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Structural disorder of monomeric α-synuclein persists in mammalian cells

Nature volume 530, pages 45–50 (2016)Cite this article

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Abstract

Intracellular aggregation of the human amyloid protein α-synuclein is causally linked to Parkinson’s disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here we use nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to derive atomic-resolution insights into the structure and dynamics of α-synuclein in different mammalian cell types. We show that the disordered nature of monomeric α-synuclein is stably preserved in non-neuronal and neuronal cells. Under physiological cell conditions, α-synuclein is amino-terminally acetylated and adopts conformations that are more compact than when in buffer, with residues of the aggregation-prone non-amyloid-β component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results establish that different types of crowded intracellular environments do not inherently promote α-synuclein oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells.

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Figure 1: Delivery of αSyn into mammalian cells.
Figure 2: αSyn in-cell NMR samples and spectra.
Figure 3: αSyn dynamics in cells and crowded solutions.
Figure 4: αSyn interactions and conformations in cells.
Figure 5: Compact αSyn structures in cells and crowded solutions.

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References

  1. Theillet, F. X. et al. Physicochemical properties of cells and their effects on intrinsically disordered proteins (IDPs). Chem. Rev. 114, 6661–6714 (2014)

    Article  CAS  Google Scholar 

  2. Goedert, M., Spillantini, M. G., Del Tredici, K. & Braak, H. 100 years of Lewy pathology. Nature Rev. Neurol. 9, 13–24 (2013)

    Article  CAS  Google Scholar 

  3. Iwai, A. et al. The precursor protein of non-A beta component of Alzheimer’s disease amyloid is a presynaptic protein of the central nervous system. Neuron 14, 467–475 (1995)

    Article  CAS  Google Scholar 

  4. Lashuel, H. A., Overk, C. R., Oueslati, A. & Masliah, E. The many faces of alpha-synuclein: from structure and toxicity to therapeutic target. Nature Rev. Neurosci. 14, 38–48 (2013)

    Article  CAS  Google Scholar 

  5. Dettmer, U., Selkoe, D. & Bartels, T. New insights into cellular α-synuclein homeostasis in health and disease. Curr. Opin. Neurobiol. 36, 15–22 (2015)

    Article  Google Scholar 

  6. Bartels, T., Choi, J. G. & Selkoe, D. J. α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Nature 477, 107–110 (2011)

    Article  ADS  CAS  Google Scholar 

  7. Wang, W. et al. A soluble α-synuclein construct forms a dynamic tetramer. Proc. Natl Acad. Sci. USA 108, 17797–17802 (2011)

    Article  ADS  CAS  Google Scholar 

  8. Binolfi, A., Theillet, F. X. & Selenko, P. Bacterial in-cell NMR of human alpha-synuclein: a disordered monomer by nature? Biochem. Soc. Trans. 40, 950–954 (2012)

    Article  CAS  Google Scholar 

  9. Burré, J. et al. Properties of native brain α-synuclein. Nature 498, E4–E6 (2013)

    Article  Google Scholar 

  10. Fauvet, B. et al. Characterization of semisynthetic and naturally N-alpha-acetylated α-synuclein in vitro and in intact cells: implications for aggregation and cellular properties of alpha-synuclein. J. Biol. Chem. 287, 28243–28262 (2012)

    Article  CAS  Google Scholar 

  11. Fauvet, B. et al. α-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer. J. Biol. Chem. 287, 15345–15364 (2012)

    Article  CAS  Google Scholar 

  12. Dettmer, U., Newman, A. J., Luth, E. S., Bartels, T. & Selkoe, D. In vivo cross-linking reveals principally oligomeric forms of α-synuclein and β-synuclein in neurons and non-neural cells. J. Biol. Chem. 288, 6371–6385 (2013)

    Article  CAS  Google Scholar 

  13. Dettmer, U. et al. Parkinson-causing alpha-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation. Nature Commun. 6, 7314 (2015)

    Article  ADS  Google Scholar 

  14. Luth, E. S., Bartels, T., Dettmer, U., Kim, N. C. & Selkoe, D. J. Purification of α-synuclein from human brain reveals an instability of endogenous multimers as the protein approaches purity. Biochemistry 54, 279–292 (2015)

    Article  CAS  Google Scholar 

  15. Selkoe, D. et al. Defining the native state of α-synuclein. Neurodegener. Dis. 13, 114–117 (2014)

    Article  CAS  Google Scholar 

  16. Uversky, V. N. Intrinsically disordered proteins and their (disordered) proteomes in neurodegenerative disorders. Front. Aging Neurosci. 7, 18 (2015)

    Article  Google Scholar 

  17. Paris, I. et al. The catecholaminergic RCSN-3 cell line: a model to study dopamine metabolism. Neurotox. Res. 13, 221–230 (2008)

    Article  CAS  Google Scholar 

  18. Wilhelm, B. G. et al. Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins. Science 344, 1023–1028 (2014)

    Article  ADS  CAS  Google Scholar 

  19. Okochi, M. et al. Constitutive phosphorylation of the Parkinson’s disease associated α-synuclein. J. Biol. Chem. 275, 390–397 (2000)

    Article  CAS  Google Scholar 

  20. Starheim, K. K., Gevaert, K. & Arnesen, T. Protein N-terminal acetyltransferases: when the start matters. Trends Biochem. Sci. 37, 152–161 (2012)

    Article  CAS  Google Scholar 

  21. Kang, L. et al. N-terminal acetylation of alpha-synuclein induces increased transient helical propensity and decreased aggregation rates in the intrinsically disordered monomer. Protein Sci. 21, 911–917 (2012)

    Article  CAS  Google Scholar 

  22. Maltsev, A. S., Ying, J. F. & Bax, A. Impact of N-terminal acetylation of α-synuclein on its random coil and lipid binding properties. Biochemistry 51, 5004–5013 (2012)

    Article  CAS  Google Scholar 

  23. Dikiy, I. & Eliezer, D. N-terminal acetylation stabilizes N-terminal helicity in lipid- and micelle-bound alpha-synuclein and increases its affinity for physiological membranes. J. Biol. Chem. 289, 3652–3665 (2014)

    Article  CAS  Google Scholar 

  24. Li, C. et al. Differential dynamical effects of macromolecular crowding on an intrinsically disordered protein and a globular protein: Implications for in-cell NMR spectroscopy. J. Am. Chem. Soc. 130, 6310–6311 (2008)

    Article  CAS  Google Scholar 

  25. Fusco, G. et al. Direct observation of the three regions in α-synuclein that determine its membrane-bound behaviour. Nature Commun. 5, 3827 (2014)

    Article  ADS  CAS  Google Scholar 

  26. Martorana, A. et al. Probing protein conformation in cells by EPR distance measurements using Gd3+ spin labelling. J. Am. Chem. Soc. 136, 13458–13465 (2014)

    Article  CAS  Google Scholar 

  27. Bertoncini, C. W. et al. Release of long-range tertiary interactions potentiates aggregation of natively unstructured α-synuclein. Proc. Natl Acad. Sci. USA 102, 1430–1435 (2005)

    Article  ADS  CAS  Google Scholar 

  28. Dedmon, M. M., Lindorff-Larsen, K., Christodoulou, J., Vendruscolo, M. & Dobson, C. M. Mapping long-range interactions in α-synuclein using spin-label NMR and ensemble molecular dynamics simulations. J. Am. Chem. Soc. 127, 476–477 (2005)

    Article  CAS  Google Scholar 

  29. Wu, K. P. & Baum, J. Detection of transient interchain interactions in the intrinsically disordered protein α-synuclein by NMR paramagnetic relaxation enhancement. J. Am. Chem. Soc. 132, 5546–5547 (2010)

    Article  CAS  Google Scholar 

  30. Kostka, M. et al. Single particle characterization of iron-induced pore-forming α-synuclein oligomers. J. Biol. Chem. 283, 10992–11003 (2008)

    Article  CAS  Google Scholar 

  31. Danielsson, J. et al. Thermodynamics of protein destabilization in live cells. Proc. Natl Acad. Sci. USA 112, 12402–12407 (2015)

    Article  ADS  CAS  Google Scholar 

  32. Monteith, W. B., Cohen, R. D., Smith, A. E., Guzman-Cisneros, E. & Pielak, G. J. Quinary structure modulates protein stability in cells. Proc. Natl Acad. Sci. USA 112, 1739–1742 (2015)

    Article  ADS  CAS  Google Scholar 

  33. Cremades, N. et al. Direct observation of the interconversion of normal and toxic forms of α-synuclein. Cell 149, 1048–1059 (2012)

    Article  CAS  Google Scholar 

  34. Galvagnion, C. et al. Lipid vesicles trigger α-synuclein aggregation by stimulating primary nucleation. Nature Chem. Biol. 11, 229–234 (2015)

    Article  CAS  Google Scholar 

  35. Lamberto, G. R. et al. Structural and mechanistic basis behind the inhibitory interaction of PcTS on α-synuclein amyloid fibril formation. Proc. Natl Acad. Sci. USA 106, 21057–21062 (2009)

    Article  ADS  CAS  Google Scholar 

  36. Ulmer, T. S. & Bax, A. Comparison of structure and dynamics of micellebound human α-synuclein and Parkinson disease variants. J. Biol. Chem. 280, 43179–43187 (2005)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Burre, T. Sudhof, D. Jovin, D. Mulvihill, P. Caviedes and T. Maritzen for plasmids and reagents, and for critical discussions. V. Subramanian, D. Eliezer, J. Clark and J. Dyson for carefully reading the manuscript and helpful comments. M. Herzig for essential input in the development of the electroporation protocol and M. Beerbaum and P. Schmieder for maintenance of the NMR infrastructure. RCSN-3 cells are made available by P. Caviedes upon request (pcaviede@med.uchile.cl). F.-X.T. acknowledges funding from the Association pour la Recherche sur le Cancer (ARC). D.G. acknowledges support by the Israel Science Foundation (ISF) F.I.R.S.T. program (grant 1114/12). P.S. was funded by the Deutsche Forschungsgemeinschaft (DFG) via an Emmy Noether Project Grant (SE1794/1-1). This work is supported by the European Research Council (ERC) Consolidator Grant (CoG) ‘NeuroInCellNMR’ (647474) awarded to P.S.

Author information

Author notes

  1. Francois-Xavier Theillet & Andres Binolfi

    Present address: † Present addresses: Department of Biochemistry, Biophysics and Structural Biology, Institute for Integrative Biology of the Cell (I2BC), UMR9198 CNRS, Bât 144, CEA Saclay, 91191 Gif-sur-Yvette, France (F.-X.T.); Max Planck Laboratory for Structural Biology, Chemistry and Molecular Biophysics of Rosario (MPLbioR-UNR) and Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario (IIDEFAR-CONICET), 27 de Febrero 210 bis; S2002LRK-Rosario, Argentina (A.B.).,

  2. Francois-Xavier Theillet, Andres Binolfi and Beata Bekei: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of NMR-supported Structural Biology, In-Cell NMR Laboratory, Leibniz Institute of Molecular Pharmacology (FMP Berlin), Robert-Rössle Strasse 10, Berlin, 13125, Germany

    Francois-Xavier Theillet, Andres Binolfi, Beata Bekei, Honor May Rose, Marchel Stuiver, Silvia Verzini, Marleen van Rossum & Philipp Selenko

  2. Department of Chemical Physics, Weizmann Institute of Science, Rehovot, 76100, Israel

    Andrea Martorana & Daniella Goldfarb

  3. Department of Molecular Physiology and Cell Biology, Leibniz Institute of Molecular Pharmacology (FMP Berlin), Robert-Rössle Strasse 10, Berlin, 13125, Germany

    Dorothea Lorenz

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Contributions

F.-X.T. and A.B. performed in-cell and in vitro NMR experiments. B.B., H.M.R., S.V., M.v.R. and M.S. developed the electroporation protocol, manipulated and handled cells, and performed light microscopy, cell and molecular biology experiments. A.M. and D.G. designed and performed EPR experiments, and analysed data. D.L. performed electron microscopy experiments. F.-X.T., A.B. and P.S. conceived the study, analysed the data and wrote the paper. F.-X.T., A.B. and B.B. contributed equally. All authors discussed the results and commented on the manuscript.

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Correspondence to Philipp Selenko.

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Extended data figures and tables

Extended Data Figure 1 Quality control experiments and full 2D in-cell NMR spectra.

a, Flow cytometry scatter-plots of electroporated HeLa, RCSN-3 and B65 cells carrying Atto488-tagged αSyn (red, x axis) counterstained with 7-AAD (y axis). Percentages of viable αSyn-positive (bottom quadrant) and apoptotic (top quadrant) cells are indicated. Mock-electroporated cells are shown in black. Immunofluorescence imaging of αSyn (red) in electroporated HeLa, RCSN-3 and B65 cells. Phalloidin staining shows actin filaments (green), DAPI staining identifies cell nuclei (blue). b, Cryo-electron microscopy and anti-αSyn immunogold-labelling of mock-electroporated (top) and αSyn-electroporated (bottom) HeLa and RCSN-3 cells confirms organelle intactness and uniform distribution of delivered αSyn. Arrowheads indicate positions of gold-particles indicative of intracellular αSyn. c, Representative 1D 1H–15N amide-envelope traces of αSyn in-cell NMR signals (Seff shown in red) in HeLa (Ncell = 2 × 107), RCSN-3 (Ncell = 7 × 107) and B65 (Ncell = 7 × 107) cells (electroporated with 400 μM αSyn). Reference traces of 5 μM 15N isotope-enriched, N-terminally acetylated αSyn in buffer are shown in black. 1D NMR traces of leakage tests after the respective in-cell NMR experiments, acquired with the same NMR acquisition parameters and spectrometer settings as for the in-cell NMR samples, are shown in blue (see Supplementary Methods). d, Exemplary Trypan blue cell-viability tests on in-cell NMR specimens. Average percentages of dead cells are indicated for the different cell lines. e, Time course western blot of αSyn leakage from HeLa cells (that is, highest percentage of dead cells after in-cell NMR experiments) reveals negligible amounts of extracellular protein. f, Overlay of 2D in-cell NMR spectra of αSyn in A2780, HeLa, RCSN-3, B65 and SK-N-SH cells (red, full spectral region) and of N-terminally acetylated αSyn in buffer (black). Selected regions of NMR spectra shown in Fig. 2b are shaded in grey. Sites of N-terminal line broadening are boxed. Arrows denote growth medium-specific metabolite signals (background). Chemical shift differences of non-proline backbone amide (1H–15N) resonances between αSyn in buffer and in cells were calculated as Δδ = [(Δδ1H)2+ (Δδ15N × 0.2)2]1/2 (in p.p.m.) and are shown next to the corresponding in-cell NMR spectra. Residues without Δδ values were not analysed owing to spectral overlap. Δδ of His50 is not included because of the known pH sensitivity of this resonance signal. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Figure 2 Reproducibility of in-cell NMR samples and spectra.

Representative in-cell NMR spectra (red) of independent replicate samples in the different mammalian cell types (electroporation concentrations of αSyn: 400 μM). 1D 1H–15N traces of in-cell amide-envelope signals (Seff red) are shown above the respective 2D NMR spectra (5 μM reference trace: black; leakage trace: blue). Ncell and NMR tube diameters are indicated. 2D reference NMR spectra of N-terminally acetylated αSyn (5 μM) are shown in black. Sites of N-terminal line broadening are boxed. Growth medium-specific natural abundance metabolite signals (background) are marked with arrows.

Extended Data Figure 3 Low contour level in-cell and lysate NMR spectra.

a, Same in-cell NMR spectra as in Extended Data Fig. 1f plotted at lower contour levels (red). Boxes and arrows indicate broadened NMR signals of N-terminal αSyn residues, including acetylated Met1. N-terminally acetylated αSyn (5 μM) is shown in the respective reference NMR spectra (black). Asterisks denote natural abundance signals of background metabolites (growth medium specific). b, Same samples as in a after cell lysis. NMR spectra were directly acquired on the soluble fractions of cleared lysates (red), alleviating N-terminal line broadening and displaying signal overlap with the acetylated reference state of αSyn. Chemical shift changes of His50 indicate minor pH changes. c, Left, fractionation, SDS–PAGE separation and western blotting of HeLa and RCSN-3 in-cell NMR sample lysates indicate recovery of intracellular αSyn in soluble lysate fractions. Right, separation of SK-N-SH αSyn in-cell NMR sample lysates by native PAGE yields monomeric αSyn. Covalent low- and high-molecular mass (MW) cytochrome c/H2O2 aggregates of αSyn and non-covalent αSyn fibrils serve as input controls for differently aggregated forms of the protein (see Supplementary Methods). For gel source data, see Supplementary Fig. 1.

Extended Data Figure 4 NMR characterization of N-terminally acetylated αSyn and in-cell relaxation data.

a, Secondary structure propensity (SSP) scores and Cα, Cβ chemical shift deviations from random coil values indicate levels of residual helicity within the first ten residues of non-acetylated (left) and N-terminally acetylated (right) αSyn. b, Dynamic light scattering (DLS) of SUVs prepared from pig brain polar lipids (average diameter ~60 nm) and schematic depiction of one possible αSyn conformation when bound to SUVs (according to ref. 36). αSyn membrane binding is mediated by its first ~100 residues forming either a continuous or a broken α-helix. Residue-resolved signal intensity ratios (I/I0) of free αSyn(I0) versus αSyn in the presence of SUVs (I), obtained with 15N isotope-enriched, N-terminally acetylated αSyn and saturating amounts of SUVs (16 g l−1). αSyn membrane binding results in uniform line broadening of its first ~100 residues. c, Line-broadening profiles (I/I0) of αSyn residues in HeLa, RCSN-3 and B65 cells are similar to A2780 and SK-N-SH cells (Fig. 3a), irrespective of different intracellular αSyn concentrations. For simplification, profiles show values averaged over three consecutively resolved residues. d, Representative residue-resolved T1 relaxation curves acquired on intracellular αSyn in A2780 and SK-N-SH cells (red), and in buffer (grey). Residues of the NAC are shaded in grey. e, Experimental T2 relaxation curves for residues in different regions of intracellular αSyn in A2780 (left) and SK-N-SH (right) cells. All in-cell NMR relaxation data were acquired on two independent samples. Measurement points indicate the mean of two independent experiments. Error bars show the range of lowest and highest values.

Source data

Extended Data Figure 5 αSyn relaxation rates in A2780 and SK-N-SH cells and in crowded solutions.

Residue-resolved 15N longitudinal (R1) and transverse (R2) relaxation rates, 15N–1H NOE data, and proton (1H) R2 values of N-terminally acetylated αSyn in buffer (grey), A2780 and SK-N-SH cells (red), in the presence of 200 g l−1 Ficoll (blue), 200 g l−1 BSA (green), 10 g l−1 lysozyme (orange), sub-saturating amounts of SUVs (3.2 g l−1, purple) and 8 M urea (grey). All in vitro data were acquired on samples at pH 6.4. Relaxation data from measurements at pH 7.4 are available in the respective Source Data. Relaxation measurements were performed on two independent in vitro or in-cell NMR samples with data points representing their mean. Error bars show the range of lowest and highest values. Residues of the NAC region are shaded in grey.

Source data

Extended Data Figure 6 Residue-resolved τc and Rex profiles of αSyn in cells and in crowded solutions.

a, Rotational correlation times (τc) of N-terminally acetylated αSyn in buffer (grey) and in A2780 and SK-N-SH cells (red). In-cell to in-buffer τc ratios show average values of three consecutively resolved residues. Overall mobility of intracellular αSyn is reduced by a factor of ~1.5, which is similar to αSyn in the presence of 200 g l−1 glycerol (available in Source Data). Additional mobility profiles and τc ratios of N-terminally acetylated αSyn in solutions containing 200 g l−1 Ficoll (blue), 200 g l−1 BSA (green), 10 g l−1 lysozyme (orange) and 8 M urea (light grey) at pH 6.4 (data from measurements at pH 7.4 are available in the respective Source Data). b, Calculated Rex profiles of N-terminally acetylated αSyn in A2780 and SK-N-SH cells, and in the presence of sub-saturating amounts of SUVs (3.2 g l−1, pH 6.4). Regions exhibiting pronounced exchange behaviours are marked with arrows. c, d, Rex profiles of N-terminally acetylated αSyn in the presence of 200 g l−1 BSA (green) (c) and 10 g l−1 lysozyme (orange) (d) at pH 6.4 (data from measurements at pH 7.4 are available in the respective Source Data). Insets depict surface representations of BSA and lysozyme, colour-coded according to their electrostatic surface potentials. Isoelectric points (pI) of BSA and lysozyme are 4.7 and 11.35, respectively. N- and C-terminal line broadening effects observed in cells are recapitulated with SUVs and BSA, and with lysozyme, respectively. e, NMR relaxation data, τc and Rex profiles of N-terminally acetylated αSyn in lysozyme-crowded solutions (10 g l−1) at 300 mM salt (pH 6.4) reveal diminished exchange contributions for C-terminal αSyn residues. Data points with error bars represent the mean of two independent experiments. Error bars show the range of lowest and highest values. Residues of the NAC region are shaded in grey.

Source data

Extended Data Figure 7 Relaxation data of αSyn(F4A;Y39A) in cells and crowded solutions.

a, Residue-resolved relaxation data of N-terminally acetylated αSyn(F4A;Y39A) in buffer (grey), in A2780 and SK-N-SH cells (light red) and in the presence of 200 g l−1 of BSA (light green) at pH 6.4 (relaxation data from measurements at pH 7.4 are available in the respective Source Data). b, Residue-resolved relaxation data of N-terminally acetylated αSyn(F4A;Y39A) in the presence of sub-saturating amounts of SUVs (3.2 g l−1, pH 6.4). Rex panels show the comparison between wild-type (WT) and mutant (F4A;Y39A) αSyn in all cases. All relaxation measurements were performed on two independent samples. Error bars show the range of lowest and highest values. For simplification, profiles of τc ratios (τc/τc-buffer) depict values averaged over three consecutively resolved residues. SSP scores indicating residual helicity within the first ten residues of N-terminally acetylated αSyn(F4A;Y39A) are shown on top of the comparison of signal intensity ratios (I/I0) of wild-type (dark blue) and mutant (light blue) protein in the presence of SUVs.

Source data

Extended Data Figure 8 PRE data of αSyn in cells and crowded solutions.

Top, PRE profiles of S42C Gd(iii)-DOTA tagged N-terminally acetylated αSyn in buffer, cells and different crowded solutions at pH 6.4 (data obtained at pH 7.4 are available in the respective Source Data). Intensity ratios (Ipara/Idia) of αSyn in mixtures of 70% Gd(iii)-DOTA tagged paramagnetic αSyn and 30% Lu(iii)-DOTA tagged diamagnetic αSyn. 100% diamagnetic Lu(iii)-DOTA tagged N-terminally acetylated αSyn was used to measure reference intensities (that is, Idia). Red bars indicate residues in the vicinity of the Gd(iii)-DOTA tag experiencing proximal PRE effects and line broadening beyond the detection limit. Measurement points with error bars indicate the mean of two independent experiments. Error bars show the range of lowest and highest values. Paramagnetic R2 (1H) rates and calculated PRE distance averages (< d6>1/6) in comparison to values measured in buffer (grey) are indicated. PRE-derived distances were obtained assuming that every Gd(iii)-1H vector fluctuation rate scales linearly with that of the backbone N–H vector and the Gd(iii)-DOTA complex. Distance ratios (Dcell/crowding agent/Dbuffer) indicate levels of αSyn compaction and represent average ratios over three consecutively resolved residues. Middle, same PRE experiments with N122C Gd(iii)-DOTA tagged, N-terminally acetylated αSyn. Bottom, PRE experiments with S42C and N122C Gd(iii)-DOTA tagged, N-terminally acetylated αSyn in the presence of 8 M urea.

Source data

Extended Data Figure 9 Stable Gd(iii)-DOTA tagged αSyn for in-cell PRE and EPR-DEER experiments.

a, Size-exclusion chromatography (SEC) profiles of single- (that is, Q24C, S42C and N122C) and double- (that is, Q24C;N122C and S42C;N122C) Gd(iii)/Lu(iii)-DOTA-tagged αSyn (top) and corresponding dynamic light scattering (DLS) of monomeric protein fractions (bottom). b, SDS–PAGE separation of Gd(iii)-DOTA αSyn reveals characteristic molecular mass differences of single- and double-tagged samples (left). Western blot of exemplary A2780 in-cell EPR samples (20 × 106 cells) electroporated with 125 μM (low) or 250 μM (high) Gd(iii)-DOTA αSyn (right). For gel source data, see Supplementary Fig. 1. c, Echo-detected in-cell EPR spectrum of S42C-N122C Gd(iii)-DOTA αSyn in A2780 cells (left). EPR signals of intracellular Mn(ii) are indicated with asterisks. Red and black arrows denote microwave pump (v2) and observe (v1) frequencies, respectively. In-cell echo decays of the single- and double- Gd(iii)-DOTA tagged αSyn samples are shown on the right. DEER traces of S42C;N122C and Q24C;N122C Gd(iii)-DOTA αSyn in A2780 cells (red), and in buffer (grey) after background correction. Calculated distance distributions are shown on the right. d, DEER traces of S42C;N122C Gd(iii)-DOTA αSyn in the presence of 8 M urea (black) and in buffer (grey). Data were recorded on a different protein batch sample to that shown in c. e, Intermolecular DEER measurements of three A2780 in-cell EPR samples carrying single Gd(iii)-DOTA αSyn (that is, N122C, S42C and Q24C) at intracellular concentrations of 40 ± 10 μM reveal absence of detectable spin–spin coupling, as evident from straight decays of the corresponding DEER traces. Cartoon illustrations on the right depict possible αSyn–αSyn contacts with orientations chosen arbitrarily. f, Intermolecular PRE profiles of mixtures (50:50) of 15N isotope-enriched αSyn and of Gd(iii)-DOTA (S42C or N122C) αSyn containing natural abundance nitrogen in A2780 cells (red, intracellular αSyn concentrations ~100 μM), and in buffer (grey). Cartoon illustrations depict possible αSyn–αSyn contacts with orientations chosen arbitrarily. Intermolecular PRE effects are manifested by selective signal attenuations (I/I0) of the isotope-enriched, NMR-visible portion of the αSyn mixture (I) over reference samples containing only 15N isotope-enriched αSyn (I0). For simplification, I/I0 profiles show values averaged over three consecutively resolved residues. Experiments with reconstituted mixtures of αSyn (100 μM) in the presence of Ficoll (blue, 200 g l−1), BSA (green, 200 g l−1) and lysozyme (orange, 10 g l−1) are shown below. Insets depict magnified regions of preferred αSyn–αSyn contacts. Intermolecular PRE effects are largest for solutions containing Ficoll and BSA, whereas they are not detected in A2780 cells. All αSyn constructs are N-terminally acetylated.

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Theillet, FX., Binolfi, A., Bekei, B. et al. Structural disorder of monomeric α-synuclein persists in mammalian cells. Nature 530, 45–50 (2016). https://doi.org/10.1038/nature16531

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