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. 2014 Jan 24;9(1):e86495.
doi: 10.1371/journal.pone.0086495. eCollection 2014.

Diverse metastable structures formed by small oligomers of α-synuclein probed by force spectroscopy

Krishna Neupane  1 , Allison Solanki  1 , Iveta Sosova  2 , Miro Belov  2 , Michael T Woodside  3
Affiliations

Diverse metastable structures formed by small oligomers of α-synuclein probed by force spectroscopy

Krishna Neupane et al. PLoS One. .

Abstract

Oligomeric aggregates are widely suspected as toxic agents in diseases caused by protein aggregation, yet they remain poorly characterized, partly because they are challenging to isolate from a heterogeneous mixture of species. We developed an assay for characterizing structure, stability, and kinetics of individual oligomers at high resolution and sensitivity using single-molecule force spectroscopy, and applied it to observe the formation of transient structured aggregates within single oligomers of α-synuclein, an intrinsically-disordered protein linked to Parkinson's disease. Measurements of the molecular extension as the proteins unfolded under tension in optical tweezers revealed that even small oligomers could form numerous metastable structures, with a surprisingly broad range of sizes. Comparing the structures formed in monomers, dimers and tetramers, we found that the average mechanical stability increased with oligomer size. Most structures formed within a minute, with size-dependent rates. These results provide a new window onto the complex α-synuclein aggregation landscape, characterizing the microscopic structural heterogeneity and kinetics of different pathways.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Engineered α-synuclein dimer and tetramer constructs.
Schematic of the monomer and multimeric protein constructs, the later containing an N-terminal His-tag and enterokinase cleavage site (EK). Monomers are connected via linkers with three amino acids. Cys residues (red) are used to attach DNA handles.
Figure 2
Figure 2. Force spectroscopy of α-synuclein monomers.
(A) Inset: A single protein molecule was attached at its ends to DNA handles, bound to beads and held under tension between two optical traps. Most FECs of a single monomer display no structure (cyan) and fit well to the WLC model expected for the unfolded-state (red). Some reveal discrete unfolding transitions (black, orange, blue) with different contour lengths, as found from WLC fits (grey). (B) Histogram of ΔL c for all identifiable transitions in FECs of the monomer.
Figure 3
Figure 3. FECs of α-synuclein dimers.
(A, B) Representative FECs of a dimer show unfolding of stable structures with a wide range of sizes and unfolding forces. WLC fits to determine contour length changes are displayed as dashed lines (grey: folded states, red: unfolded state). Inset: the dimer contains two monomers connected by short, flexible peptides linkers. (C) Histogram of ΔL c for all identifiable transitions in dimer FECs.
Figure 4
Figure 4. FECs of α-synuclein tetramers.
(A, B) Representative FECs of a tetramer reveal many structures with different sizes and unfolding forces. WLC fits are shown as dashed lines (grey: folded states, red: unfolded state). Inset: the tetramer contains four α-synuclein domains connected by short, flexible peptide linkers. (C) Histogram of ΔL c for all identifiable transitions in FECs of the tetramer.
Figure 5
Figure 5. Contour length and unfolding force distributions.
(A) Histogram of ΔL c for all identifiable transitions in FECs of the tetramer (red), dimer (blue), and monomer (black). (B) Scatterplot of F u vs ΔL c for tetramer (red), dimer (blue), and monomer (black). Arrows indicate ΔL c values consistent with a β-sandwich structure, asterisks indicate ΔL c values expected from a helical multimer structure (blue: dimer and tetramer, red: tetramer only). Dashed lines indicate the contour lengths of the entire monomer (black), dimer (blue), or tetramer (red). (C) Histograms of F u for the tetramer (red), dimer (blue), and monomer (black) show an increase in F u with increasing oligomer size.
Figure 6
Figure 6. Dynamic force spectroscopy.
Loading rate dependence of the average unfolding force for the two most frequent transitions: ΔL c = 11–13 nm for the dimer (blue) and ΔL c = 16–18 nm for the tetramer (red). Fits to Equation 2 yield the unfolding rates at zero force, ∼0.1 s−1, and the distance to the barrier for unfolding, ∼1 nm.
Figure 7
Figure 7. Size-dependent structure formation rates.
The rate at which structures of a given total contour length change (including all intermediates) occur is similar for all constructs (tetramer: red, dimer: blue, monomer: black), but declines roughly exponentially with increasing length. Wait time at zero force was 5 sec. Rates were estimated from the occurrence frequency of specific ΔL c values, binned in 15-nm increments to improve the statistics.
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