The effects of biological crowders on fibrillization, structure, diffusion, and conformational dynamics of α-synuclein

Abstract

α-synuclein is an intrinsically disordered protein (IDP) whose aggregation in presynaptic neuronal cells is a pathological hallmark of Lewy body formation and Parkinson's disease. This aggregation process is likely affected by the crowded macromolecular cellular environment. In this study, α-synuclein was studied in the presence of both a synthetic crowder, Ficoll70, and a biological crowder composed of lysed cells that better mimics the biocomplexity of the cellular environment. ¹⁵ N-¹ H HSQC NMR results show similar α-synuclein chemical shifts in non-crowded and all crowded conditions implying that it remains similarly unstructured in all conditions. Nevertheless, both HSQC NMR and fluorescence measurements indicate that, only in the cell lysate, α-synuclein forms aggregates over a timescale of 48 h. ¹⁵ N-edited diffusion measurements indicate that all crowders slow down the α-synuclein's diffusivity. Interestingly, at high concentrations, α-synuclein diffuses faster in cell lysate than in Ficoll70, possibly due to additional soft (e.g., electrostatic or hydrophobic) interactions. ¹⁵ N-edited relaxation measurements show that some residues are more mobile in cell lysate than in Ficoll70; the rates that are most different are predominantly in hydrophobic residues. We thus examined cell lysates with reduced hydrophobicity and found slower dynamics (higher relaxation rates) in several α-synuclein residues. Taken together, these experiments suggest that while cell lysate does not substantially affect α-synuclein structure (HSQC spectra), it does affect chain dynamics and translational diffusion, and strongly affects aggregation over a timescale of days, in a manner that is different from either no crowder or an artificial crowder: soft hydrophobic interactions are implicated.

Keywords: NMR; intrinsically disordered protein; macromolecular crowding; relaxation; translational diffusion; α-synuclein.

FIGURE 1

α‐synuclein fibrillation. ThT fluorescence assay with 0.04 mM α‐synuclein (green squares), 0.04 mM α‐synuclein in 280 mg/mL Ficoll70 (purple pentagons), 0.04 mM α‐synuclein in 280 mg/mL ultracentrifuged cell lysate (brown pentagons), 280 mg/mL Ficoll70 (purple diamonds), 280 mg/mL ultracentrifuged cell lysate (brown diamonds) and water (black circles) at room temperature at pH 7. The fluorescence signal is reported as fluorescence intensity per μL ThT buffer. 69.2 μL ThT buffer was used with Ficoll, 27.2 μL ThT buffer was used with ultracentrifuged cell lysate, and 237.2 μL with α‐synuclein alone. The cell lysate experiments have been duplicated.

FIGURE 2

(a) ¹⁵N‐¹H HSQC spectra of 0.2 mM α‐synuclein in the absence (green) and presence of 200 mg/mL Ficoll70 (purple) and bacterial cell lysate (blue) at 0 h, 37°C, and pH 7. The contour levels are the same in all spectra, and the spectra are obtained at a ¹H frequency of 500 MHz with 128 scans. (b) Combined ¹⁵N and ¹H chemical shift $\left(∆\mathcal{V}=\sqrt{\frac{1}{2}\left[{\left(\alpha ·∆{\mathcal{V}}_N\right)}^2+∆{\mathcal{V}}_H^2\right]}\right)$ of assigned residues. The gray shaded region below 0.02 ppm indicates chemical shift changes that are too small to be significant, and α = 0.14 (Williamson, 2013). Some peaks in (a) could not be assigned.

FIGURE 3

Translational diffusion of 0.2 mM α‐synuclein without crowder and in the presence of 200 mg/mL bacterial cell lysate and Ficoll70 at 25°C and pH 7. Diffusion coefficients of α‐synuclein are obtained by ¹⁵N‐¹H HSQC‐DOSY experiments at a ¹H frequency of 500 MHz NMR with 2560 scans. (a) 1D ¹⁵N‐¹H DOSY spectrum of ¹⁵N‐enriched α‐synuclein without crowder. The intensities of the integrated peaks between 7.73 and 8.70 ppm are used to plot the logarithm of the attenuated signal versus the gradient strength. Gradient strength ranged from 0.48 to 45.74 G/cm. (b) Attenuated signal versus the gradient strength parameter with and without 200 mg/mL crowders. The fit is biexponential decay. (c) Bar graphs depict the comparison of α‐synuclein diffusion value (the slow components of panel b) in different crowded conditions. The error bar represents the standard deviation of the fit to the biexponential.

FIGURE 4

¹⁵N‐¹H HSQC transverse relaxation rate (R₂) of 0.2 mM α‐synuclein (a) in the absence of crowders (green), presence of 200 mg/mL unmanipulated bacterial cell lysate (blue), and Ficoll70 (purple), (b) in the presence of 200 mg/mL less hydrophobic cell lysate (red) and unmanipulated bacterial cell lysate (blue) (c) in the absence of crowders versus in the 200 mg/mL unmanipulated cell lysate, (d) in the absence of crowders versus in the 200 mg/mL Ficoll70, (e) in the 200 mg/mL of less hydrophobic cell lysate versus 200 mg/mL of unmanipulated cell lysate at 25°C and pH 7. The R₂ values were derived from the peak height (intensity), and the error bars represent the standard deviation of the fit. The relaxation experiments are done at 600 MHz with 44 scans.