Probing Alpha-Synuclein Conformations by Electron Paramagnetic Resonance (EPR) Spectroscopy

  • Julia Cattani
  • Theresa Braun
  • Malte DrescherEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1948)


Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling is ideally suited to study structure, dynamics, and interactions of intrinsically disordered proteins as alpha-synuclein.

Here we describe all steps required for a corresponding study: the spin labeling procedure, sample preparation, spectroscopic experimental procedure, and data analysis.

Key words

Electron paramagnetic resonance (EPR) spectroscopy Protein structure Protein dynamics Site-directed spin labeling Alpha-synuclein Intrinsically disordered protein 



This work was supported by the DFG within SFB 969, project C03.


  1. 1.
    Drescher M, Veldhuis G, van Rooijen BD et al (2008) Antiparallel arrangement of the helices of vesicle-bound α-synuclein. J Am Chem Soc 130:7796–7797. Scholar
  2. 2.
    Kumar P, Segers-Nolten IMJ, Schilderink N et al (2015) Parkinson’s protein α-synuclein binds efficiently and with a novel conformation to two natural membrane mimics. PLoS One 10:e0142795. Scholar
  3. 3.
    Robotta M, Braun P, van Rooijen B et al (2011) Direct evidence of coexisting horseshoe and extended helix conformations of membrane-bound alpha-synuclein. ChemPhysChem 12:267–269. Scholar
  4. 4.
    Jao CC, Hegde BG, Chen J et al (2008) Structure of membrane-bound α-synuclein from site-directed spin labeling and computational refinement. Proc Natl Acad Sci 105:19666–19671. Scholar
  5. 5.
    Georgieva ER, Ramlall TF, Borbat PP et al (2008) Membrane-bound α-Synuclein forms an extended helix: long-distance pulsed ESR measurements using vesicles, bicelles, and rodlike micelles. J Am Chem Soc 130:12856–12857. Scholar
  6. 6.
    Trexler AJ, Rhoades E (2009) α-Synuclein binds large unilamellar vesicles as an extended helix. Biochemistry (Mosc) 48:2304–2306. Scholar
  7. 7.
    Robotta M, Hintze C, Schildknecht S et al (2012) Locally resolved membrane binding affinity of the N-terminus of α-synuclein. Biochemistry (Mosc) 51:3960–3962. Scholar
  8. 8.
    Robotta M, Gerding HR, Vogel A et al (2014) Alpha-synuclein binds to the inner membrane of mitochondria in an α-helical conformation. Chembiochem 15:2499–2502. Scholar
  9. 9.
    Robotta M, Cattani J, Martins JC et al (2017) Alpha-synuclein disease mutations are structurally defective and locally affect membrane binding. J Am Chem Soc 139:4254–4257. Scholar
  10. 10.
    Theillet F-X, Binolfi A, Bekei B et al (2016) Structural disorder of monomeric α-synuclein persists in mammalian cells. Nature 530:45–50CrossRefGoogle Scholar
  11. 11.
    Cattani J, Subramaniam V, Drescher M (2017) Room-temperature in-cell EPR spectroscopy: alpha-synuclein disease variants remain intrinsically disordered in the cell. Phys Chem Chem Phys 19:18147. Scholar
  12. 12.
    Li Q, Fung LW-M (2009) Structural and dynamic study of the tetramerization region of non-erythroid α-spectrin: a frayed Helix revealed by site-directed spin labeling electron paramagnetic resonance. Biochemistry (Mosc) 48:206–215. Scholar
  13. 13.
    Watanabe Y, Inanami O, Horiuchi M et al (2006) Identification of pH-sensitive regions in the mouse prion by the cysteine-scanning spin-labeling ESR technique. Biochem Biophys Res Commun 350:549–556. Scholar
  14. 14.
    Sahu ID, Craig AF, Dunagan MM et al (2015) Probing structural dynamics and topology of the KCNE1 membrane protein in lipid bilayers via site-directed spin labeling and electron paramagnetic resonance spectroscopy. Biochemistry (Mosc) 54:6402. Scholar
  15. 15.
    Koteiche HA, Reeves MD, Mchaourab HS (2003) Structure of the substrate binding pocket of the multidrug transporter EmrE: site-directed spin labeling of transmembrane segment 1. Biochemistry (Mosc) 42:6099–6105. Scholar
  16. 16.
    Isas JM, Langen R, Haigler HT, Hubbell WL (2002) Structure and dynamics of a helical hairpin and loop region in Annexin 12: a site-directed spin labeling study. Biochemistry (Mosc) 41:1464–1473. Scholar
  17. 17.
    Stoll S, Schweiger A (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J Magn Reson 178:42–55. Scholar
  18. 18.

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Chemistry and Konstanz Research School Chemical BiologyUniversity of KonstanzKonstanzGermany

Personalised recommendations