Monitoring methanol-induced protein unfolding by fluorescence anisotropy measurements of covalently labelled rhodamine probe

  • Antonin Soleilhac
  • Franck Bertorelle
  • Philippe Dugourd
  • Marion Girod
  • Rodolphe Antoine
Regular Article
Part of the following topical collections:
  1. Topical Issue: Dynamics of Systems at the Nanoscale


We describe the use of an extrinsic fluorophore (rhodamine B isothiocyanate) as a versatile probe to measure rotational motions of proteins. To illustrate the usefulness of this probe, we describe the fluorescence anisotropy values of this fluorophore covalently linked to myoglobin protein measured in aqueous solutions of increased methanol content. Methanol-induced unfolding is revealed by the transition from constrained to free rotation of the covalently attached rhodamine B fluorophore.

Graphical abstract

Supplementary material


  1. 1.
    J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd edn. (Springer Science+Business Media, LLC, Singapore, 2006)Google Scholar
  2. 2.
    A.S. Ladokhin, in Encyclopedia of Analytical Chemistry, edited by R.A. Meyers (Wiley, New York, 2000), pp. 5762–5779Google Scholar
  3. 3.
    T. Ha, T. Enderle, D.F. Ogletree, D.S. Chemla, P.R. Selvin, S. Weiss, Proc. Natl. Acad. Sci. 93, 6264 (1996)ADSCrossRefGoogle Scholar
  4. 4.
    D. Canet, K. Doering, C.M. Dobson, Y. Dupont, Biophys. J. 80, 1996 (2001)CrossRefGoogle Scholar
  5. 5.
    A. Ghisaidoobe, S. Chung, Int. J. Mol. Sci. 15, 22518 (2014)CrossRefGoogle Scholar
  6. 6.
    A. Hawe, M. Sutter, W. Jiskoot, Pharm. Res. 25, 1487 (2008)CrossRefGoogle Scholar
  7. 7.
    V. LeTilly, C.A. Royer, Biochemistry 32, 7753 (1993)CrossRefGoogle Scholar
  8. 8.
    A. Marczak, Bioelectrochemistry 74, 236 (2009)CrossRefGoogle Scholar
  9. 9.
    S. Weiss, Nat. Struct. Mol. Biol. 7, 724 (2000)CrossRefGoogle Scholar
  10. 10.
    G. Hungerford, J. Benesch, J.F. Mano, R.L. Reis, Photochem. Photobiol. Sci. 6, 152 (2007)CrossRefGoogle Scholar
  11. 11.
    M. Brinkley, Bioconjug. Chem. 3, 2 (1992)CrossRefGoogle Scholar
  12. 12.
    J.C. Kendrew, G. Bodo, H.M. Dintzis, R.G. Parrish, H. Wyckoff, D.C. Phillips, Nature 181, 662 (1958)ADSCrossRefGoogle Scholar
  13. 13.
    A. Castro-Forero, D. Jiménez, J. López-Garriga, M. Torres-Lugo, J. Appl. Polym. Sci. 107, 881 (2008)CrossRefGoogle Scholar
  14. 14.
    L.F. Mottram, S. Forbes, B.D. Ackley, B.R. Peterson, Beilstein, J. Org. Chem. 8, 2156 (2012)CrossRefGoogle Scholar
  15. 15.
    M. Girod, X. Dagany, R. Antoine, P. Dugourd, Int. J. Mass Spectrom. 308, 41 (2011)ADSCrossRefGoogle Scholar
  16. 16.
    M. Girod, X. Dagany, V. Boutou, M. Broyer, R. Antoine, P. Dugourd, A. Mordehai, C. Love, M. Werlich, J. Fjeldsted, G. Stafford, Phys. Chem. Chem. Phys. 14, 9389 (2012)CrossRefGoogle Scholar
  17. 17.
    A. Soleilhac, X. Dagany, P. Dugourd, M. Girod, R. Antoine, Anal. Chem. 87, 8210 (2015)CrossRefGoogle Scholar
  18. 18.
    A. Soleilhac, M. Girod, P. Dugourd, B. Burdin, J. Parvole, P.-Y. Dugas, F. Bayard, E. Lacôte, E. Bourgeat-Lami, R. Antoine, Langmuir 32, 4052 (2016)CrossRefGoogle Scholar
  19. 19.
    D.M. Jameson, J.A. Ross, Chem. Rev. 110, 2685 (2010)CrossRefGoogle Scholar
  20. 20.
    D. Lavalette, C. Tétreau, M. Tourbez, Y. Blouquit, Biophys. J. 76, 2744 (1999)CrossRefGoogle Scholar
  21. 21.
    M.T. Tyn, T.W. Gusek, Biotechnol. Bioeng. 35, 327 (1990)CrossRefGoogle Scholar
  22. 22.
    S.Z. Mikhail, W.R. Kimel, J. Chem. Eng. Data 6, 533 (1961)CrossRefGoogle Scholar
  23. 23.
    T.T. Herskovits, B. Gadegbeku, H. Jaillet, J. Biol. Chem. 245, 2588 (1970)Google Scholar
  24. 24.
    Q. Shao, J. Phys. Chem. B 118, 6175 (2014)CrossRefGoogle Scholar
  25. 25.
    A. Fernández, O. Sinanoglu, Biophys. Chem. 21, 163 (1985)CrossRefGoogle Scholar
  26. 26.
    J. Seo, W. Hoffmann, S. Warnke, M.T. Bowers, K. Pagel, G. von Helden, Angew. Chem. Int. Ed. 55, 14173 (2016)CrossRefGoogle Scholar
  27. 27.
    Y.Y. Gottlieb, P. Wahl, J. Chim. Phys. Phys. Chim. Biol. 60, 849 (1963)CrossRefGoogle Scholar
  28. 28.
    K. Kinosita, S. Kawato, A. Ikegami, Biophys. J. 20, 289 (1977)CrossRefGoogle Scholar
  29. 29.
    G.F. Schröder, U. Alexiev, H. Grubmüller, Biophys. J. 89, 3757 (2005)CrossRefGoogle Scholar

Copyright information

© EDP Sciences, SIF, Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Institut lumière matière, UMR5306 Université Claude Bernard Lyon1-CNRS, Université de LyonVilleurbanne CedexFrance
  2. 2.Univ Lyon, CNRS, Université Claude Bernard Lyon 1, Ens de Lyon, Institut des Sciences Analytiques, UMR 5280VilleurbanneFrance

Personalised recommendations