Abstract
Lifetimes of fluorescent states and their fluorescence intensities are strictly coupled and very sensitive to the environment of the fluorophores. The advantage of measuring lifetimes, next to intensities, comes from the fact that it can reveal heterogeneity and dynamic properties of this environment. In this way lifetime analysis can be used to characterize static and dynamic conformational properties and heterogeneity of fluorescent groups in different areas of a protein and as a function of time for an evolving protein. The phenomena that determine the lifetime of a label are its intrinsic properties, dynamic quenching by neighboring groups, exposure to the solvent, as well as Förster resonance energy transfer (FRET) between different groups. The basic principles of these fluorescence phenomena can be found extensively described in the excellent book of Lakowicz (Principles of fluorescence spectroscopy, 3rd edn. Springer, New York, 2006). The fluorescent groups involved are either natural amino acid side chains like tryptophan (Trp) or tyrosine (Tyr), or fluorescent labels covalently engineered into the protein. Even a single fluorescent group can show indications of heterogeneity in the local environment. If several natural fluorescent groups are present, the properties of the individual groups can be separated using site-directed mutagenesis, and additivity of their contributions can be analyzed (Engelborghs, Spectrochim Acta A Mol Biomol Spectrosc 57(11):2255–2270, 2001). If no fluorescent group is naturally present, site-directed mutagenesis can be used to introduce either a fluorescent amino acid or a cysteine allowing chemical labeling.
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References
Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, New York
Engelborghs Y (2001) The analysis of time resolved protein fluorescence in multi-tryptophan proteins. Spectrochim Acta A Mol Biomol Spectrosc 57(11):2255–2270
Wu PG, Brand L (1994) Resonance energy transfer: methods and applications [review]. Anal Biochem 218:1–13
Ferreon AC, Gambin Y, Lemke EA, Deniz AA (2009) Interplay of alpha-synuclein binding and conformational switching probed by single-molecule fluorescence. Proc Natl Acad Sci USA 106(14):5645–5650
Ulrih NP, Barry CH, Fink AL (2008) Impact of Tyr to Ala mutations on alpha-synuclein fibrillation and structural properties. Biochim Biophys Acta 1782(10):581–585
Van Rooijen BD, van Leijenhorst-Groener KA, Claessens MM, Subramaniam V (2009) Tryptophan fluorescence reveals structural features of alpha-synuclein oligomers. J Mol Biol 394(5):826–833
Lee JC, Langen R, Hummel PA, Gray HB, Winkler JR (2004) Alpha-synuclein structures from fluorescence energy-transfer kinetics: implications for the role of the protein in Parkinson’s disease. Proc Natl Acad Sci USA 101(47):16466–16471
Winkler GR, Harkins SB, Lee JC, Gray HB (2006) Alpha-synuclein structures probed by 5-fluorotryptophan fluorescence and 19F NMR spectroscopy. J Phys Chem B 110(13):7058–7061
Gerard M, Debyser Z, Desender L, Kahle PJ, Baert J, Baekelandt V, Engelborghs Y (2006) The aggregation of alpha-synuclein is stimulated by FK506 binding proteins as shown by fluorescence correlation spectroscopy. FASEB J 20:524–526
Neuweiler H, Banachewicz W, Fersht AR (2010) Kinetics of chain motions within a protein-folding intermediate. Proc Natl Acad Sci USA 107(51):22106–22110
Boens N et al (2007) Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy. Anal Chem 79(5):2137–2149
Kaylor J, Bodner N, Edridge S, Yamin G, Hong D-P, Fink A (2005) Characterisation of oligomeric intermediates in α-synuclein fibrillation: FRET studies of Y125W/Y133F/Y136Fα-synuclein. J Mol Biol 353:357–372
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Schreurs, S., Kluba, M., Meuvis, J., Engelborghs, Y. (2012). Fluorescence Lifetime Measurements of Intrinsically Unstructured Proteins: Application to α-Synuclein. In: Uversky, V., Dunker, A. (eds) Intrinsically Disordered Protein Analysis. Methods in Molecular Biology, vol 895. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-927-3_27
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DOI: https://doi.org/10.1007/978-1-61779-927-3_27
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