Single-fluorophore membrane transport activity sensors with dual-emission read-out

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...

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Detalles Bibliográficos
Autores principales: Ast, Cindy, De Michele, Roberto, Kumke, Michael U, Frommer, Wolf B
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2015
Materias:
Biophysics and Structural Biology
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491562/
https://www.ncbi.nlm.nih.gov/pubmed/26090909
http://dx.doi.org/10.7554/eLife.07113
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author Ast, Cindy
De Michele, Roberto
Kumke, Michael U
Frommer, Wolf B
author_facet Ast, Cindy
De Michele, Roberto
Kumke, Michael U
Frommer, Wolf B
author_sort Ast, Cindy
collection PubMed
description 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 is important for the successful generation of single-fluorophore-based dual-emission sensors. DOI: http://dx.doi.org/10.7554/eLife.07113.001
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spelling pubmed-44915622015-07-07 Single-fluorophore membrane transport activity sensors with dual-emission read-out Ast, Cindy De Michele, Roberto Kumke, Michael U Frommer, Wolf B eLife Biophysics and Structural Biology 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 is important for the successful generation of single-fluorophore-based dual-emission sensors. DOI: http://dx.doi.org/10.7554/eLife.07113.001 eLife Sciences Publications, Ltd 2015-06-19 /pmc/articles/PMC4491562/ /pubmed/26090909 http://dx.doi.org/10.7554/eLife.07113 Text en © 2015, Ast et al http://creativecommons.org/licenses/by/4.0/ This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Biophysics and Structural Biology
Ast, Cindy
De Michele, Roberto
Kumke, Michael U
Frommer, Wolf B
Single-fluorophore membrane transport activity sensors with dual-emission read-out
title Single-fluorophore membrane transport activity sensors with dual-emission read-out
title_full Single-fluorophore membrane transport activity sensors with dual-emission read-out
title_fullStr Single-fluorophore membrane transport activity sensors with dual-emission read-out
title_full_unstemmed Single-fluorophore membrane transport activity sensors with dual-emission read-out
title_short Single-fluorophore membrane transport activity sensors with dual-emission read-out
title_sort single-fluorophore membrane transport activity sensors with dual-emission read-out
topic Biophysics and Structural Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491562/
https://www.ncbi.nlm.nih.gov/pubmed/26090909
http://dx.doi.org/10.7554/eLife.07113
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