Effects of curvature and composition on α-synuclein binding to lipid vesicles

Abstract

Parkinson's disease is characterized by the presence of intracellular aggregates composed primarily of the neuronal protein α-synuclein (αS). Interactions between αS and various cellular membranes are thought to be important to its native function as well as relevant to its role in disease. We use fluorescence correlation spectroscopy to investigate binding of αS to lipid vesicles as a function of the lipid composition and membrane curvature. We determine how these parameters affect the molar partition coefficient of αS, providing a quantitative measure of the binding energy, and calculate the number of lipids required to bind a single protein. Specific anionic lipids have a large effect on the free energy of binding. Lipid chain saturation influences the binding interaction to a lesser extent, with larger partition coefficients measured for gel-phase vesicles than for fluid-phase vesicles, even in the absence of anionic lipid components. Although we observe variability in the binding of the mutant proteins, differences in the free energies of partitioning are less dramatic than with varied lipid compositions. Vesicle curvature has a strong effect on the binding affinity, with a >15-fold increase in affinity for small unilamellar vesicles over large unilamellar vesicles, suggesting that αS may be a curvature-sensing protein. Our findings provide insight into how physical properties of the membrane may modulate interactions of αS with cellular membranes.

Figure 1

Schematic of αS illustrating three major regions. Shown are the three PD-associated mutations (A30P, E46K, and A53T) and the site of the cysteine mutation used for labeling (E130C).

Figure 2

Binding of αS to vesicles. (A) Normalized autocorrelation curves show a shift to longer times as αS binds to vesicles (from left to right with increasing lipid concentration). (B) αS binding curve derived from FCS measurements using Eq. 1 in the text (squares) is fit well by a hyperbolic equation (Eq. 2 in the text; line).

Figure 3

Molar partition coefficients (K_p) of αS. Comparison of K_p values measured for (A) WT αS with 93 nm LUVs of six different lipid compositions, (B) WT with 1:1 POPS/POPC vesicles covering a range of diameters, and (C) WT and disease-associated αS mutants with 93 nm 1:1 POPS/POPC vesicles.

Figure 4

Determination of the number of lipids in a binding site. αS was added to 93 nm (squares) or 46 nm (circles) 1:1 POPS/POPC vesicles at pH 5. The curve saturates at an accessible lipid/bound protein molar ratio of ∼20:1 (dashed line).