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Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing

Nature Methods volume 8, pages 239–241 (2011)Cite this article

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Abstract

We combined rapid microfluidic mixing with single-molecule fluorescence resonance energy transfer to study the folding kinetics of the intrinsically disordered human protein α-synuclein. The time-resolution of 0.2 ms revealed initial collapse of the unfolded protein induced by binding with lipid mimics and subsequent rapid formation of transient structures in the encounter complex. The method also enabled analysis of rapid dissociation and unfolding of weakly bound complexes triggered by massive dilution.

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Figure 1: Microfluidic setup for kinetic smFRET measurements.
Figure 2: Folding and unfolding of α-synuclein.

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Acknowledgements

We thank R.L. Nussbaum (US National Institutes of Health) for the wild-type α-synuclein plasmid. This work was supported by grants RO1 GM066833 and R21 AG033382 (US National Institutes of Health to A.A.D.) and PHY-0750049 (National Science Foundation to A.A.D. and A.G.). A.C.M.F. acknowledges a US National Institutes of Health fellowship, and E.A.L. acknowledges Deutsche Forschungsgemeinschaft funding.

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  1. Yann Gambin, Virginia VanDelinder & Edward A Lemke

    Present address: Present addresses: Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia (Y.G.) and Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany (V.V. and E.A.L.).,

  2. Virginia VanDelinder and Allan Chris M Ferreon: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA

    Yann Gambin, Allan Chris M Ferreon, Edward A Lemke & Ashok A Deniz

  2. Department of Physics, University of California San Diego, San Diego, California, USA

    Virginia VanDelinder & Alex Groisman

Authors
  1. Yann Gambin
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  2. Virginia VanDelinder
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  3. Allan Chris M Ferreon
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  4. Edward A Lemke
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  6. Ashok A Deniz
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Contributions

Y.G., A.G. and A.A.D. designed research; Y.G. performed smFRET experiments; Y.G. and V.V. characterized the device; A.C.M.F. provided α-synuclein expertise and samples; E.A.L. provided instrumentation support; and all authors contributed to writing the paper.

Corresponding authors

Correspondence to Yann Gambin, Alex Groisman or Ashok A Deniz.

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The authors declare no competing financial interests.

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Supplementary Figures 1–23 and Supplementary Note 1 (PDF 6680 kb)

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Gambin, Y., VanDelinder, V., Ferreon, A. et al. Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing. Nat Methods 8, 239–241 (2011). https://doi.org/10.1038/nmeth.1568

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