Fluorescence Spectroscopy and Microscopy pp 597-615

Part of the Methods in Molecular Biology book series (MIMB, volume 1076) | Cite as

PET-FCS: Probing Rapid Structural Fluctuations of Proteins and Nucleic Acids by Single-Molecule Fluorescence Quenching

  • Markus Sauer
  • Hannes Neuweiler


Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van der Waals contact and can be used as reporter for structural fluctuations at the 1-nm scale in proteins, peptides, and nucleic acids. The combination of PET with fluorescence correlation spectroscopy (FCS) establishes a powerful method (PET-FCS) to study equilibrium dynamics at the single-molecule level on time scales from nano- to milliseconds. We delineate the fundamentals of PET-based fluorescence quenching, reporter engineering, instrumental and experimental design, and provide examples.

Key words

Fluorescence quenching Photoinduced electron transfer Fluorescence correlation spectroscopy Protein folding and dynamics Intrinsically disordered proteins 


  1. 1.
    Dyson HJ, Wright PE (2005) Intrinsically unstructured proteins and their functions. Nat Rev Mol Cell Biol 6(3):197–208CrossRefPubMedGoogle Scholar
  2. 2.
    Dunker AK, Silman I, Uversky VN, Sussman JL (2008) Function and structure of inherently disordered proteins. Curr Opin Struct Biol 18(6):756–764CrossRefPubMedGoogle Scholar
  3. 3.
    Fersht AR (2008) From the first protein structures to our current knowledge of protein folding: delights and scepticisms. Nat Rev Mol Cell Biol 9(8):650–654CrossRefPubMedGoogle Scholar
  4. 4.
    Henzler-Wildman K, Kern D (2007) Dynamic personalities of proteins. Nature 450(7172):964–972CrossRefPubMedGoogle Scholar
  5. 5.
    Kapanidis AN, Strick T (2009) Biology, one molecule at a time. Trends Biochem Sci 34(5):234–243CrossRefPubMedGoogle Scholar
  6. 6.
    Sauer M, Hofkens J, Enderlein J (2011) Handbook of fluorescence spectroscopy and imaging: from single molecules to ensembles. Wiley-VCH, Weinheim, GermanyCrossRefGoogle Scholar
  7. 7.
    Weiss S (1999) Fluorescence spectroscopy of single biomolecules. Science 283(5408):1676–1683CrossRefPubMedGoogle Scholar
  8. 8.
    Selvin PR (2000) The renaissance of fluorescence resonance energy transfer. Nat Struct Biol 7(9):730–734CrossRefPubMedGoogle Scholar
  9. 9.
    Schuler B, Eaton WA (2008) Protein folding studied by single-molecule FRET. Curr Opin Struct Biol 18(1):16–26CrossRefPubMedGoogle Scholar
  10. 10.
    Neuweiler H, Sauer M (2004) Using photoinduced charge transfer reactions to study conformational dynamics of biopolymers at the single-molecule level. Curr Pharm Biotechnol 5(3):285–298CrossRefPubMedGoogle Scholar
  11. 11.
    Doose S, Neuweiler H, Sauer M (2009) Fluorescence quenching by photoinduced electron transfer: a reporter for conformational dynamics of macromolecules. Chemphyschem 10(9–10):1389–1398CrossRefPubMedGoogle Scholar
  12. 12.
    Vaiana AC et al (2003) Fluorescence quenching of dyes by tryptophan: interactions at atomic detail from combination of experiment and computer simulation. J Am Chem Soc 125(47):14564–14572CrossRefPubMedGoogle Scholar
  13. 13.
    Neuweiler H, Doose S, Sauer M (2005) A microscopic view of miniprotein folding: enhanced folding efficiency through formation of an intermediate. Proc Natl Acad Sci USA 102(46):16650–16655CrossRefPubMedGoogle Scholar
  14. 14.
    Neuweiler H, Lollmann M, Doose S, Sauer M (2007) Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy. J Mol Biol 365(3):856–869CrossRefPubMedGoogle Scholar
  15. 15.
    Bollmann S et al (2011) Conformational flexibility of glycosylated peptides. Chemphyschem 12(16):2907–2911CrossRefPubMedGoogle Scholar
  16. 16.
    Neuweiler H, Johnson CM, Fersht AR (2009) Direct observation of ultrafast folding and denatured state dynamics in single protein molecules. Proc Natl Acad Sci USA 106(44):18569–18574CrossRefPubMedGoogle Scholar
  17. 17.
    Neuweiler H, Banachewicz W, Fersht AR (2010) Kinetics of chain motions within a protein-folding intermediate. Proc Natl Acad Sci USA 107(51):22106–22110CrossRefPubMedGoogle Scholar
  18. 18.
    Teufel DP, Johnson CM, Lum JK, Neuweiler H (2011) Backbone-driven collapse in unfolded protein chains. J Mol Biol 409(2):250–262CrossRefPubMedGoogle Scholar
  19. 19.
    Lum JK, Neuweiler H, Fersht AR (2012) Long-range modulation of chain motions within the intrinsically disordered transactivation domain of tumor suppressor p53. J Am Chem Soc 134(3):1617–1622CrossRefPubMedGoogle Scholar
  20. 20.
    Jensen MH, Sukumaran M, Johnson CM et al (2011) Intrinsic motions in the N-terminal domain of an ionotropic glutamate receptor detected by fluorescence correlation spectroscopy. J Mol Biol 414(1):96–105CrossRefPubMedGoogle Scholar
  21. 21.
    Kim J, Doose S, Neuweiler H, Sauer M (2006) The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy. Nucleic Acids Res 34(9):2516–2527CrossRefPubMedGoogle Scholar
  22. 22.
    Schüttpelz M et al (2008) Changes in conformational dynamics of mRNA upon AtGRP7 binding studied by fluorescence correlation spectroscopy. J Am Chem Soc 130(29):9507–9513CrossRefPubMedGoogle Scholar
  23. 23.
    Rehm D, Weller A (1969) Kinetik und Mechanismus der Elektronenübertragung bei der Fluoreszenzlöschung in Acetonitril. BerBunsenges Phys Chem 73:834–839Google Scholar
  24. 24.
    Adams DM et al (2003) Charge transfer on the nanoscale: current status. J Phys Chem B 107(28):6668–6697CrossRefGoogle Scholar
  25. 25.
    Zhong D, Zewail AH (2001) Femtosecond dynamics of flavoproteins: charge separation and recombination in riboflavin (vitamin B2)-binding protein and in glucose oxidase enzyme. Proc Natl Acad Sci USA 98(21):11867–11872CrossRefPubMedGoogle Scholar
  26. 26.
    Li X, Zhu R, Yu A, Zhao XS (2011) Ultrafast photoinduced electron transfer between tetramethylrhodamine and guanosine in aqueous solution. J Phys Chem B 115(19):6265–6271CrossRefPubMedGoogle Scholar
  27. 27.
    Doose S, Neuweiler H, Sauer M (2005) A close look at fluorescence quenching of organic dyes by tryptophan. Chemphyschem 6(11):2277–2285CrossRefPubMedGoogle Scholar
  28. 28.
    Marme N, Knemeyer JP, Sauer M, Wolfrum J (2003) Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan. Bioconjug Chem 14(6):1133–1139CrossRefPubMedGoogle Scholar
  29. 29.
    Heinlein T, Knemeyer JP, Piestert O, Sauer M (2003) Photoinduced electron transfer between fluorescent dyes and guanosine residues in DNA-hairpins. J Phys Chem B 107(31):7957–7964CrossRefGoogle Scholar
  30. 30.
    Krichevsky O, Bonnet G (2002) Fluorescence correlation spectroscopy: the technique and its applications. Rep Prog Phys 65(2):251–297CrossRefGoogle Scholar
  31. 31.
    Hess ST, Huang S, Heikal AA, Webb WW (2002) Biological and chemical applications of fluorescence correlation spectroscopy: a review. Biochemistry 41(3):697–705CrossRefPubMedGoogle Scholar
  32. 32.
    Haustein E, Schwille P (2007) Fluorescence correlation spectroscopy: novel variations of an established technique. Annu Rev Biophys Biomol Struct 36:151–169CrossRefPubMedGoogle Scholar
  33. 33.
    Selo I, Negroni L, Creminon C et al (1996) Preferential labeling of alpha-amino N-terminal groups in peptides by biotin: application to the detection of specific anti-peptide antibodies by enzyme immunoassays. J Immunol Methods 199(2):127–138CrossRefPubMedGoogle Scholar
  34. 34.
    Yin J, Lin AJ, Golan DE, Walsh CT (2006) Site-specific protein labeling by Sfp phosphopantetheinyl transferase. Nat Protoc 1(1):280–285CrossRefPubMedGoogle Scholar
  35. 35.
    Lallana E, Riguera R, Fernandez-Megia E (2011) Reliable and efficient procedures for the conjugation of biomolecules through Huisgen azide-alkyne cycloadditions. Angew Chem Int Ed Engl 50(38):8794–8804CrossRefPubMedGoogle Scholar
  36. 36.
    Vousden KH, Lane DP (2007) p53 in health and disease. Nat Rev Mol Cell Biol 8(4):275–283CrossRefPubMedGoogle Scholar
  37. 37.
    Joerger AC, Fersht AR (2008) Structural biology of the tumor suppressor p53. Annu Rev Biochem 77:557–582CrossRefPubMedGoogle Scholar
  38. 38.
    Mayor U et al (2003) The complete folding pathway of a protein from nanoseconds to microseconds. Nature 421(6925):863–867CrossRefPubMedGoogle Scholar
  39. 39.
    Religa TL, Markson JS, Mayor U et al (2005) Solution structure of a protein denatured state and folding intermediate. Nature 437(7061):1053–1056CrossRefPubMedGoogle Scholar
  40. 40.
    Wells M et al (2008) Structure of tumor suppressor p53 and its intrinsically disordered N-terminal transactivation domain. Proc Natl Acad Sci USA 105(15):5762–5767CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Markus Sauer
    • 1
  • Hannes Neuweiler
    • 1
  1. 1.Department of Biotechnology & BiophysicsBiozentrum, Julius-Maximilians-University WürzburgWürzburgGermany

Personalised recommendations