Abstract
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.
Highlights
Ceded by assembly into protofibrillar aggregation intermediates as shown by chromatography and by an investigation of the aggregation kinetics [9]
We have shown recently, using isothermal titration calorimetry and CD spectroscopy, that the protein interacts strongly with small unilamellar vesicles (SUV) composed of neutral phospholipids such as 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) below the respective chain melting temperatures (Tm) [15]
Titration of the vesicles at T Ͻ Tm into an ␣S solution leads to a considerable heat release that is accompanied by a coil-helix transition as shown by CD spectroscopy
Summary
Chemicals—DMPC, DPPC, 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC), egg sphingomyelin, phosphatidylcholine spin labels 1-palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3-phosphocholine (5-PCSL), and 1-palmitoyl-2-stearoyl-(14-doxyl)-sn-glycero-3-phosphocholine (14-PCSL) were purchased from Avanti Polar Lipids (Alabaster, AL). Vesicle Preparation—Phospholipids (20 mg) were hydrated in 1 ml of buffer containing 20 mM sodium phosphate, 100 mM KCl, and 0.2 g/liter NaN3 (pH 7.20) for 15 min at 30 °C. SUV were prepared by sonication of the phospholipid suspensions for 20 min under argon at temperatures ϳ5 °C above the respective transition temperature (Tm) of the lipids. Samples containing ␣S were prepared by mixing 50 l of the vesicle suspension (25 mM phospholipid) with 91 g of the lyophilized protein to obtain a lipid/protein molar ratio of 200:1. Excitation and emission wavelengths of 360 and 430 nm, respectively, were employed for measurements of the fluorescence anisotropy of DPH. The steady-state fluorescence anisotropy is defined as r ϭ (Iʈ Ϫ IЌ)/(Iʈ ϩ 2IЌ), where the symbols ʈ and Ќ refer to light intensity obtained with parallel and perpendicular polarizers, respectively. The temperature was controlled with an accuracy of Ϯ0.5 °C
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