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pHlameleons: A Family of FRET-Based Protein Sensors for Quantitative pH Imaging†

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Laboratory for Molecular and Cellular Systems, Department of Neuro- and Sensory Physiology, University Medicine Göttingen, Humboldtalee 23, 37073 Göttingen, Germany
†F.S.W. is member of, and financed by, the “Molecular Microscopy” section and the Excellence Cluster 171 “Microscopy on the Nanometer Scale” of the DFG-funded (German Research Council) Center for Molecular Physiology of the Brain (CMPB). Additional financing from the German Federal Ministry for Education and Research (BMBF) for the project “FLI-Cam” in the Biophotonik III program is acknowledged. M.G. is funded by the Fritz Thyssen Foundation.
* To whom correspondence should be addressed. Phone: +44-1223-334193. Fax: +44-1223-334796. E-mail: [email protected]
‡Present address: Laser Analytics Group, Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, U.K.
§Present address: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103 Leipzig, Germany.
Cite this: Biochemistry 2008, 47, 49, 13115–13126
Publication Date (Web):November 13, 2008
https://doi.org/10.1021/bi8009482
Copyright © 2008 American Chemical Society
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    Abstract

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    Intracellular pH is an important indicator for cellular metabolism and pathogenesis. pH sensing in living cells has been achieved using a number of synthetic organic dyes and genetically expressible sensor proteins, even allowing the specific targeting of intracellular organelles. Ideally, a class of genetically encodeable sensors need to cover relevant cellular pH ranges. We present a FRET-based pH sensor platform, based on the pH modulation of YFP acceptor fluorophores in a fusion construct with ECFP. The concurrent loss of the overlap integral upon acidification results in a proportionally reduced FRET coupling. The readout of FRET over the sensitized YFP fluorescence lifetime yields a highly sensitive and robust pH measurement that is self-calibrated. The principle is demonstrated in the existing high-efficiency FRET fusion Cy11.5, and tunability of the platform design is demonstrated by genetic alteration of the pH sensitivity of the acceptor moiety.

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