1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

Visualizing conformational dynamics of proteins in solution and at the cell membrane

  1. Sharona E Gordon  Is a corresponding author
  2. Mika Munari
  3. William N Zagotta  Is a corresponding author
  1. University of Washington, United States
https://doi.org/10.7554/eLife.37248
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  1. Sharona E Gordon
  2. Mika Munari
  3. William N Zagotta
(2018)
Visualizing conformational dynamics of proteins in solution and at the cell membrane
eLife 7:e37248.
https://doi.org/10.7554/eLife.37248
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Abstract

Conformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins.

Data availability

Data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Sharona E Gordon

    Department of Physiology and Biophysics, University of Washington, Seattle, United States
    For correspondence
    seg@uw.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0914-3361

  2. Mika Munari

    Department of Physiology and Biophysics, University of Washington, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. William N Zagotta

    Department of Physiology and Biophysics, University of Washington, Seattle, United States
    For correspondence
    zagotta@uw.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7631-8168

Funding

National Eye Institute (R01EY017564)

  • Sharona E Gordon

National Institute of Mental Health (R01MH102378)

  • William N Zagotta

National Institute of General Medical Sciences (R01GM100718)

  • William N Zagotta

National Eye Institute (R01EY010329)

  • William N Zagotta

National Institute of General Medical Sciences (R01GM125351)

  • William N Zagotta

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

Copyright

© 2018, Gordon et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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