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Protein folding stability and dynamics imaged in a living cell

Nature Methods volume 7, pages 319–323 (2010)Cite this article

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Abstract

Biomolecular dynamics and stability are predominantly investigated in vitro and extrapolated to explain function in the living cell. We present fast relaxation imaging (FreI), which combines fluorescence microscopy and temperature jumps to probe biomolecular dynamics and stability inside a single living cell with high spatiotemporal resolution. We demonstrated the method by measuring the reversible fast folding kinetics as well as folding thermodynamics of a fluorescence resonance energy transfer (FRET) probe-labeled phosphoglycerate kinase construct in two human cell lines. Comparison with in vitro experiments at 23–49 °C showed that the cell environment influences protein stability and folding rate. FReI should also be applicable to the study of protein-protein interactions and heat-shock responses as well as to comparative studies of cell populations or whole organisms.

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Figure 1: Schematic of the temperature-jump fluorescence imaging microscope.
Figure 2: Heating laser profiles, resulting temperature profiles and folding kinetics of the low-Tm PGK construct.
Figure 3: Temperature-dependent FRET and thermal denaturation of the low-Tm PGK fusion construct.
Figure 4: Normalized D/A relaxation kinetics of the PGK fusion protein in vivo and in vitro track the denaturation curves.
Figure 5: Comparison of folding kinetics of the less stable PGK mutant in two cell lines.

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Acknowledgements

We acknowledge funding from the US National Science Foundation (MCB 0613643). A.D. was supported by the National Science Foundation Center for Physics of Living Cells (Illinois Physics Department) while part of this work was carried out. S.E. and M.G. acknowledge support from the Alexander von Humboldt Foundation. We thank Z. Shen and K.V. Prasanth for growing the U2OS and HeLa cell lines.

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Authors and Affiliations

  1. Department of Chemistry, University of Illinois, Urbana, Illinois, USA

    Simon Ebbinghaus, Apratim Dhar, J Douglas McDonald & Martin Gruebele

  2. Department of Physics, University of Illinois, Urbana, Illinois, USA

    Martin Gruebele

  3. Center for Biophysics and Computational Biology, University of Illinois, Urbana, Illinois, USA

    Martin Gruebele

Authors
  1. Simon Ebbinghaus
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  2. Apratim Dhar
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  4. Martin Gruebele
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Contributions

A.D. and S.E. designed and implemented instrumentation and software, performed experiments, analyzed data and wrote the paper. J.D.M. designed experimental components. M.G. designed the experiment, analyzed data and wrote the paper.

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Correspondence to Martin Gruebele.

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The authors declare no competing financial interests.

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Ebbinghaus, S., Dhar, A., McDonald, J. et al. Protein folding stability and dynamics imaged in a living cell. Nat Methods 7, 319–323 (2010). https://doi.org/10.1038/nmeth.1435

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