Methionine oxidation in α-synuclein inhibits its propensity for ordered secondary structure

Abstract

α-Synuclein (AS) is an intrinsically disordered protein highly expressed in dopaminergic neurons. Its amyloid aggregates are the major component of Lewy bodies, a hallmark of Parkinson's disease (PD). AS is particularly exposed to oxidation of its methionine residues, both in vivo and in vitro Oxidative stress has been implicated in PD and oxidized α-synuclein has been shown to assemble into soluble, toxic oligomers, rather than amyloid fibrils. However, the structural effects of methionine oxidation are still poorly understood. In this work, oxidized AS was obtained by prolonged incubations with dopamine (DA) or epigallocatechin-3-gallate (EGCG), two inhibitors of AS aggregation, indicating that EGCG promotes the same final oxidation product as DA. The conformational transitions of the oxidized and non-oxidized protein were monitored by complementary biophysical techniques, including MS, ion mobility (IM), CD, and FTIR spectroscopy assays. Although the two variants displayed very similar structures under conditions that stabilize highly disordered or highly ordered states, differences emerged in the intermediate points of transitions induced by organic solvents, such as trifluoroethanol (TFE) and methanol (MeOH), indicating a lower propensity of the oxidized protein for forming either α- or β-type secondary structures. Furthermore, oxidized AS displayed restricted secondary-structure transitions in response to dehydration and slightly amplified tertiary-structure transitions induced by ligand binding. This difference in susceptibility to induced folding could explain the loss of fibrillation potential observed for oxidized AS. Finally, site-specific oxidation kinetics point out a minor delay in Met-127 modification, likely due to the effects of AS intrinsic structure.

Keywords: Fourier transform IR (FTIR); amyloid; circular dichroism (CD); dopamine; epigallocatechin-3-gallate; ion mobility (IM); mass spectrometry (MS); methionine oxidation; neurodegenerative disease; α-synuclein (α-synuclein).

Figure 1.

Oxidation kinetics of intact AS. A–C, ESI-MS peaks corresponding to the 15+ charge state of AS after a 24-h incubation in the absence of ligands (A), in the presence of DA (B), and in the presence of EGCG (C). In each panel the peaks are labeled by the number of oxidized residues. (D and E): AS oxidation extent (0, black; 1, red; 2, blue; 3, green; 4, magenta, oxidized methionine residues) as a function of the incubation time in the presence of DA (D) or EGCG (E). Error bars indicate the standard deviation from three independent experiments.

Figure 2.

Oxidation kinetics of AS methionine residues. A–C, ESI-MS peaks corresponding to the 4+ charge state of the AS chymotryptic peptide containing Met-116, after 24-h incubation in the absence of ligands (A), in the presence of DA (B), and in the presence of EGCG (C). D and E, oxidation fraction of each methionine residue (M₁, black; M₅, red; M₁₁₆, blue; M₁₂₇, green) as a function of the incubation time in the presence of DA (D) or EGCG (E). Quantification for Met-116 and Met-127 has been performed on MS data on chymotryptic peptides. Quantification for Met-1 and Met-5 has been performed on MS/MS data on tryptic peptides. The black and red symbols are mostly overlaid for the 40-h time point and beyond. Error bars indicate the S.D. from at least three independent experiments.

Figure 3.

Secondary-structure transitions by CD spectroscopy. Far-UV CD spectra of oMet₀ (black lines) and oMet₄ (red lines) AS in the absence of organic solvents (A); in the presence of 10% (B) and 15% (C) TFE; in the presence of 35% (E) and 40% (F) MeOH. D, SDS-PAGE analysis of supernatants (s) and pellets (p) obtained from the following samples: AS before incubation (ref); AS after 72 h incubation in the absence of ligands (AS); AS after 72 h incubation in the presence of DA (+DA); AS after 72 h incubation in the presence of EGCG (+EGCG). The bands corresponding to AS monomer, fragments, and oligomers are labeled. Representative data from at least three independent experiments are shown.

Figure 4.

Secondary-structure transitions by FTIR spectroscopy. Absorption (A) and second-derivative (B) spectra of oMet₀ (black lines) and oMet₄ (red lines) AS measured in the form of protein films obtained after solvent evaporation. C, absorption spectra of oMet₀ and oMet₄ AS measured in D₂O solution at 30 °C. Second-derivative spectra of oMet₀ (D) and oMet₄ (E) AS measured in D₂O solution at different temperatures from 25 to 100 °C. Arrows point to the spectral changes observed at increasing temperatures. The peak positions of the main components are shown. F, ratio of the intensity at 1655 and 1642 cm⁻¹ taken from the second-derivative spectra of D and E. Representative data from 3 (A and B) or 2 (C–F) independent experiments are shown. Error bars indicate the standard deviation.

Figure 5.

Ligand binding by native MS. A and B, ESI-MS spectra of ternary mixtures of oMet₀, oMet₄, and either DA (A) or EGCG (B). The 11+ and 8+ peaks are labeled by the corresponding charge state. C and D, magnification of A and B in the region of the 11+ (C) and 8+ (D) charge state. The peaks of oMet₀ (black symbols) and oMet₄ (red symbols) are labeled by the number of ligand molecules bound in the complex. Representative data from at least three independent experiments are shown.

Figure 6.

Tertiary-structure transitions induced by ligand binding to AS by IM-MS. Collision cross-section obtained from the 11+ (A) or 8+ (B) charge states of oMet₀ and oMet₄ complexes with ligands by IM-MS experiments, as a function of the number of bound ligand molecules (oMet₀, black symbols; oMet₄, red symbols). The relative intensities of the signals are expressed by a color scale (inset). Conformers are named by lowercase, italic letters. Representative data from at least three replicas are shown.