α-Synuclein Interaction with Lipid Bilayer Discs

Abstract

α-Synuclein (aSyn) is a 140 residue long protein present in presynaptic termini of nerve cells. The protein is associated with Parkinson's disease, in which case it has been found to self-assemble into long amyloid fibrils forming intracellular inclusions that are also rich in lipids. Furthermore, its synaptic function is proposed to involve interaction with lipid membranes, and hence, it is of interest to understand aSyn-lipid membrane interactions in detail. In this paper we report on the interaction of aSyn with model membranes in the form of lipid bilayer discs. Using a combination of cryogenic transmission electron microscopy and small-angle neutron scattering, we show that circular discs undergo a significant shape transition after the adsorption of aSyn. When aSyn self-assembles into fibrils, aSyn molecules desorb from the bilayer discs, allowing them to recover to their original shape. Interestingly, the desorption process has an all-or-none character, resulting in a binary coexistence of circular bilayer discs with no adsorbed aSyn and deformed bilayer discs having a maximum amount of adsorbed protein. The observed coexistence is consistent with the recent finding of cooperative aSyn adsorption to anionic lipid bilayers.

Figure 1

(a) Cryo-TEM image of a lipid bilayer disc suspension at a lipid concentration of 7.5 mM. (b) The same image as shown in panel (a) with discs highlighted with orange color for better visualization. (c) Size distribution of the lipid bilayer disc radius obtained from cryo-TEM images compared with a log-normal distribution (red line). The diameter of the tilted discs was set to be the longest axis. (d) Scattering profile of a 2.1 mM disc dispersion (black squares) and the disc model that provides the best agreement with the data (red line). The inset shows a schematic illustration of a lipid bilayer disc, where the red color indicates the lipid chain region, and the blue filled circles represent the lipid headgroups.

Figure 2

(a) Scattering profiles of a pure disc dispersion (filled black squares), of discs during the first 15 min after addition of aSyn (open red squares), and the parallelepiped model (solid red line). The best model was obtained with a length of shorter edge a = 13 nm and a length of longer edge b = 550 nm. The inset shows an illustration of the parallelepiped, where the red color illustrates the lipid chain region, and the blue represents the lipid head groups. (b) Cryo-TEM image taken 13 min after mixing lipid bilayer discs and aSyn. Arrows labeled with number 1 indicate elongated structures, and arrows labeled with number 2 indicate elongated structures whose normal is not perpendicular to the plane of view. (c) The same image as shown in panel b with deformed lipid bilayer discs highlighted in orange for better visualization. Scale bars in panels b and c correspond to 200 nm. The protein and lipid concentrations in all of the samples were 0.40 and 2.1 mM, respectively.

Figure 3

(a) ThT fluorescence intensity versus time. (b) Time-resolved SANS profiles, I(q) vs q, plotted for a total time period of ca. 22 h. with a time resolution of 1 h. The inset shows a plot of the intensity obtained at q = 0.002 Å ^–1 as an additional illustration of the time evolution of the scattered intensity. (c) Scattering profiles obtained at different time points (open squares) were modeled as a superposition (solid lines) of the scattering profile obtained during the first 15 min after mixing and the pure disc dispersion. The data are shifted for easier comparison. (d) Fraction of undeformed circular lipid bilayer discs versus time. The protein and lipid concentrations in all of the samples were 0.40 mM and 2.1 mM, respectively.

Figure 4

Cryo-TEM images taken (a) 3 h after mixing, (b) 7 h after mixing, and (c) 21 h after mixing. Arrows labeled with the number 1 indicate elongated structures, arrows labelled with the number 2 elongated structures whose normal is not perpendicular to the plane of view, arrows labeled with the number 3 partially recovered structures, arrows labeled with the number 4 structures recovered to the circular shape, and arrows labeled with the number 5 aSyn fibrils. More cryo-TEM images are shown in the SI (Figure S3).

Figure 5

Illustration of adsorbed amount, Γ, versus monomer concentration. The recovery parameter reaches its equilibrium value f_eq = 0.7 at the monomer concentration which corresponds to the solubility value.

Figure 6

An illustration of the overall events suggested by the experimental data. Top: circular lipid bilayer discs (blue) are incubated with aSyn monomers (red). Bottom left: adsorption of aSyn monomers onto lipid bilayer discs is a fast process that results in formation of elongated structures that can be represented by parallelepipeds. Bottom right: aSyn desorption is a cooperative and slow process that results in a recovery of discs to their original shape.