Skip to main content
SpringerLink
Log in

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

  • Protocol
  • First Online:
SNAREs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1860))

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Wang Y, Bugge K, Kragelund BB, Lindorff-Larsen K (2018) Role of protein dynamics in transmembrane receptor signaling. Curr Opin Struct Biol 48:74–82

    Article  CAS  Google Scholar 

  2. Guo J, Zhou HX (2016) Protein Allostery and conformational dynamics. Chem Rev 116(11):6503

    Article  CAS  Google Scholar 

  3. Boehr DD, Nussinov R, Wright PE (2009) The role of dynamic conformational ensembles in biomolecular recognition. Nat Chem Biol 5(11):789–796

    Article  CAS  Google Scholar 

  4. Peter Lu H (2005) Probing single-molecule protein conformational dynamics. Acc Chem Res 38(7):557–565

    Article  Google Scholar 

  5. Michalet X, Weiss S, Jäger M (2006) Single-molecule fluorescence studies of protein folding and conformational dynamics. Chem Rev 106(5):1785–1813

    Article  CAS  Google Scholar 

  6. Jarymowycz VA, Stone MJ (2006) Fast time scale dynamics of protein backbones: NMR relaxation methods, applications, and functional consequences. Chem Rev 106(5):1624–1671

    Article  CAS  Google Scholar 

  7. Sze KH, Lai PM (2011) Probing protein dynamics by nuclear magnetic resonance. Protein Pept Lett 18(4):373–379

    Article  CAS  Google Scholar 

  8. Watt ED, Rienstra CM (2014) Recent advances in solid-state nuclear magnetic resonance techniques to quantify biomolecular dynamics. Anal Chem 86(1):58–64

    Article  CAS  Google Scholar 

  9. Magde D, Elson E, Webb WW (1972) Thermodynamic fluctuations ina reacting system - measurement by fluorescence correlation spectroscopy. Phys Rev Lett 29:705–708.302

    Article  CAS  Google Scholar 

  10. Berland KM, So PTC, Gratton E (1995) Two-photon fluorescence correlation spectroscopy, method and application to the intracellular environment. Biophys J 68:694–701

    Article  CAS  Google Scholar 

  11. Schwille P (2001) Cross-correlation analysis in FCS. In: Rigler R, Elson ES (eds) Fluorescence correlationspectroscopy. Springer, New York, pp 360–378

    Google Scholar 

  12. Schwille P, Meyer-Almes FJ, Rigler R (1997) Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. Biophys J 72:1878–1886

    Article  CAS  Google Scholar 

  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 77:2251–2265

    Article  CAS  Google Scholar 

  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 97:151–156

    Article  CAS  Google Scholar 

  15. Eid, J. S. (2002) Two-photon dual channel fluctuation correlation spectroscopy: theory and application. Ph.D. Dissertation, Urbana, Illinois

    Google Scholar 

  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 100:15516–15521

    Article  CAS  Google Scholar 

  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 114:5895–5902

    Article  Google Scholar 

  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 96:893–898

    Article  CAS  Google Scholar 

  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 11:135–141

    Article  CAS  Google Scholar 

  20. Sako Y, Minoghchi S, Yanagida T (2000) Single-molecule imaging of EGFR signalling on the surface of living cells. Nat Cell Biol 2:168–172

    Article  CAS  Google Scholar 

  21. Lesoine JF, Holmberg B, Maloney P, Wang X, Novotny L, Knauf PA (2006) Acta Physiol 187:141–147

    Article  CAS  Google Scholar 

  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 100:15516–15521

    Article  CAS  Google Scholar 

  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 100:14800–14805

    Article  CAS  Google Scholar 

  24. Tochio H, Tsui MM, Banfield DK, Zhang M (2001) An autoinhibitory mechanism for nonsyntaxin SNARE proteins revealed by the structure of Ykt6p. Science 293:698–702

    Article  CAS  Google Scholar 

  25. Wenyu Wen JY (2001) Lipid-induced conformational switch controls fusion activity of longin domain SNARE Ykt6. Mol Cell 37:383–395

    Article  Google Scholar 

  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 6:30282

    Article  CAS  Google Scholar 

  27. Hunte C (2005) Specific protein-lipid interactions in membrane proteins. Biochem Soc Trans 33(5):938–942

    Article  CAS  Google Scholar 

  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–L13

    Article  CAS  Google Scholar 

  29. Watkins LP, Yang H (2005) Detection of intensity change points in time-resolved single-molecule measurements. J Phys Chem B 109:617–628

    Article  CAS  Google Scholar 

  30. Krüger AC, Birkedal V (2013) Single molecule FRET data analysis procedures for FRET efficiency determination: probing the conformations of nucleic acid structures. Methods 64:36–42

    Article  Google Scholar 

  31. Förster T (1948) Zwischenmolekulare energiewanderung und fluoreszenz. Ann Phys 437:55–75

    Article  Google Scholar 

  32. Bacia K, Kim SA, Schwille P (2006) Fluorescence cross-correlation spectroscopy in living cells. Nat Methods 3:83–89

    Article  CAS  Google Scholar 

  33. Bacia K, Schwille P (2007) Practical guidelines for dual-colour fluorescence cross-correlation spectroscopy. Nat Protoc 2:2842–2856

    Article  CAS  Google Scholar 

  34. Hess ST, Huang S, Heikal AA, Webb WW (2002) Biological and chemical applications of fluorescence correlation spectroscopy: a review. Biochemistry (Mosc) 41:697–705

    Article  CAS  Google Scholar 

  35. Krichevsky O, Bonnet G (2002) Fluorescence correlation spectroscopy: the technique and its applications. Rep Prog Phys 65:251

    Article  CAS  Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

  1. State Key Laboratory of Surface Physics and Department of Physics, Fudan University, Shanghai, China

    Pei Li, Yawei Dai & Yan-Wen Tan

  2. Department of Physics, The University of Hong Kong, Hong Kong, China

    Yawei Dai

  3. Biological Imaging Center, Technische Universität München, München, Germany

    Markus Seeger

Authors
  1. Pei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yawei Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Markus Seeger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yan-Wen Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yan-Wen Tan .

Editor information

Editors and Affiliations

  1. Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA

    Rutilio Fratti

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Li, P., Dai, Y., Seeger, M., Tan, YW. (2019). Quantifying Intramolecular Protein Conformational Dynamics Under Lipid Interaction Using smFRET and FCCS. In: Fratti, R. (eds) SNAREs. Methods in Molecular Biology, vol 1860. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8760-3_23

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8760-3_23

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8759-7

  • Online ISBN: 978-1-4939-8760-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation