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The intracellular deposition of fibrillar aggregates of alpha-synuclein is a characteristic feature of Parkinson disease. Alternatively, as a result of its unusual conformational plasticity, alpha-synuclein may fold into an amphipathic helix upon contact with a lipid-water interface. Using spin label ESR and fluorescence spectroscopy, we show here that alpha-synuclein affects the lipid packing in small unilamellar vesicles. The ESR hyperfine splittings of spin-labeled phospholipid probes revealed that alpha-synuclein induces chain ordering at carbon 14 of the acyl chains below the chain melting phase transition temperature but not in the liquid crystalline state of electroneutral vesicle membranes. Binding of alpha-synuclein leads to an increase in the temperature and cooperativity of the phase transition according to the fluorescence anisotropy of the hydrophobic polyene 1,6-diphenylhexatriene and of the fluorescence emission maxima of the amphiphilic probe 6-dodecanoyl-2-dimethylaminonaphthalene. Binding parameters were obtained from the fluorescence anisotropy measurements in combination with our previous determinations by titration calorimetry (Nuscher, B., Kamp, F., Mehnert, T., Odoy, S., Haass, C., Kahle, P. J., and Beyer, K. (2004) J. Biol. Chem. 279, 21966-21975). We also show that alpha-synuclein interacts with vesicle membranes containing sphingomyelin and cholesterol. We propose that the protein is capable of annealing defects in curved vesicle membranes, which may prevent synaptic vesicles from premature fusion.

A number of neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are characterized by the intracellular deposition of fibrillar aggregates that contain a high proportion of α-synuclein (αS). The interaction with the membrane-water interface strongly modulates folding and aggregation of the protein. The present study investigates the lipid binding and the coil-helix transition of αS, using titration calorimetry, differential scanning calorimetry, and circular dichroism spectroscopy. Titration of the protein with small unilamellar vesicles composed of zwitterionic phospholipids below the chain melting temperature of the lipids yielded exceptionally large exothermic heat values. The sigmoidal titration curves were evaluated in terms of a simple model that assumes saturable binding sites at the vesicle surface. The cumulative heat release and the ellipticity were linearly correlated as a result of simultaneous binding and helix folding. There was no heat release and folding of αS in the presence of large unilamellar vesicles, indicating that a small radius of curvature is necessary for the αS-membrane interaction. The heat release and the negative heat capacity of the protein-vesicle interaction could not be attributed to the coil-helix transition of the protein alone. We speculate that binding and helix folding of αS depends on the presence of defect structures in the membrane-water interface, which in turn results in lipid ordering in the highly curved vesicular membranes. This will be discussed with regard to a possible role of the protein for the stabilization of synaptic vesicle membranes.

Cells require a constant influx of free fatty acids for lipid resynthesis and metabolic energy. Fatty acids also act as second messengers and modulate channel activities. In the pancreatic beta-cell, fatty acids have both acute and chronic effects on insulin secretion. We show that the addition of fatty acid to pancreatic beta-cells in vitro produces a persistent decrease in intracellular pH, which begins immediately after the addition of fatty acid and has an exponential time course with t1/2 approximately 60 s. The pH drop can be largely reversed by the addition of albumin. The observed pH effect can be explained by passive diffusion ("flip-flop") of un-ionized fatty acid across the plasma membrane. Acidification by a fatty acid dimer and alkalinization by an alkylamine also favor the flip-flop mechanism of transport rather than a protein-mediated mechanism. Our method provides for the first time a real-time measurement of fatty acid import into cells. The significant pH change may be important in mediating some of the regulatory effects of fatty acid, such as inhibition of glycolysis.