pHlameleons: a family of FRET-based protein sensors for quantitative pH imaging

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biotechnology / methods*
  • Cell Line
  • Cells, Cultured
  • Chemistry Techniques, Analytical / methods*
  • Fluorescence Resonance Energy Transfer / methods*
  • Hydrogen-Ion Concentration
  • Image Processing, Computer-Assisted
  • Luminescent Proteins / analysis
  • Luminescent Proteins / chemistry*
  • Sensitivity and Specificity

Substances

  • Luminescent Proteins