Fig 1.
Effect of SDS on the secondary structure of aS and Ac-aS.
(A and B) Far-UV CD spectra were recorded at 25°C in 20 mM HEPES buffer pH 7.2, in presence of increasing SDS concentrations for aS (A) and Ac-aS (B). The protein concentration was 100 μM in all measurements. The spectra were normalized to mean residue molar ellipticity. (C) Percentage of α-helix structure of 100 μM aS (filled symbols) and Ac-aS (open symbols) at different SDS concentrations. The values have been calculated from the mean residue ellipticity at 222 nm measured in the far-UV CD spectra [45]. The error bars correspond to at least two independent measurements.
Fig 2.
Changes in molecular size of aS (A) and Ac-aS (B) as a result of interactions with SDS.
The apparent hydrodynamic radius (Rh) of 100 μM protein in presence of different SDS concentrations was measured by DLS at 25°C. The black symbols represent the Rh of particles (left scale) corresponding to more than 2% of the protein mass and more than 10% of the scattered intensity. Measurements reporting two different particle sizes (circles and triangles) are represented with filled symbols. The black solid line corresponds to the intensity-averaged Rh at each SDS concentration. The red symbols represent the mass percent of each type of particle (right scale).
Fig 3.
Hydrodynamic properties of aS and Ac-aS.
Size-exclusion chromatography analysis aS (A) and Ac-aS (B) in presence of different SDS concentrations, as indicated in different colors. (C) Elution volumes of each peak maximum at each SDS concentration.
Fig 4.
Effect of SDS on the NMR spectra of aS and Ac-aS.
Aromatic and aliphatic regions of the 1D 1H NMR spectra of aS (A) and Ac-aS (B) in 100% D2O, 5 mM sodium phosphate buffer pH 7.2, in the presence of different SDS concentrations. Spectra were acquired at 25°C at 100 μM protein concentration. The intensities of each spectral region have been adapted for proper visualization. The Hε1 resonance of the His50 is identified with arrows at 0 and 10 mM SDS. The signal marked with a cross corresponds to an impurity. The positions of the free SDS resonances have been marked with asterisks. (C) Signal intensity at the position of the resonance of α-methylene protons of free SDS at different SDS concentrations in presence of 100 μM aS and Ac-aS. The intensity values correspond to the integral of the spectra between 3.80 and 3.95 ppm.
Fig 5.
Hydrodynamic properties of SDS, aS and Ac-aS measured by DOSY NMR.
(A) Diffusion coefficients of SDS, aS and Ac-aS in free SDS solutions and protein-SDS mixtures at different SDS concentrations. DOSY experiments were carried out at 25°C in 100% D2O, 5 mM sodium phosphate buffer pH 7.2. Protein concentration in the mixtures was 100 μM. Errors correspond to 95% confidence intervals of the fittings of the DOSY intensity decays. The CMC of SDS (3.8 mM) was estimated from the intercept of the two observed linear tendencies. (B) Apparent hydrodynamic radius of aS and Ac-aS in presence of different SDS concentrations calculated from the diffusion coefficients shown in (A).
Fig 6.
Amyloid aggregation induced by SDS.
(A and B) Representative aggregation kinetics of aS (left panel) and Ac-aS (right panel) at 37°C followed by ThT fluorescence in presence of different SDS concentrations. (C) Scale expansion of some of the kinetics showing incipient lag phases. Numbers alongside each trace indicate the SDS concentration for aS (blue) and Ac-aS (red). (D) Initial aggregation rates at 37°C of WT aS and Ac-aS in presence of different SDS concentrations. The rates have been determined from the initial slopes of the ThT fluorescence kinetics. Error bars correspond to standard deviations from several independent measurements. (E-H) Transmission electron microscopy images of SDS-induced aggregates of aS (E and F) and Ac-aS (G and H). Samples at 100 μM concentration were incubated at 37°C in presence of SDS during the times lengths and at the SDS concentrations indicated on top of each image.
Fig 7.
Changes in secondary structure in early-onset PD variants.
Percentage of α-helix structure of early-onset PD variants of aS (filled symbols) and Ac-aS (open symbols) at different SDS concentrations. The values have been calculated from the mean residue ellipticity at 222 nm measured in the far-UV CD spectra [45].
Fig 8.
Changes in molecular size of early-onset PD variants.
The apparent hydrodynamic radius (Rh) of 100 μM aS variants (left panels) or Ac-aS variants (right panels) in presence of different SDS concentrations was measured by DLS at 25°C. The open symbols represent the Rh of particles (left scale) corresponding to more than 2% of the protein mass and more than 10% the scattered intensity. Measurements reporting two different particle sizes (circles and triangles) are represented in red. The black solid line corresponds to the intensity-averaged Rh at each SDS concentration. Closed symbols represent the mass percent of each type of particle (right scale).
Fig 9.
Amyloid aggregation rates of early-onset PD variants.
Initial aggregation rates at 37°C of aS and Ac-aS PD variants in presence of different SDS concentrations were measured by ThT fluorescence. The rates were determined from the initial slopes of the ThT fluorescence kinetics. Error bars correspond to standard deviations from several independent measurements.
Fig 10.
Schematic illustration of the proposed conformational-oligomerization equilibria of aS in the presence of different concentrations of SDS.
The N-terminal lipid-binding region of aS is colored in blue, the NAC region in green and the acidic C-terminal tail in red. Spherical SDS micelles have been depicted for simplicity. The effect of N-acetylation and PD mutations on the amyloid aggregation rate and on the stability of α-helix-rich oligomers is indicated with + or − symbols.