Specificity and Kinetics of α-Synuclein Binding to Model Membranes Determined with Fluorescent Excited State Intramolecular Proton Transfer (ESIPT) Probe

Abstract

Parkinson disease is characterized cytopathologically by the deposition in the midbrain of aggregates composed primarily of the presynaptic neuronal protein ␣-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.

Effects of impaired membrane interactions on α-synuclein aggregation and neurotoxicity

“…Binding curves generated by plotting [ θ ] MR ,222 versus the lipid concentration were analyzed by fitting to the following equation: $$R=R_0-false(R_0-R_{f}false)\frac{K_d+C+L/N-\sqrt{{false(K_d+C+L/Nfalse)}^2-4CL/N}}{2C}$$ where R is the measured [ θ ] MR ,222 at a given lipid concentration, R 0 is [ θ ] MR ,222 in the absence of lipid, R f is [ θ ] MR ,222 in the presence of saturating lipid, L is the total lipid concentration, C is the total protein concentration, K d is the apparent macroscopic dissociation equilibrium constant, and N is the binding stoichiometry (lipids/protein) (Shvadchak et al, 2011a). The data obtained for WT aSyn were fit to equation 2 via non-linear regression with R 0 , R f , K d , and N as unconstrained parameters.…”

Unraveling amyloid formation paths of Parkinson's disease protein α-synuclein triggered by anionic vesicles

“…Stronger attractive interactions will result in higher molecular order in the polar head-group region of DOPS relative to DOPG lipid layers (Lindblom et al 1991) and increased exposure of hydrophobic parts of DOPG relative to DOPS lipid bilayers. It has been previously described that αS binds with higher affinity to model membranes with defects, indicating that lipid packing strongly affects αS-membrane interactions (Nuscher et al 2004;Ouberai et al 2013;Shvadchak et al 2011a). Thus, DOPG may expose more hydrophobic areas and αS may insert deeper into the DOPG membrane than the DOPS membrane.…”

Oligomerization and Membrane-binding Properties of Covalent Adducts Formed by the Interaction of α-Synuclein with the Toxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde (DOPAL)

“…Lipid titration curves generated by plotting [] MR, 222 versus the lipid concentration were analyzed as described previously (24,25) by fitting to Equation 2,…”