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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Winter R, Noll F (1998) Methoden der Biophysikalischen Chemie. Springer, Stuttgart
Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer, New York
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.2013.06.005
Burstein EA, Vedenkina NS, Ivkova MN (1973) Fluorescence and the location of tryptophan residues in protein molecules. Photochem Photobiol 18(4):263–279
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)75824-9
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.21857
Chen Y, Barkley MD (1998) Toward understanding tryptophan fluorescence in proteins. Biochemistry 37(28):9976–9982
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.M007021
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/bi9803933
Eisinger J (1969) Intramolecular energy transfer in adrenocorticotropin. Biochemistry 8(10):3902–3908
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.2005.08.046
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/bi0107694
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.5560041120
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/ac801676j
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.2016.01.007
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Hellmann, N., Schneider, D. (2019). Hands On: Using Tryptophan Fluorescence Spectroscopy to Study Protein Structure. In: Kister, A. (eds) Protein Supersecondary Structures. Methods in Molecular Biology, vol 1958. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9161-7_20
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9161-7_20
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9160-0
Online ISBN: 978-1-4939-9161-7
eBook Packages: Springer Protocols