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pH sensitivity of FRET reporters based on cyan and yellow fluorescent proteins

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Abstract

It is generally acknowledged that the popular cyan and yellow fluorescent proteins carried by genetically encoded reporters suffer from strong pH sensitivities close to the physiological pH range. We studied the consequences of these pH responses on the intracellular signals of model Förster resonant energy transfer (FRET) tandems and FRET-based reporters of cAMP-dependent protein kinase activity (AKAR) expressed in the cytosol of living BHK cells, while changing the intracellular pH by means of the nigericin ionophore. Although the simultaneous pH sensitivities of the donor and the acceptor may mask each other in some cases, the magnitude of the perturbations can be very significant, as compared to the functional response of the AKAR biosensor. Replacing the CFP donor by the spectrally identical, but pH-insensitive Aquamarine variant (pK1/2 = 3.3) drastically modifies the biosensor pH response and gives access to the acid transition of the yellow acceptor. We developed a simple model of pH-dependent FRET and used it to describe the expected pH-induced changes in fluorescence lifetime and ratiometric signals. This model qualitatively accounts for most of the observations, but reveals a complex behavior of the cytosolic AKAR biosensor at acid pHs, associated to additional FRET contributions. This study underlines the major and complex impact of pH changes on the signal of FRET reporters in the living cell.

The complex behaviour of a cytosolic FRET construct carrying cyan and yellow fluorescent proteins in conditions of varying pH is well described by a simple analytical model. Changing the ECFP donor for an Aquamarine unveils the strong pH sensitivity of the yellow acceptor

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Acknowledgments

D.B.B. was the recipient of a doctoral grant from Region Ile-de-France (DIM Nanosciences IdF). We acknowledge supports from the Fondation pour la Recherche Médicale, the Centre National de la Recherche Scientifique through the program NEEDS and Paris-Sud University.

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Authors and Affiliations

  1. CNRS, Laboratoire de Chimie Physique, UMR 8000 and Université Paris-Sud, Avenue Jean Perrin 15, 91405, Orsay, France

    Dahdjim-Benoît Betolngar, Marie Erard, Hélène Pasquier, Yasmina Bousmah, Awa Diop-Sy & Fabienne Mérola

  2. CNRS, Biological Adaptation and Ageing, UMR 8256 and Université Pierre et Marie Curie, Place Jussieu 4, 75252, Paris, France

    Elvire Guiot & Pierre Vincent

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  1. Dahdjim-Benoît Betolngar
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  2. Marie Erard
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  3. Hélène Pasquier
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  4. Yasmina Bousmah
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  6. Elvire Guiot
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  8. Fabienne Mérola
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Correspondence to Fabienne Mérola.

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Betolngar, DB., Erard, M., Pasquier, H. et al. pH sensitivity of FRET reporters based on cyan and yellow fluorescent proteins. Anal Bioanal Chem 407, 4183–4193 (2015). https://doi.org/10.1007/s00216-015-8636-z

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  • DOI: https://doi.org/10.1007/s00216-015-8636-z

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