Single molecule characterization of α-synuclein in aggregation-prone states

Abstract

α-Synuclein (αS) is an intrinsically disordered protein whose aggregation into ordered, fibrillar structures underlies the pathogenesis of Parkinson's disease. A full understanding of the factors that cause its conversion from soluble protein to insoluble aggregate requires characterization of the conformations of the monomer protein under conditions that favor aggregation. Here we use single molecule Förster resonance energy transfer to probe the structure of several aggregation-prone states of αS. Both low pH and charged molecules have been shown to accelerate the aggregation of αS and induce conformational changes in the protein. We find that at low pH, the C-terminus of αS undergoes substantial collapse, with minimal effect on the N-terminus and central region. The proximity of the N- and C-termini and the global dimensions of the protein are relatively unaffected by the C-terminal collapse. Moreover, although compact at low pH, with restricted chain motion, the structure of the C-terminus appears to be random. Low pH has a dramatically different effect on αS structure than the molecular aggregation inducers spermine and heparin. Binding of these molecules gives rise to only minor conformational changes in αS, suggesting that their mechanism of aggregation enhancement is fundamentally different from that of low pH.

Figure 1

Schematic of αS primary structure. αS is divided into three regions: the N-terminal membrane binding region (residues 1–100), a central hydrophobic, NAC region (61–95), and the highly acidic C-terminal region (95–140). Positions of residues mutated to cysteines for smFRET labeling in this study are labeled.

Figure 2

Collapse of the C-terminus of αS at low pH. (A–E) ET_eff histograms for αS double mutants, labeling sites displayed in each panel. (Solid lines) Gaussian fits to the ET_eff distribution. Panels B–E show that the mean values of the histograms (x_c) shift to higher values at low pH, indicating a decrease in the separation between the C-terminus and the central (∼30–100) region of the protein. Panel A shows that, in contrast to other regions of the protein, there is very little change in the distance between the N- and C-termini at neutral and low pH. FCS measurements support this finding, as the ratio of average αS diffusion time at pH 3.0 to pH 7.4 is 0.98 (by the Student's t-test, p = 0.001, no significant difference). Although smFRET and FCS are sensitive to different dimensional properties, radius of gyration and hydrodynamic radius, respectively, both measurements indicate that the global dimensions of αS at pH 7.4 and 3.0 are similar. (Lower right-hand panel) Summary of the mean values (x_c) and full width at half-maximum (σ) taken from the Gaussian fits of all constructs at both pHs.

Figure 3

Effects of aggregation inducers on conformation of αS. (A, C, and E) Spermine (300 μM) causes only minor conformational changes throughout αS, as seen by slight shifts in the ET_eff histograms for constructs probing the C-terminus, NAC region, and N-terminus, respectively, in the presence (columns) and absence (solid lines) of spermine. (B, D, and F) Heparin (500 nM) shows similar results for the same αS constructs. For comparison, the solid lines in each panel are a Gaussian fit to the same αS construct in pH 7.4 buffer in the absence of molecular aggregation inducers.

Figure 4

Denaturation of the low pH state monitored by smFRET. Solid squares are mean value, x_c, from the Gaussian fit to the ET_eff histogram for αS-labeled at residues 72 and 130, measured at pH 3.0 with increasing concentrations of GdnHCl. Error bars are standard deviation of three experiments on three separate labeling preparations. (Open square) Mean value of the ET_eff histogram of the same αS construct at pH 7.4. The widths of the Gaussian fits to the ET_eff histograms were invariant up to 3 M GdnHCl (Table S2).

Figure 5

Cartoon highlighting differential distance changes between residues 9–130 and 33–130 at pH 7.4 and 3.0. The solid arrows, which span residues 9–130, are the same size in both figures, whereas the dashed arrow, between residues 33–130, in the pH 3.0 figure is shorter than the dashed arrow in the pH 7.4 figure, reflecting the relative closer proximity between these two residues at pH 3.0. The circles are also the same size in both figures, indicating that the overall dimensions of the protein remain the same at both pH values (based on our FCS measurements and the hydrodynamic radius measurements from other groups).