A: SDS-PAGE, RP-HPLC and MALDI-TOF analyses of the pure semisynthetic pY125 α-syn. MALDI analysis pY125 α-syn (expected mass: 14541 Da ([M+H]), a sinapinic matrix adduct is also observed as well as the double-charged peak. B: Western blot analyses of WT and pY125 α-syn confirmed that the phosphorylated semisynthetic protein could be detected by the anti-phosphorylated α-syn primary antibody, as well as by a non-phospho-specific antibody C: Circular dichroism spectra of pY125 α-syn in the absence (circles) and presence (triangles) of POPG vesicles. Open and closed symbols indicate WT recombinant α-syn and pY125 α-syn, respectively.

A: Paramagnetic relaxation enhancement profiles for A107C (upper panel) and A107C/pY125 (lower panel) α-syn, derived from the intensity ratios of equivalent resonances between ¹H, ¹⁵N HSQC spectra collected with and without an attached spin-label. A ratio of 1 indicates no PRE effect, while smaller ratios indicated a PRE effect, resulting from the proximity of the corresponding residue to the site of spin-label attachment. B: Overlaid ¹H, ¹⁵N HSQC spectra of SDS micelle bound WT (black) and A107C/pY125 (red) α-syn (upper panel) and plots of the normalized chemical shift changes between the two spectra as a function of residue number (lower panel). The solid line in panel B is a three-residue average. Note that each red peak corresponds closely to a black peak, indicating that the structures of the two variants are highly similar in the micelle-bound state. Black peaks without corresponding red peaks arise from residues 108 and beyond, where the semisynthetic A107C/pY125 protein is not ¹⁵N-labeled and therefore does not give rise to signals in this spectrum.

A: ThT fluorescence of recombinant WT and pY125 α-syn monitored over 48 h. B: SDS-PAGE analysis of the remaining WT and pY125 α-syn monomers prior to and after aggregation for 72 h and filtration through a 100 kD membrane. C: TEM images of WT and pY125 α-syn after 7 days of aggregation. The scale bar represents 500 nm. The images are representative of 5 independent experiments. The scale bar represents 500 nm.

A: Confocal images of HeLa cells transfected with WT α-syn plasmid and treated or not with pervanadate for 30min. The scale bar represents 20 µm. Subsequently, the cells were fixed and stained with the indicated antibodies (respectively rabbit anti-α-syn from Abcam ab51252 and mouse anti-pY125 α-syn from BD Pharmingen). B: Detection of pY125 α-syn in primary neurons. Confocal images of rat hippocampal neurons microinjected with 1 µM pY125 α-syn (25 fl/injection) together with the visible marker dye Faster Green (100 µg/ml) to identify the injected cells, the cells were treated with medium containing pervanadate for 30 min. Subsequently, the cells were fixed and stained with the indicated antibodies (respectively rabbit anti-α-syn from Abcam ab51252 and mouse anti-pY125 α-syn from BD Pharmingen). C: Immunoblots of HEK and HeLa cell lysates untreated or treated with pervanadate for 0, 5, 10 and 20min. The membranes were probed with total α-syn and pY125-specific antibodies (respectively rabbit anti-α-syn ab51252 from Abcam and mouse anti-pY125 α-syn from BD Pharmingen) D: Immunoblots of pY125 α-syn dephosphorylated from HeLa cell lysate. Membranes were probed with total α-syn and pY125-specific antibodies. The dephosphorylation was inhibited using different concentrations of sodium orthovanadate. E: Immuboblots of the subcellular fractionation of HEK cells over-expressing WT α-syn and treated with pervanadate for 20 min. Markers for cytoplasmic, membrane and nuclear fraction were used to assess for the purity of each fractions (Hsp90α, calnexin and PARP-1).

A: Immunoblots of pY125 α-syn phosphorylated by CK1 and PLK3. Membranes were probed using pS129, pY125, and α-syn antibodies. B: MALDI-TOF-MS analysis of the phosphorylation reaction after 12h. C: Immunoblots of pS129 α-syn phosphorylated by Syk, Lyn, Fyn, c-Src, and c-Fgr. Membranes were probed for pY125, pS129, and α-syn immunoreactivity. D: MALDI-TOF analysis of pS129 α-syn phosphorylated by (a) Syk, (b) Lyn, (c) Fyn, (d) c-Src, (e) and c-Fgr (f) after 14h of reaction. In all MALDI-TOF-MS spectra, the symbol ‘n’ indicates the peak corresponding to the starting material, the numbers above the other peaks correspond to the number of phosphorylation events, which were each detected by a +80 Da mass shift. The symbol ‘*’ indicates a sinapinic acid matrix adduct.

Immunoblots of HeLa cells over-expressing WT α-syn and treated with pervanadate for 0, 5, 10 and 20min. Membranes were probed with pS129 and pY125 α-syn and WT α-syn antibodies.

A: Schematic depiction of the sequence of α-syn with its 3 domains. The putative and identified sites of C-terminal phosphorylation are indicated with arrows. B: Sequence alignment of the sequences of the C-terminal domain of α-syn from 5 different species. The conserved positions of the phosphorylation sites are highlighted in red.

Top: A peptide thioester and a peptide fragment bearing an N-terminal Cys undergo a thioester exchange followed by an S to N acyl migration to create an amide bond between two unprotected peptides in aqueous solution. Bottom: Semisynthetic strategy to generate derivatives of α-syn site-specifically phosphorylated within the C-terminal region. The protein thioester α-syn(1–106)SR generated by recombinant protein expression is ligated by NCL to the synthetic C-terminal fragment α-syn(A107C-140) bearing the desired phosphorylated side chain. A final desulfurization step restores the native Ala-107.

A: Expressed Protein Ligation (EPL) approach to generate WT α-syn and pY125 α-syn. The α-syn(1–106)SR fragment was generated using intein-mediated thioester formation. MALDI-TOF analyses of 1) α-syn(1–106)SR (expected mass: 10744 Da [M+H]); The synthetic peptides α-syn (A107C-140) and α-syn(A107C-140)_pY125 were generated by Fmoc-based SPPS. MALDI-TOF analyses of 2) α-syn(A107C-140)_pY125 (expected mass: 3970 Da [M+H]);. Full-length semisynthetic α-synA107C and α-synA107C_pY125 were generated by a NCL step between α-syn(1–106)SR and synthetic peptide α-syn(A107C-140)_pY125 or α-syn(A107C-140). Radical desulfurization of the purified ligation product generated pY125 α-syn and α-syn WT. The final purification step involved a cation-exchange chromatographic step. B: MALDI-TOF analyses of 3) α-synA107C_pY125 (expected mass: 14573 Da [M+H]); and 4) pY125 α-syn (expected mass: 14541 Da ([M+H])). C: Coomassie blue stained SDS-PAGE analysis of the NCL reaction to generate pY125 α-syn. Lane 1, 2, 3, and 4 represent α-syn(1–106)SR; ligation reaction after 45 min, 90 min, and 140 min, respectively.