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Biochemistry. 2008 Dec 9;47(49):13115-26. doi: 10.1021/bi8009482.

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 Gottingen, Humboldtalee 23, 37073 Gottingen, Germany.


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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