In situ single-molecule investigations of the impacts of biochemical perturbations on conformational intermediates of monomeric α-synuclein

Abstract

α-Synuclein aggregation is a common trait in synucleinopathies, including Parkinson's disease. Being an unstructured protein, α-synuclein exists in several distinct conformational intermediates, contributing to both its function and pathogenesis. However, the regulation of these monomer conformations by biochemical factors and potential drugs has remained elusive. In this study, we devised an in situ single-molecule manipulation approach to pinpoint kinetically stable conformational intermediates of monomeric α-synuclein and explore the effects of various biochemical factors and drugs. We uncovered a partially folded conformation located in the non-amyloid-β component (NAC) region of monomeric α-synuclein, which is regulated by a preNAC region. This conformational intermediate is sensitive to biochemical perturbations and small-molecule drugs that influencing α-synuclein's aggregation tendency. Our findings reveal that this partially folded intermediate may play a role in α-synuclein aggregation, offering fresh perspectives for potential treatments aimed at the initial stage of higher-order α-synuclein aggregation. The single-molecule approach developed here can be broadly applied to the study of disease-related intrinsically disordered proteins.

FIG. 1.

In situ magnetic-tweezer-based single-molecule approach. (a) Illustrated sequence map of wild-type α-synuclein (1 − 140 a.a.), alongside potential conformational intermediates of α-synuclein. (b) Schematic diagram of the experimental setup for in situ magnetic-tweezer-based single-molecule manipulation, comprising a magnetic tweezer for precise mechanical manipulation of proteins in the piconewton range with nanometer resolution, an in situ buffer exchange chamber with membrane wells, and a temperature control system. (c) Elaborate schematics showcasing that the recombinant monomeric α-synuclein was tethered to the glass substrate and the superparamagnetic beads through specific SpyTag–SpyCatcher chemistry and biotin–neutravidin bond, respectively.

FIG. 2.

Two major conformational intermediates of monomeric $\alpha$-synuclein. (a) Typical force−bead height curves of a disordered monomeric α-synuclein during force-increase scan (red, loading rate of 5 pN s⁻¹) and subsequent force-decrease scan (blue, loading rate of −1 pN s⁻¹). (b) Typical force−bead height curves of partially folded (PF) α-synuclein during force-increase and force-decrease scans at the same corresponding loading rates as in (a). (c) Unfolding and refolding force-step size distribution of PF α-synuclein at loading rates of 5 pN s⁻¹ (red dots) and −1 pN s⁻¹ (blue dots), respectively. A worm-like chain model was used to fit the data (red line), and the optimal fitting persistence length (A) and contour length (Lc) were obtained as A = 0.50 ± 0.05 and Lc = 15.50 ± 1.50 nm. ± indicates S.E. (d) Normalized unfolding and refolding step size distributions of PF α-synuclein, respectively. The data in (c) and (d) are from 124 (unfolding) and 268 (refolding) events, which were collected from more than 30 independent α-synuclein tethers. (e) Conformation fractions of monomeric α-synuclein (N = 131 molecules) based on their distinct mechanical signatures (supplementary material Fig. 8).

FIG. 3.

Effects of truncations of $\alpha$-synuclein. (a) Illustrated domain maps of wild-type and truncated $\alpha$-synucleins. (b) PF population fractions of wild-type α-synuclein (WT) and various truncations based on the unfolding signature of the PF species. The numbers of tested molecules are 131, 35, 20, 74, 60, 48, 51, 47, and 52 for WT, NAC, NAC + preNAC, ΔC, ΔN1, ΔN2, ΔN3, ΔNAC, and ΔpreNAC, respectively. Error bars indicate mean ± S.E.M. (c) The average unfolding forces during force-increase scans at a loading rate of 5 pN s⁻¹. The numbers of unfolding events are: 124, 153, 193, 199, 161, 237, 16, 0, and 21 for WT, NAC, NAC + preNAC, $\Delta$C, $\Delta$N1, $\Delta$N2, $\Delta$N3, $\Delta$NAC, and $\Delta$preNAC, respectively. (d) The average folding forces during force-decrease scans at a loading rate of −1 pN s⁻¹. The numbers of folding events are: 268, 0, 76, 55, 98, 89, 0, 0, and 0 for WT, NAC, NAC + preNAC, $\Delta$C, $\Delta$N1, $\Delta$N2, $\Delta$N3, $\Delta$NAC, and $\Delta$preNAC, respectively. Error bars in Fig. 3(c) and 3(d) indicate mean ± S.D. N.A. means not applicable (no PF conformation is observed). ^**p < 0.01; ^***p < 0.001 (95% of confidence intervals). p values were determined using an unpaired two-tailed Student's t-test.

FIG. 4.

In situ perturbations of the disordered-to-PF conversion. (a) Schematic illustration of the in situ single-molecule experiments. Single disordered α-synuclein was identified at 23 °C (left panel), and the seed (small α-synuclein aggregates) was introduced or the temperature was increased to 37 °C (right panel). (b) Representative force-bead height traces of the disordered α-synuclein at increasing temperatures from 23 °C (bottom) to 37 °C (top). Similar results were observed in three independent tethered proteins. (c) Representative force-bead height traces of the α-synuclein before (bottom) and after (top) flowing in 10 nM α-synuclein seed at 23 °C. Similar results were observed in four independent tethered proteins. (d) Average times required for the conversion from the disordered to PF conformations of α-synuclein under the corresponding in situ perturbations. Error bars indicate mean ± S.E.M. ^*For comparison, the disordered-to-PF transition at 23 °C without seed was not observed over 10 h.

FIG. 5.

Effects of small-molecule drugs. (a) Chemical structures of six small-molecule drugs reported to inhibit the in vitro aggregation of $\alpha$-synulcein. (b) PF fraction of wild-type and small-molecule pretreated $\alpha$-synulcein. The total molecule numbers are N = 131, 62, 61, 61, 45, 55, and 54 for wild type, six small-molecule (dopamine, baicalein, curcumin, EGCG, anle138b, and fasudil) treated $\alpha$-synulcein, respectively. Error bars indicate mean ± S.E.M. ns: p > 0.05; ^**p < 0.01; ^***p < 0.001; and ^****p < 0.0001 (95% of confidence intervals). p values were determined by unpaired two-tailed Student's t-test. (c) and (d) In situ analysis of the PF-to-disordered transition by dopamine and baicalein. (c) Representative force−bead height curves of a PF α-synuclein before (bottom) and after (top) introducing 0.1 mM dopamine solution. Similar results were repeated on three independent α-synucleins. (d) Representative force-bead height curves of a PF α-synuclein before (bottom) and after (top) introducing 0.1 mM baicalein solution. Similar results were observed on five independent tethered proteins.