Identification of a helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation

Abstract

Because oligomers and aggregates of the protein α-synuclein (αS) are implicated in the initiation and progression of Parkinson's disease, investigation of various αS aggregation pathways and intermediates aims to clarify the etiology of this common neurodegenerative disorder. Here, we report the formation of short, flexible, β-sheet-rich fibrillar species by incubation of αS in the presence of intermediate (10-20% v/v) concentrations of 2,2,2-trifluoroethanol (TFE). We find that efficient production of these TFE fibrils is strongly correlated with the TFE-induced formation of a monomeric, partly helical intermediate conformation of αS, which exists in equilibrium with the natively disordered state at low [TFE] and with a highly α-helical conformation at high [TFE]. This partially helical intermediate is on-pathway to the TFE-induced formation of both the highly helical monomeric conformation and the fibrillar species. TFE-induced conformational changes in the monomer protein are similar for wild-type αS and the C-terminal truncation mutant αS1-102, indicating that TFE-induced structural transitions involve the N terminus of the protein. Moreover, the secondary structural transitions of three Parkinson's disease-associated mutants, A30P, A53T, and E46K, are nearly identical to wild-type αS, but oligomerization rates differ substantially among the mutants. Our results add to a growing body of evidence indicating the involvement of helical intermediates in protein aggregation processes. Given that αS is known to populate both highly and partially helical states upon association with membranes, these TFE-induced conformations imply relevant pathways for membrane-induced αS aggregation both in vitro and in vivo.

Fig. 1.

WT αS aggregate characteristics as a function of [TFE]. (A–D): TEM micrographs of structures grown from 50 μM WT αS, after 2 wk incubation at 37 °C with shaking in the presence of (A) 0%, (B) 5%, (C) 10%, and (D) 15% TFE. (Scale bar: 200 nm). (E) For the samples in (A–D), the percentage of total protein incorporated into large aggregates (white bars, left axis) and the thioflavin-T enhancement (gray bars, right axis). The error bars reflect the standard deviations for three samples and the uncertainty in volume due to evaporation.

Fig. 2.

Secondary structural changes induced by TFE for WT αS at 25 °C. (A) Far-UV CD spectra for 0.5 μM WT αS variant in 0%–60% TFE. The TFE concentrations for spectra with increasing negative ellipticity at 222 nm are 0%, 2%, 5%, 8%, 7%, 9%, 10%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 20%, 22%, 24%, 26%, 30%, 40%, 45%, 50%, and 60%. The insets show selected curves from the main plot, which correspond to TFE ranges where the spectra share an isodichroic point. (Inset axes units are the same as the main plot). (B) Transition diagrams constructed from the ellipticity values at 222 nm and 198 nm using the data in (A). For clarity, some points are labeled with their [TFE]. The dotted lines show linear fits to sets of points whose CD spectra share isodichroic points.

Fig. 3.

Reconstructions of the I state spectra. (A) Predicted spectra for WT αS. The solid lines show the spectrum calculated via PCA. The dashed lines show results of MLE analysis, which were calculated using spectra that shared the low-TFE isodichroic points. The dotted line shows the MLE results calculated from spectra that shared the high-TFE isodichroic. The points (circles) show the I state reference points from Table S1. (B) Comparison of the results of the PCA calculations for all five αS variants.

Fig. 4.

Calculations based on linear combinations of the pure U, I, and F states. (A) Fit results (black lines) for WT αS CD spectra (open circles, data as in Fig. 2A), where the fitted curves were calculated from linear combinations of the 0%, 60% and the estimated I state spectra (see SI Text). The TFE concentrations for spectra with increasing negative ellipticity at 222 nm are 5%, 13%, 15%, 17%, 20%, 30%, and 50% TFE. (B) Fractions of monomer protein in the three states U, I, and F as a function of [TFE], obtained from fits of CD spectra to linear combinations of the pure states (SI Text). Black symbols: f_U. White symbols: f_I. Gray symbols: f_F. Data is shown for WT (circles), αS102 (down triangles), A30P (squares), A53T (diamonds) and E46K (up triangles) αS.

Fig. 5.

Oligomer formation kinetics 15% TFE. (A) Far-UV CD spectra taken at various time points for 2 μM WT, A30P, A53T, and E46K αS in 15% TFE at 25 °C. The initial time point for each plot has the least negative [θ] at 216 nm. (B) Kinetics of the oligomerization reaction for WT, A30P, A53T, and E46K αS. Filled circles: [θ]₂₁₆ for the curves in (A–D) plotted vs. time. Open triangles: [θ]₂₁₆ vs. time for 5 μM protein in 15% TFE. Lines: Results of fits to a single exponential model (SI Text). The error bars reflect the uncertainty in time due to mixing and experimental dead time, as well as signal fluctuations.