Advertisement

Hands On: Using Tryptophan Fluorescence Spectroscopy to Study Protein Structure

  • Nadja HellmannEmail author
  • Dirk Schneider
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1958)

Abstract

Fluorescence spectroscopy is well suited to obtain information about the structure and function of proteins. The major advantage of this spectroscopic technique is the pronounced dependence of the fluorescence emission characteristics of fluorophores on their distinct local environment and the rather inexpensive equipment required. In particular, the use of intrinsic tryptophan fluorescence offers the possibility to study structure and function of proteins without the need to modify the protein. While fluorescence spectroscopy is technically not demanding, a number of factors can artificially alter the results. In this article, we systematically describe the most common applications in fluorescence spectroscopy of proteins, i.e., how to gain information about the local environment of tryptophan residues and how to employ changes in the environment to monitor an interaction with other substances. In particular, we discuss pitfalls and wrong and/or misleading interpretations of gained data together with potential solutions.

Key words

Tryptophan Intrinsic fluorescence Inner filter effect Quenching Energy transfer Protein fluorescence 

References

  1. 1.
    Winter R, Noll F (1998) Methoden der Biophysikalischen Chemie. Springer, StuttgartCrossRefGoogle Scholar
  2. 2.
    Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer, New YorkCrossRefGoogle Scholar
  3. 3.
    Tognotti D, Gabellieri E, Morelli E et al (2013) Temperature and pressure dependence of azurin stability as monitored by tryptophan fluorescence and phosphorescence. The case of F29A mutant. Biophys Chem 182:44–50.  https://doi.org/10.1016/j.bpc.2013.06.005CrossRefPubMedGoogle Scholar
  4. 4.
    Burstein EA, Vedenkina NS, Ivkova MN (1973) Fluorescence and the location of tryptophan residues in protein molecules. Photochem Photobiol 18(4):263–279CrossRefGoogle Scholar
  5. 5.
    Reshetnyak YK, Burstein EA (2001) Decomposition of protein tryptophan fluorescence spectra into log-normal components. II. The statistical proof of discreteness of tryptophan classes in proteins. Biophys J 81(3):1710–1734.  https://doi.org/10.1016/S0006-3495(01)75824-9CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Shen C, Menon R, Das D et al (2008) The protein fluorescence and structural toolkit: Database and programs for the analysis of protein fluorescence and structural data. Proteins 71(4):1744–1754.  https://doi.org/10.1002/prot.21857CrossRefPubMedGoogle Scholar
  7. 7.
    Chen Y, Barkley MD (1998) Toward understanding tryptophan fluorescence in proteins. Biochemistry 37(28):9976–9982CrossRefGoogle Scholar
  8. 8.
    Weinberg RB, Cook VR (2010) Distinctive structure and interfacial activity of the human apolipoprotein A-IV 347S isoprotein. J Lipid Res 51(9):2664–2671.  https://doi.org/10.1194/jlr.M007021CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Santos NC, Prieto M, Castanho MA (1998) Interaction of the major epitope region of HIV protein gp41 with membrane model systems. A fluorescence spectroscopy study. Biochemistry 37(24):8674–8682.  https://doi.org/10.1021/bi9803933CrossRefPubMedGoogle Scholar
  10. 10.
    Eisinger J (1969) Intramolecular energy transfer in adrenocorticotropin. Biochemistry 8(10):3902–3908CrossRefGoogle Scholar
  11. 11.
    Kaylor J, Bodner N, Edridge S et al (2005) Characterization of oligomeric intermediates in alpha-synuclein fibrillation: FRET studies of Y125W/Y133F/Y136F alpha-synuclein. J Mol Biol 353(2):357–372.  https://doi.org/10.1016/j.jmb.2005.08.046CrossRefPubMedGoogle Scholar
  12. 12.
    Eisenhawer M, Cattarinussi S, Kuhn A et al (2001) Fluorescence resonance energy transfer shows a close helix−helix distance in the transmembrane M13 procoat protein. Biochemistry 40(41):12321–12328.  https://doi.org/10.1021/bi0107694CrossRefPubMedGoogle Scholar
  13. 13.
    Pace CN, Vajdos F, Fee L et al (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4(11):2411–2423.  https://doi.org/10.1002/pro.5560041120CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Gu Q, Kenny JE (2009) Improvement of inner filter effect correction based on determination of effective geometric parameters using a conventional fluorimeter. Anal Chem 81(1):420–426.  https://doi.org/10.1021/ac801676jCrossRefPubMedGoogle Scholar
  15. 15.
    Root KT, Glover KJ (2016) Reconstitution and spectroscopic analysis of caveolin-1 residues 62-178 reveals that proline 110 governs its structure and solvent exposure. Biochim Biophys Acta 1858(4):682–688.  https://doi.org/10.1016/j.bbamem.2016.01.007CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Institute for Pharmacy and BiochemistryJohannes Gutenberg-University MainzMainzGermany

Personalised recommendations