Brief Communication | Published:

Detecting the conformation of individual proteins in live cells

Nature Methods volume 7, pages 203205 (2010) | Download Citation

Abstract

We combined single-molecule fluorescence resonance energy transfer (smFRET) with single-particle tracking in live cells to detect the in vivo conformation of individual proteins. We site-specifically labeled recombinant SNARE proteins with a FRET donor and acceptor before microinjecting them into cultured cells. Individual proteins rapidly incorporated into folded complexes at the cell membrane, demonstrating the potential of this method to reveal dynamic interactions within cells.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , , , & Annu. Rev. Biochem. 77, 51–76 (2008).

  2. 2.

    et al. Nature 458, 106–109 (2009).

  3. 3.

    & Methods 46, 131–140 (2008).

  4. 4.

    , & Nat. Cell Biol. 2, 168–172 (2000).

  5. 5.

    et al. Proc. Natl. Acad. Sci. USA 101, 7317–7322 (2004).

  6. 6.

    , , & J. Am. Chem. Soc. 130, 17664–17665 (2008).

  7. 7.

    & Nat. Rev. Mol. Cell Biol. 7, 631–643 (2006).

  8. 8.

    & Science 306, 1042–1046 (2004).

  9. 9.

    , & Science 312, 228–230 (2006).

  10. 10.

    , , & Proc. Natl. Acad. Sci. USA 100, 14800–14805 (2003).

  11. 11.

    & J. Biol. Chem. 277, 34303–34309 (2002).

  12. 12.

    , , & Biochem. J. 392, 283–289 (2005).

  13. 13.

    et al. FEBS Lett. 582, 3563–3568 (2008).

  14. 14.

    , , & J. Cell Biol. 158, 751–760 (2002).

  15. 15.

    et al. Science 300, 2061–2065 (2003).

  16. 16.

    , , & Biophys. J. 87, 3569–3584 (2004).

  17. 17.

    , , , & Biophys. J. 89, 690–702 (2005).

  18. 18.

    , , , & Structure 16, 308–320 (2008).

  19. 19.

    et al. J. Biol. Chem. 273, 34171–34179 (1998).

  20. 20.

    , , , & Proc. Natl. Acad. Sci. USA 98, 14038–14043 (2001).

  21. 21.

    , & Biophys. J. 93, 2178–2187 (2007).

  22. 22.

    , & J. Phys. Chem. B 105, 12165–12170 (2001).

  23. 23.

    , & Nat. Methods 5, 507–516 (2008).

  24. 24.

    & in Live Cell Imaging: A laboratory manual (eds. Goldman, R.D. & Spector, D.L.) 585–602 (Cold Springs Harbor Laboratory Press, Woodbury, 2005).

  25. 25.

    , & J. Am. Chem. Soc. 131, 5018–5019 (2009).

  26. 26.

    , , & Nature 395, 347–353 (1998).

  27. 27.

    Q. Rev. Biophys. 38, 1–47 (2005).

  28. 28.

    , & Nat. Methods 3, 891–893 (2006).

Download references

Acknowledgements

We thank B. Choi and T. Anderson for assistance with protein preparation, G. Ribeill and E. Cady for software development, G. Feng and Z. Lu for assistance with PC-12 cells, Z.-H. Sheng (US National Institutes of Health) for the gift of a SNAP-29 expression vector, and M. Bowen, J. Ernst and G. Augustine for useful discussions. K.R.W. is supported in part by a Career Award at the Scientific Interface from the Burroughs Wellcome Fund. This work was funded by the US National Institutes of Health (GM076039).

Author information

Affiliations

  1. Department of Physics, North Carolina State University, Raleigh, North Carolina, USA.

    • John J Sakon
    •  & Keith R Weninger

Authors

  1. Search for John J Sakon in:

  2. Search for Keith R Weninger in:

Contributions

J.J.S. performed all measurements and data analysis. J.J.S. and K.R.W. contributed substantially to all other aspects.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Keith R Weninger.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Note

Videos

  1. 1.

    Supplementary Video 1

    Microinjection of Cy3-Cy5 labeled SNAP-25–CC into a BS-C-1 cell under 'near'-total internal reflection (Online Methods) with green illumination. The left half of the screen is the acceptor channel while the right half is the donor channel. Recorded at 10 frames s−1.

  2. 2.

    Supplementary Video 2

    Microinjection of Cy3-Cy5 labeled SNAP-25–CC into a PC-12 cell under full total internal reflection with lower protein concentration injected than in Supplementary Video 1. Recorded at 10 frames s−1.

  3. 3.

    Supplementary Video 3

    Microinjection of Alexa Fluor 555-Alexa Fluor 647 labeled G43D mutant of SNAP-25–CC into a BS-C-1 cell with the same conditions as in Supplementary Video 1. Recorded at 10 frames s−1.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmeth.1421

Further reading