1. Structural Biology and Molecular Biophysics
  2. Cell Biology
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Single-fluorophore membrane transport activity sensors with dual-emission read-out

  1. Cindy Ast
  2. Roberto De Michele
  3. Michael U Kumke
  4. Wolf B Frommer  Is a corresponding author
  1. Carnegie Institution for Science, United States
  2. Italian National Research Council, Italy
  3. University of Potsdam, Germany
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  • Cited 9
  • Views 1,787
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Cite this article as: eLife 2015;4:e07113 doi: 10.7554/eLife.07113

Abstract

We recently described a series of genetically encoded, single-fluorophore-based sensors, termed AmTrac and MepTrac, which monitor membrane transporter activity in vivo (De Michele et al., 2013). However, being intensiometric, AmTrac and Meptrac are limited in their use for quantitative studies. Here, we characterized the photophysical properties (steady-state and time-resolved fluorescence spectroscopy as well as anisotropy decay analysis) of different AmTrac sensors with diverging fluorescence properties in order to generate improved, ratiometric sensors. By replacing key amino acid residues in AmTrac we constructed a set of dual-emission AmTrac sensors named deAmTracs. deAmTracs show opposing changes of blue and green emission with almost doubled emission ratio upon ammonium addition. The response ratio of the deAmTracs correlated with transport activity in mutants with altered capacity. Our results suggest that partial disruption of distance-dependent excited-state proton transfer (ESPT) is important for the successful generation of single-fluorophore-based dual-emission sensors.

Article and author information

Author details

  1. Cindy Ast

    Department of Plant Biology, Carnegie Institution for Science, Stanford California, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Roberto De Michele

    Institute of Biosciences and Bioresources, Italian National Research Council, Palermo, Italy
    Competing interests
    The authors declare that no competing interests exist.
  3. Michael U Kumke

    Department of Physical Chemistry, Institute of Chemistry, University of Potsdam, Potsdam, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Wolf B Frommer

    Department of Plant Biology, Carnegie Institution for Science, Stanford California, United States
    For correspondence
    wfrommer@carnegiescience.edu
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Richard Aldrich, The University of Texas at Austin, United States

Publication history

  1. Received: February 20, 2015
  2. Accepted: June 18, 2015
  3. Accepted Manuscript published: June 19, 2015 (version 1)
  4. Version of Record published: July 6, 2015 (version 2)

Copyright

© 2015, Ast 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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