; Spectrophotometry, Infrared; Surface Properties

Interactions of monomeric alpha-synuclein (αS) with lipid membranes have been suggested to play an important role in initiating aggregation of αS. We have systematically analyzed the distribution and self-assembly of monomeric αS on supported lipid bilayers. We observe that at protein/lipid ratios higher than 1:10, αS forms micrometer-sized clusters, leading to observable membrane defects and decrease in lateral diffusion of both lipids and proteins. An αS deletion mutant lacking amino-acid residues 71-82 binds to membranes, but does not observably affect membrane integrity. Although this deletion mutant cannot form amyloid, significant amyloid formation is observed in the wild-type αS clusters. These results suggest that the process of amyloid formation, rather than binding of αS on membranes, is crucial in compromising membrane integrity.

Topics: Adsorption; alpha-Synuclein; Amyloid; Benzothiazoles; Lipid Bilayers; Liposomes; Mutant Proteins; Phosphatidylcholines; Phosphatidylglycerols; Protein Aggregates; Protein Binding; Staining and Labeling; Thiazoles

We have investigated the membrane remodeling capacity of the N-terminal membrane-binding domain of α-synuclein (α-Syn100). Using fluorescence correlation spectroscopy and vesicle clearance assays, we show that α-Syn100 fully tubulates POPG vesicles, the first demonstration that the amphipathic helix on its own is capable of this effect. We also show that at equal density of membrane-bound protein, α-Syn has dramatically reduced affinity for, and does not tubulate, vesicles composed of a 1:1 POPG:POPC mixture. Coarse-grained molecular dynamics simulations suggested that the difference between the pure POPG and mixture results may be attributed to differences in the protein's partition depth, the membrane's hydrophobic thickness, and disruption of acyl chain order. To explore the importance of these attributes compared with the role of the reduced binding energy, we created an α-Syn100 variant in which we removed the hydrophobic core of the non-amyloid component (NAC) domain and tested its impact on pure POPG vesicles. We observed a substantial reduction in binding affinity and tubulation, and simulations of the NAC-null protein suggested that the reduced binding energy increases the protein mobility on the bilayer surface, likely impacting the protein's ability to assemble into organized pretubule structures. We also used simulations to explore a potential role for interleaflet coupling as an additional driving force for tubulation. We conclude that symmetry across the leaflets in the tubulated state maximizes the interaction energy of the two leaflets and relieves the strain induced by the hydrophobic void beneath the amphipathic helix.

Topics: alpha-Synuclein; Lipids; Membranes, Artificial; Phosphatidylcholines; Phosphatidylglycerols

The fibrillization of α-synuclein (α-syn) is involved in Parkinson's disease, a neurodegenerative disorder that affects four million people in the world. The amino acid sequence 71-82 of this protein (VTGVTAVAQKTV) has appeared to be essential for fibril formation. In the present study, we have investigated the secondary structure and thermal stability of the peptide fragment 71-82, α-syn71-82, as a function of concentration and temperature, as well as its interactions with phospholipid model membranes using various spectroscopic techniques. The data show that α-syn71-82 is mainly disordered in solution with the presence of a few β-sheet structure elements. The peptide reversibly forms intermolecular β-sheets with increasing concentration and decreasing temperature, suggesting that it is subjected to a thermodynamic equilibrium between a monomeric and an oligomeric form. This equilibrium seems to be affected by the presence of zwitterionic membranes. Conversely, the influence of the peptide on zwitterionic lipid bilayers is small and concentration-dependent. By contrast, α-syn71-82 is strongly affected by anionic vesicles. The peptide indeed exhibits a dramatic conformational change, reflecting an extensive and irreversible self-aggregation, the majority of the amino acids being involved in a parallel β-sheet conformation. The aggregates appear to be located near the membrane surface but do not perturb significantly the membrane order. Comparing these results with the literature, it appears that α-syn71-82 shares several general properties and structural similarities with its parent protein. These common points suggest that the sequence 71-82 may overall contribute to the behavior and properties of α-syn.

Topics: alpha-Synuclein; Amyloid; Circular Dichroism; Humans; Lipid Bilayers; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Osmolar Concentration; Peptide Fragments; Phosphatidylcholines; Phosphatidylglycerols; Protein Aggregation, Pathological; Protein Conformation; Protein Interaction Domains and Motifs; Protein Stability; Protein Structure, Secondary; Protein Unfolding; Solubility; Temperature

α-Synuclein (αS) is a membrane-binding protein with sequence similarity to apolipoproteins and other lipid-carrying proteins, which are capable of forming lipid-containing nanoparticles, sometimes referred to as "discs." Previously, it has been unclear whether αS also possesses this property. Using cryo-electron microscopy and light scattering, we found that αS can remodel phosphatidylglycerol vesicles into nanoparticles whose shape (ellipsoidal) and dimensions (in the 7-10-nm range) resemble those formed by apolipoproteins. The molar ratio of αS to lipid in nanoparticles is ∼1:20, and αS is oligomeric (including trimers and tetramers). Similar nanoparticles form when αS is added to vesicles of mitochondrial lipids. This observation suggests a mechanism for the previously reported disruption of mitochondrial membranes by αS. Circular dichroism and four-pulse double electron electron resonance experiments revealed that in nanoparticles αS assumes a broken helical conformation distinct from the extended helical conformation adopted when αS is bound to intact vesicles or membrane tubules. We also observed αS-dependent tubule and nanoparticle formation in the presence of oleic acid, implying that αS can interact with fatty acids and lipids in a similar manner. αS-related nanoparticles might play a role in lipid and fatty acid transport functions previously attributed to this protein.

Topics: alpha-Synuclein; Cholesterol; Chromatography, Gel; Cryoelectron Microscopy; Fluorescence Resonance Energy Transfer; Humans; Lipoproteins; Membranes, Artificial; Mitochondrial Membranes; Nanoparticles; Particle Size; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Protein Structure, Quaternary; Protein Structure, Secondary

α-Synuclein is abundantly present in Lewy bodies, characteristic of Parkinson's disease. Its exact physiological role has yet to be determined, but mitochondrial membrane binding is suspected to be a key aspect of its function. Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling allowed for a locally resolved analysis of the protein-membrane binding affinity for artificial phospholipid membranes, supported by a study of binding to isolated mitochondria. The data reveal that the binding affinity of the N-terminus is nonuniform.

Topics: alpha-Synuclein; Cell Membrane; Electron Spin Resonance Spectroscopy; Humans; Lewy Bodies; Membranes, Artificial; Mutation; Phosphatidylcholines; Phosphatidylglycerols; Phospholipids

α-Synuclein (αS) is a protein with multiple conformations and interactions. Natively unfolded in solution, αS accumulates as amyloid in neurological tissue in Parkinson disease and interacts with membranes under both physiological and pathological conditions. Here, we used cryoelectron microscopy in conjunction with electron paramagnetic resonance (EPR) and other techniques to characterize the ability of αS to remodel vesicles. At molar ratios of 1:5 to 1:40 for protein/lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol), large spherical vesicles are converted into cylindrical micelles ~50 Å in diameter. Other lipids of the same charge (negative) exhibit generally similar behavior, although bilayer tubes of 150-500 Å in width are also produced, depending on the lipid acyl chains. At higher protein/lipid ratios, discoid particles, 70-100 Å across, are formed. EPR data show that, on cylindrical micelles, αS adopts an extended amphipathic α-helical conformation, with its long axis aligned with the tube axis. The observed geometrical relationship between αS and the micelle suggests that the wedging of its long α-helix into the outer leaflet of a membrane may cause curvature and an anisotropic partition of lipids, leading to tube formation.

Topics: alpha-Synuclein; Electron Spin Resonance Spectroscopy; Humans; Lipid Bilayers; Micelles; Parkinson Disease; Phosphatidylglycerols; Protein Folding; Protein Structure, Secondary

The question of how the aggregation of the neuronal protein α-synuclein contributes to neuronal toxicity in Parkinson's disease has been the subject of intensive research over the past decade. Recently, attention has shifted from the amyloid fibrils to soluble oligomeric intermediates in the α-synuclein aggregation process. These oligomers are hypothesized to be cytotoxic and to permeabilize cellular membranes, possibly by forming pore-like complexes in the bilayer. Although the subject of α-synuclein oligomer-membrane interactions has attracted much attention, there is only limited evidence that supports the pore formation by α-synuclein oligomers. In addition the existing data are contradictory.. Here we have studied the mechanism of lipid bilayer disruption by a well-characterized α-synuclein oligomer species in detail using a number of in vitro bilayer systems and assays. Dye efflux from vesicles induced by oligomeric α-synuclein was found to be a fast all-or-none process. Individual vesicles swiftly lose their contents but overall vesicle morphology remains unaltered. A newly developed assay based on a dextran-coupled dye showed that non-equilibrium processes dominate the disruption of the vesicles. The membrane is highly permeable to solute influx directly after oligomer addition, after which membrane integrity is partly restored. The permeabilization of the membrane is possibly related to the intrinsic instability of the bilayer. Vesicles composed of negatively charged lipids, which are generally used for measuring α-synuclein-lipid interactions, were unstable to protein adsorption in general.. The dye efflux from negatively charged vesicles upon addition of α-synuclein has been hypothesized to occur through the formation of oligomeric membrane pores. However, our results show that the dye efflux characteristics are consistent with bilayer defects caused by membrane instability. These data shed new insights into potential mechanisms of toxicity of oligomeric α-synuclein species.

Topics: Adsorption; alpha-Synuclein; Amyloid; Cell Membrane; Dextrans; Humans; Lipid Bilayers; Lipids; Microscopy, Confocal; Neurons; Permeability; Phosphatidylglycerols; Proteins

Soluble oligomeric aggregates of alpha-synuclein have been implicated to play a central role in the pathogenesis of Parkinson's disease. Disruption and permeabilization of lipid bilayers by alpha-synuclein oligomers is postulated as a toxic mechanism, but the molecular details controlling the oligomer-membrane interaction are still unknown. Here we show that membrane disruption strongly depends on the accessibility of the hydrophobic membrane core and that charge interactions play an important but complex role. We systematically studied the influence of the physical membrane properties and solution conditions on lipid bilayer disruption by oligomers using a dye release assay. Varying the lipid headgroup composition revealed that membrane disruption only occurs for negatively charged bilayers. Furthermore, the electrostatic repulsion between the negatively charged alpha-synuclein and the negative surface charge of the bilayer inhibits vesicle disruption at low ionic strength. The disruption of negatively charged vesicles further depends on lipid packing parameters. Bilayer composition changes that result in an increased lipid headgroup spacing make vesicles more prone to disruption, suggesting that the accessibility of the bilayer hydrocarbon core modulates oligomer-membrane interaction. These data shed important new insights into the driving forces governing the highly debated process of oligomer-membrane interactions.

Topics: alpha-Synuclein; Amino Acid Substitution; Cell Death; Electrophoresis, Polyacrylamide Gel; Humans; Kinetics; Lipid Bilayers; Parkinsonian Disorders; Phosphatidylglycerols; Spectrophotometry, Ultraviolet; Substantia Nigra

Alpha-synuclein, a presynaptic protein associated with Parkinson's disease, is found as both soluble cytosolic and membrane-bound forms. Although the function of alpha-synuclein is unknown, several observations suggest that its association with membranes is important. In the present study we investigated the effect of alpha-synuclein on lipid oxidation in membranes containing phospholipids with unsaturated fatty acids. The kinetics of lipid oxidation were monitored by the change in fluorescence intensity of the dye C11-BODIPY. We find that monomeric alpha-synuclein efficiently prevented lipid oxidation, whereas fibrillar alpha-synuclein had no such effect. Our data suggest that the prevention of unsaturated lipid oxidation by alpha-synuclein requires that it bind to the lipid membrane. The antioxidant function of alpha-synuclein is attributed to its facile oxidation via the formation of methionine sulfoxide, as shown by mass spectrometry. These findings suggest that the inhibition of lipid oxidation by alpha-synuclein may be a physiological function of the protein.

Topics: alpha-Synuclein; Antioxidants; Boron Compounds; Circular Dichroism; Fluorescence; Kinetics; Liposomes; Oxidation-Reduction; Phosphatidylcholines; Phosphatidylglycerols