SNAREs pp 345-359 | Cite as

Quantifying Intramolecular Protein Conformational Dynamics Under Lipid Interaction Using smFRET and FCCS

  • Pei Li
  • Yawei Dai
  • Markus Seeger
  • Yan-Wen TanEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)


Fӧrster-type resonance energy transfer (FRET) with fluorescence cross-correlation spectroscopy (FCCS) is a powerful combination for observing intramolecular conformational dynamics on the micro- to millisecond timescale. Owing to its sensitivity to various physical parameters, FRET-FCCS has also been used to detect the reagent effects on proteins dynamics. However, FRET-FCCS alone cannot acquire the exact measurements of rate constants. Moreover, this technique is highly model dependent and can be unreliable when determining too many parameters at once. On the contrary, single-molecular FRET (smFRET) can measure the conformational states and their populations directly, although it is extremely challenging for probing fast dynamics under 1 ms. In this chapter, we describe how to realize sub-millisecond conformational dynamics measurements of a SNARE protein Ykt6 under lipid environments by smFRET and FRET-FCCS. This protocol includes sample preparation, microscope designs, data acquisition, and analysis methodology.

Key words

Intramolecular conformational dynamics Lipid interaction smFRET FRET-FCCS 



We thank Song Song and Jian Chang for building the microscopes. This work was sponsored by National Natural Science Foundation of China (No. 11274076 and 21773039). The original experiments were conducted by Yawei Dai and Markus Seeger in Yan-Wen Tan’s lab at State Key Laboratory of Surface Physics and Department of Physics, Fudan University.


  1. 1.
    Wang Y, Bugge K, Kragelund BB, Lindorff-Larsen K (2018) Role of protein dynamics in transmembrane receptor signaling. Curr Opin Struct Biol 48:74–82CrossRefGoogle Scholar
  2. 2.
    Guo J, Zhou HX (2016) Protein Allostery and conformational dynamics. Chem Rev 116(11):6503CrossRefGoogle Scholar
  3. 3.
    Boehr DD, Nussinov R, Wright PE (2009) The role of dynamic conformational ensembles in biomolecular recognition. Nat Chem Biol 5(11):789–796CrossRefGoogle Scholar
  4. 4.
    Peter Lu H (2005) Probing single-molecule protein conformational dynamics. Acc Chem Res 38(7):557–565CrossRefGoogle Scholar
  5. 5.
    Michalet X, Weiss S, Jäger M (2006) Single-molecule fluorescence studies of protein folding and conformational dynamics. Chem Rev 106(5):1785–1813CrossRefGoogle Scholar
  6. 6.
    Jarymowycz VA, Stone MJ (2006) Fast time scale dynamics of protein backbones: NMR relaxation methods, applications, and functional consequences. Chem Rev 106(5):1624–1671CrossRefGoogle Scholar
  7. 7.
    Sze KH, Lai PM (2011) Probing protein dynamics by nuclear magnetic resonance. Protein Pept Lett 18(4):373–379CrossRefGoogle Scholar
  8. 8.
    Watt ED, Rienstra CM (2014) Recent advances in solid-state nuclear magnetic resonance techniques to quantify biomolecular dynamics. Anal Chem 86(1):58–64CrossRefGoogle Scholar
  9. 9.
    Magde D, Elson E, Webb WW (1972) Thermodynamic fluctuations ina reacting system - measurement by fluorescence correlation spectroscopy. Phys Rev Lett 29:705–708.302CrossRefGoogle Scholar
  10. 10.
    Berland KM, So PTC, Gratton E (1995) Two-photon fluorescence correlation spectroscopy, method and application to the intracellular environment. Biophys J 68:694–701CrossRefGoogle Scholar
  11. 11.
    Schwille P (2001) Cross-correlation analysis in FCS. In: Rigler R, Elson ES (eds) Fluorescence correlationspectroscopy. Springer, New York, pp 360–378Google Scholar
  12. 12.
    Schwille P, Meyer-Almes FJ, Rigler R (1997) Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. Biophys J 72:1878–1886CrossRefGoogle Scholar
  13. 13.
    Schwille P, Haupts U, Maiti S, Webb WWW (1999) Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation. Biophys J 77:2251–2265CrossRefGoogle Scholar
  14. 14.
    Schwille P, Kummer S, Heikal AH, Moerner WE, Webb WWW (2000) Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins. Proc Natl Acad Sci U S A 97:151–156CrossRefGoogle Scholar
  15. 15.
    Eid, J. S. (2002) Two-photon dual channel fluctuation correlation spectroscopy: theory and application. Ph.D. Dissertation, Urbana, IllinoisGoogle Scholar
  16. 16.
    Margittai M, Widengren J, Schweinberger E, Schroder GF, Felekyan S, Haustein E, Konig M, Fasshauer D, Grubmuller H, Jahn R, Seidel CA (2003) Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1. Proc Natl Acad Sci U S A 100:15516–15521CrossRefGoogle Scholar
  17. 17.
    Shane Price E, DeVore MS, Johnson CK (2010) Detecting intramolecular dynamics and multiple Fo¨rster resonance energy transfer states by fluorescence correlation spectroscopy. J Phys Chem B 114:5895–5902CrossRefGoogle Scholar
  18. 18.
    Ha T, Ting AY, Liang J, Caldwell WB, Deniz AA, Chemla DS, Schultz PG, Weiss S (1999) Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism. Proc Natl Acad Sci U S A 96:893–898CrossRefGoogle Scholar
  19. 19.
    Diez M, Zimmermann B, Borsch M, Konig M, Schweinberger E, Steigmiller S, Reuter R, Felekyan S, Kudryavtsev V, Seidel CA, Graber P (2004) Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase. Nat Struct Mol Biol 11:135–141CrossRefGoogle Scholar
  20. 20.
    Sako Y, Minoghchi S, Yanagida T (2000) Single-molecule imaging of EGFR signalling on the surface of living cells. Nat Cell Biol 2:168–172CrossRefGoogle Scholar
  21. 21.
    Lesoine JF, Holmberg B, Maloney P, Wang X, Novotny L, Knauf PA (2006) Acta Physiol 187:141–147CrossRefGoogle Scholar
  22. 22.
    Margittai M, Widengren J, Schweinberger E, Schroder GF, Felekyan S, Haustein E, Konig M, Fasshauer D, Grubmuller H, Jahn R, Seidel CA (2003) Development of an spFRET method to measure structure changes in ion exchange proteins. Proc Natl Acad Sci U S A 100:15516–15521CrossRefGoogle Scholar
  23. 23.
    Weninger K, Bowen ME, Chu S, Brunger AT (2003) Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations. Proc Natl Acad Sci U S A 100:14800–14805CrossRefGoogle Scholar
  24. 24.
    Tochio H, Tsui MM, Banfield DK, Zhang M (2001) An autoinhibitory mechanism for nonsyntaxin SNARE proteins revealed by the structure of Ykt6p. Science 293:698–702CrossRefGoogle Scholar
  25. 25.
    Wenyu Wen JY (2001) Lipid-induced conformational switch controls fusion activity of longin domain SNARE Ykt6. Mol Cell 37:383–395CrossRefGoogle Scholar
  26. 26.
    Dai Y, Seeger M, Weng J, Song S, Wang W, Tan Y-W (2016) Lipid regulated intramolecular conformational dynamics of SNARE-protein Ykt6. Sci Rep 6:30282CrossRefGoogle Scholar
  27. 27.
    Hunte C (2005) Specific protein-lipid interactions in membrane proteins. Biochem Soc Trans 33(5):938–942CrossRefGoogle Scholar
  28. 28.
    Pal P, Lesoine JF, Lieb MA, Novotny L, Knauf PA (2005) A novel immobilization method for single protein spFRET studies. Biophys J 89:L11–L13CrossRefGoogle Scholar
  29. 29.
    Watkins LP, Yang H (2005) Detection of intensity change points in time-resolved single-molecule measurements. J Phys Chem B 109:617–628CrossRefGoogle Scholar
  30. 30.
    Krüger AC, Birkedal V (2013) Single molecule FRET data analysis procedures for FRET efficiency determination: probing the conformations of nucleic acid structures. Methods 64:36–42CrossRefGoogle Scholar
  31. 31.
    Förster T (1948) Zwischenmolekulare energiewanderung und fluoreszenz. Ann Phys 437:55–75CrossRefGoogle Scholar
  32. 32.
    Bacia K, Kim SA, Schwille P (2006) Fluorescence cross-correlation spectroscopy in living cells. Nat Methods 3:83–89CrossRefGoogle Scholar
  33. 33.
    Bacia K, Schwille P (2007) Practical guidelines for dual-colour fluorescence cross-correlation spectroscopy. Nat Protoc 2:2842–2856CrossRefGoogle Scholar
  34. 34.
    Hess ST, Huang S, Heikal AA, Webb WW (2002) Biological and chemical applications of fluorescence correlation spectroscopy: a review. Biochemistry (Mosc) 41:697–705CrossRefGoogle Scholar
  35. 35.
    Krichevsky O, Bonnet G (2002) Fluorescence correlation spectroscopy: the technique and its applications. Rep Prog Phys 65:251CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Pei Li
    • 1
  • Yawei Dai
    • 1
    • 2
  • Markus Seeger
    • 3
  • Yan-Wen Tan
    • 1
    Email author
  1. 1.State Key Laboratory of Surface Physics and Department of PhysicsFudan UniversityShanghaiChina
  2. 2.Department of PhysicsThe University of Hong KongHong KongChina
  3. 3.Biological Imaging CenterTechnische Universität MünchenMünchenGermany

Personalised recommendations