Abstract
The aggregation of alpha-synuclein is believed to be a critical factor in the etiology of Parkinson's disease. alpha-Synuclein is an abundant neuronal protein of unknown function, which is enriched in the presynaptic terminals of neurons. Although alpha-synuclein is found predominantly in the cytosolic fractions, membrane-bound alpha-synuclein has been suggested to play an important role in fibril formation. The effects of alpha-synuclein on lipid bilayers of different compositions were determined using fluorescent environment-specific probes located at various depths. alpha-Synuclein-membrane interactions were found to affect both protein and membrane properties. Our results indicate that in addition to electrostatic interactions, hydrophobic interactions are important in the association of the protein with the bilayer, and lead to disruption of the membrane. The latter was observed by atomic force microscopy and fluorescent dye leakage from vesicles. The kinetics of alpha-synuclein fibril formation were significantly affected by the protein association and subsequent membrane disruption, and reflected the conformation of alpha-synuclein. The ability of alpha-synuclein to disrupt membranes correlated with the binding affinity of alpha-synuclein for the particular membrane composition, and to the induced helical conformation of alpha-synuclein. Protofibrillar or fibrillar alpha-synuclein caused a much more rapid destruction of the membrane than soluble monomeric alpha-synuclein, indicating that protofibrils (oligomers) or fibrils are likely to be significantly neurotoxic.
Highlights
Parkinson’s disease (PD)1 is the second most common neurodegenerative disease in the United States, currently affecting as many as 1.5 million individuals
Our results indicate that in addition to electrostatic interactions, hydrophobic interactions are important in the association of the protein with the bilayer, and lead to disruption of the membrane
It is likely that interactions between hydrophobic residues of the amphipathic ␣-helices of ␣-synuclein play an important role in the association of the protein to the membrane
Summary
Expression and Purification of Protein—Recombinant ␣-synuclein was expressed (in Escherichia coli) and purified as described preciously [5], and stored at Ϫ80 °C. Protein was mixed with pre-formed SUVs or LUVs, and incubated for 20 min before circular dichroism, fluorescence, or kinetic measurements. Aliquots of ␣-synuclein, protofibrils, or fibrils of ␣-synuclein were titrated directly into the fluorescence cuvette, and measurements were taken after incubating the solution for 20 min. The change in the fluorescence intensity was monitored at 510 nm (excitation 490 nm) after incubating ␣-synuclein and dye-containing vesicles at 25 °C in 20 mM Tris-HCl buffer, 100 mM NaCl, pH 7.4, until the dye release reached its end point, around 30 min. Preparation of Planar Phospholipid Bilayer for AFM Measurements—Freshly cleaved mica was hydroxylated by submerging it in a solution consisting of concentrated H2SO4 (95 vol %) and 50 mM potassium dichromate for 20 min, rinsed with water, and dried with a stream of nitrogen. Fibrils were prepared using centrifugation at the completion of fibrillation, followed by washing the pellet twice in buffer, a procedure that eliminates soluble oligomers
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