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Comment
. 2013 Jun 13;498(7453):E4-6; discussion E6-7.
doi: 10.1038/nature12125.

Properties of native brain α-synuclein

Jacqueline Burré  1 , Sandro Vivona, Jiajie Diao, Manu Sharma, Axel T Brunger, Thomas C Südhof
Affiliations
Comment

Properties of native brain α-synuclein

Jacqueline Burré et al. Nature. .
No abstract available

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Figures

Figure 1
Figure 1
a & b, Immunoblotting analysis of mouse brain homogenate (input), cytosol, and membranes (a), and quantification of protein levels (b; means ± SEMs; n=3). c, Native mouse brain α-synuclein (375 μg) elutes as an apparent tetramer during gel filtration on a Superdex 200 column (top), as analyzed by α-synuclein immunoblotting (bottom). d, Analysis of purified recombinant myc-epitope-tagged α-synuclein by SDS-PAGE and immunoblotting. e, Molecular mass calibration curve for gel filtration (Rf = migration distance of proteins versus total running distance). f, Recombinant myc-tagged human α-synuclein (16 μg) also elutes as an apparent tetramer during gel filtration. g, Circular dichroism spectroscopy shows that recombinant α-synuclein (10 μg) is unstructured in solution and becomes α-helical upon liposome binding (PS = phosphatidylserine; PC = phosphatidylcholine; molar protein-to-lipid ratio = 1:530). h, Recombinant (0.5 μg) and α-synuclein in brain cytosol (12 μg total protein) run as apparent tetramers on blue-native gels without boiling or after boiling for 5 min. i, Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) reveals that recombinant α-synuclein (0.5 mg) is monomeric.
Figure 2
Figure 2
a, SDS-PAGE analysis of five stages of α-synuclein purification from mouse brain (IEX, anion exchange chromatography, HIC, hydrophobic interaction chromatography). Purified α-synuclein was also analyzed by immunoblotting and mass spectrometry as shown. b, Mass spectrometry analysis reveals N-terminal acetylation of native α-synuclein. Shown is an extracted ion chromatogram of the N-terminally acetylated α-synuclein peptide (inset: MSMS spectrum containing the sequence of the N-terminal peptide and identified b and y ions). c, SEC-MALS shows that purified brain α-synuclein (150 μg) is largely monomeric (main peak with a mass of 17±1 kDa), but includes a minor component (plateau along the left shoulder with a mass of 58±5 kDa) that contains little detectable α-synuclein (see immunoblot in insert). Calculated masses were extracted from marked areas. d, CD spectroscopy of freshly purified brain α-synuclein (7.5 μM) shows mainly disordered conformations that progressively acquire structured conformations as a result of time- and temperature-dependent aggregation. e, Purified brain α-synuclein (0.12 mg/ml) rapidly aggregates as measured by dynamic light scattering immediately (0 h) or 152 h after purification.
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