Single-Molecule Force Spectroscopy of Rapidly Fluctuating, Marginally Stable Structures in the Intrinsically Disordered Protein $\alpha$-Synuclein

Intrinsically disordered proteins form transient, fluctuating structures that are difficult to observe directly. We used optical tweezers to apply force to single $\alpha$-synuclein molecules and measure their extension, characterizing the resulting conformational transitions. Force-extension curves revealed rapid fluctuations at low force, arising from the folding of two different classes of structure that were only marginally stable. The energy landscape for these transitions was characterized via the force-dependent kinetics derived from correlation analysis of the extension trajectories. The barriers were small, only a few $k_{B}T$, but the diffusion was slow, revealing a landscape that is flat but rough.

Figure 1

Force spectroscopy of $\alpha$-synuclein. (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. FECs of $\alpha$-synuclein monomers sometimes revealed discrete unfolding rips (black), but usually showed a monotonic rise in force with extension without obvious rips (cyan). Similar behavior was seen for $\alpha$-synuclein dimers (b) and tetramers (c). FECs without rips nevertheless deviated from WLC behavior (red). (d)–(f) FECs without discrete rips were averaged (cyan) and compared to polymer models. Data did not fit a noninteracting WLC model (red; residuals in inset), but did fit a model incorporating rapid structural fluctuations, Eq. (1) (yellow; residuals in inset).

Figure 2

Autocorrelation analysis of $\alpha$-synuclein tetramer. (a) The extension autocorrelation of a tetramer construct in the high-force range ($>8\mathrm{pN}$, triangles) demonstrates a single-exponential decay, and is identical to the result for a construct containing DNA handles only, without protein (circles). (b) In the midforce range (2–8 pN), where the shoulder feature is present in FEC data, the extension autocorrelation exhibits a single-exponential decay for the handle only (circles), but a double-exponential decay for the protein construct (triangles), indicating the presence of an additional mode corresponding to structural transitions in the protein.

Figure 3

Microscopic rates and energy landscape schematic. (a),(b) The microscopic rates for tetramer unfolding (triangles) and refolding (circles) of type 1 (gray) and type 2 (black) transitions are well fit by Eq. (S3) [24], yielding parameters describing the energy landscape. (c) Schematic of the energy landscape at zero force, showing the barriers, roughness, and free-energy changes for the two transitions.