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Detecting the conformation of individual proteins in live cells

Nature Methods volume 7, pages 203–205 (2010)Cite this article

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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.

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Figure 1: smFRET detection in live cells.
Figure 2: smFRET studies of SNARE proteins in BS-C-1 cells.

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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).

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Authors and Affiliations

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

    John J Sakon & Keith R Weninger

Authors
  1. John J Sakon
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  2. Keith R Weninger
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Contributions

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

Corresponding author

Correspondence to Keith R Weninger.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Note (PDF 814 kb)

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. (MOV 2604 kb)

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. (MOV 2883 kb)

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. (MOV 2420 kb)

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Sakon, J., Weninger, K. Detecting the conformation of individual proteins in live cells. Nat Methods 7, 203–205 (2010). https://doi.org/10.1038/nmeth.1421

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