Formation of covalent di-tyrosine dimers in recombinant α-synuclein

Abstract

Parkinson's disease is associated with fibril deposition in the diseased brain. Misfolding events of the intrinsically disordered synaptic protein α-synuclein are suggested to lead to the formation of transient oligomeric and cytotoxic species. The etiology of Parkinson's disease is further associated with mitochondrial dysfunction and formation of reactive oxygen species. Oxidative stress causes chemical modification of native α-synuclein, plausibly further influencing misfolding events. Here, we present evidence for the spontaneous formation of covalent di-tyrosine α-synuclein dimers in standard recombinant protein preparations, induced without extrinsic oxidative or nitrative agents. The dimers exhibit no secondary structure but advanced SAXS studies reveal an increased structural definition, resulting in a more hydrophobic micro-environment than the highly disordered monomer. Accordingly, monomers and dimers follow distinct fibrillation pathways.

Keywords: EOM; Parkinson's disease; SAXS; amyloids; di-tyrosine dimers; α-synuclein.

Figure 1.

Initial characterization of the αSN batches after variation in the purification procedure. (A) The UV_280nm trace of the final SEC step of the purification of recombinant αSN. 2 mL eluent from the ion-exchange chromatography were injected on the HiLoad Superdex 200 SEC column in concentrations of 3.5, 5.5 8.0 and 10 mg/ml to obtain αSN type0 (black curve), type1 (red curve), type2 (blue curve) and type3 (green curve), respectively. The arrow indicates the increasing size of the late eluting shoulder (B) ANS titration of αSN type1 (red), type2 (blue) and type3 (green) depicting increasing ANS fluorescence intensity.

Figure 2.

Preliminary biophysical characterization of type0–3 preparations. (A) Far-UV CD measurements on readily dissolved αSN type1 (red curve), type2 (blue curve) and type3 (green). (B) 3D scan of Intrinsic fluorescence of type3 αSN.

Figure 3.

Fibrillation kinetics of 12 mg/ml αSN were monitored using 20 µm ThT as in situ probe. Measurements were performed in triplicate of αSN type0 (black curve), type1 (red curve), type2 (blue curve) and type3 (green curve). The averaged curves are shown as full lines and the dotted lines indicate the standard deviation.

Figure 4.

Basic parameters derived from SAXS show increasing global average dimension of αSN type0–3. The 1D scattering curves (A) were analyzed in order to obtain the R_g and I(0) (B). The R_g (filled bars, left axis) and I(0) (empty bars, right axis) of αSN are shown for type0 (black), type1 (red), type2 (blue) and type3 (green).

Figure 5.

Full ETD fragmentation spectrum of isolated F+G peptide including the Y39-Y39 cross-link. The precursor ion is indicated ([M+4]⁴⁺).

Figure 6.

Residuals from the decomposition of the SAXS data from the eluting monomer-dimer peak from the SEC column. The first axis depicts elution volumes, the second axis the q-range and the third axis depicts the scattering intensities. (A) The residuals from decomposing the data using one component. (B) The residuals from the decomposition using both monomer and dimer data. The color scaling is the same in both plots, ranging from blue to yellow, with brighter colors representing higher scattering intensities.

Figure 7.

Data from the SEC-SAXS experiments. (A) The UV_280nm trace from the concentrated sample injected on the gel-filtration column. The black and orange curves are initial Gaussian estimates of the elution of first and second components, respectively. (B) The isolated data curves from the first part of the peak corresponding to an αSN monomer (black), and the data curves from the last eluting fractions corresponding to an αSN dimer (orange). (C) The average pair distance distribution functions (PDDF) for the monomer (black) and dimer (orange). (D) Results from the EOM analysis: The distributions of maximum dimensions in the selected ensembles for the monomer (black) and dimer (orange) respectively. Error bars are the standard deviations from 12 runs. Please cf. supplementary information for a comparison between the initial random pool and the selected ensemble (Figs. S6 and S7). (E–F) Results from the EOM analysis of the monomer (E) and dimer (F) data: Maximum distance plotted as a function of the radius of gyration. The colors represent the 12 different runs and the size of the sphere represent the frequency by which the given D_max/R_g values were selected. Note that far fewer conformations are selected for the dimers, than ditto of the monomer, and the dimensions are on average larger.