1. Structural Biology and Molecular Biophysics
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Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

  1. Huaying Zhao
  2. Yan Fu
  3. Carla Glasser
  4. Eric J Andrade Alba
  5. Mark L Mayer
  6. George Patterson
  7. Peter Schuck  Is a corresponding author
  1. National Institutes of Health, United States
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Cite this article as: eLife 2016;5:e17812 doi: 10.7554/eLife.17812

Abstract

The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors.

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Author details

  1. Huaying Zhao

    Dynamics of Macromolecular Assembly Section, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.

  2. Yan Fu

    Section on Biophotonics, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Carla Glasser

    Laboratory of Cellular and Molecular Neurophysiology, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Eric J Andrade Alba

    Section on Biophotonics, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Mark L Mayer

    Laboratory of Cellular and Molecular Neurophysiology, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. George Patterson

    Section on Biophotonics, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Peter Schuck

    Dynamics of Macromolecular Assembly Section, National Institutes of Health, Bethesda, United States
    For correspondence
    schuckp@mail.nih.gov
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8859-6966

Funding

National Institute of Biomedical Imaging and Bioengineering (ZIA EB000051-09 LCIM)

  • Peter Schuck

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Antoine M van Oijen, University of Groningen, Netherlands

Publication history

  1. Received: May 13, 2016
  2. Accepted: July 19, 2016
  3. Accepted Manuscript published: July 20, 2016 (version 1)
  4. Version of Record published: August 15, 2016 (version 2)

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

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