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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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  • 1Laboratory for Molecular and Cellular Systems, Department of Neuro- and Sensory Physiology, University Medicine Gottingen, Humboldtalee 23, 37073 Gottingen, Germany. aesposito@quantitative-microscopy.org

Abstract

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.

PMID:
19007185
[PubMed - indexed for MEDLINE]
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