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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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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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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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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DOI: https://doi.org/10.1038/nmeth.1435
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