Abstract
Parkinson disease is characterized cytopathologically by the deposition in the midbrain of aggregates composed primarily of the presynaptic neuronal protein α-synuclein (AS). Neurotoxicity is currently attributed to oligomeric microaggregates subjected to oxidative modification and promoting mitochondrial and proteasomal dysfunction. Unphysiological binding to membranes of these and other organelles is presumably involved. In this study, we performed a systematic determination of the influence of charge, phase, curvature, defects, and lipid unsaturation on AS binding to model membranes using a new sensitive solvatochromic fluorescent probe. The interaction of AS with vesicular membranes is fast and reversible. The protein dissociates from neutral membranes upon thermal transition to the liquid disordered phase and transfers to vesicles with higher affinity. The binding of AS to neutral and negatively charged membranes occurs by apparently different mechanisms. Interaction with neutral bilayers requires the presence of membrane defects; binding increases with membrane curvature and rigidity and decreases in the presence of cholesterol. The association with negatively charged membranes is much stronger and much less sensitive to membrane curvature, phase, and cholesterol content. The presence of unsaturated lipids increases binding in all cases. These findings provide insight into the relation between membrane physical properties and AS binding affinity and dynamics that presumably define protein localization in vivo and, thereby, the role of AS in the physiopathology of Parkinson disease.
Highlights
The precise causes of cell death are unknown, the misfolding and aggregation of the abundant neuronal protein, ␣-synuclein (AS),4 in Parkinson disease pathogenesis is almost certainly involved [2,3,4]. This protein forms amyloid-like fibrils, which constitute the major component of pathological intraneuronal protein deposits denoted Lewy bodies
We have previously reported that FE reveals differences in the supramolecular organization of amyloid fibrils [55] and the utility of a related excited state intramolecular proton transfer (ESIPT) probe (FC) as a reporter of AS aggregation [56]
Increased levels of AS in neurons induce a defect in the reclustering of synaptic vesicles after endocytosis [6] and endoplasmic reticulum-Golgi vesicle trafficking [5]
Summary
Protein Expression and Purification—␣-Synuclein and its cysteine variants (A18C, A90C, and A140C) were expressed and purified from Escherichia coli as described elsewhere [56] (for preparation details, see supplemental material). Where FDPPC, and FDOPG, are reference spectra of the protein in DPPC and DOPG SUVs, respectively, and a is the molar fraction of AS bound to DPPC SUVs (supplemental Fig. S1) Because both lipids were present in the solution in the same concentration, the ratio a/(1 Ϫ a) is equal to the ratio of the corresponding dissociation equilibrium constants for AS, KDOPG/KDPPC. This method works well for comparison of protein binding to two types of membranes with high and comparable affinities. For recording of the initial fluorescence level (Fo), the solution of DOPG was substituted by buffer
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