Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor

Alexey A. Pakhomov, Vladimir I. Martynov, Alexander N. Orsa, Alena A. Bondarenko, Rita V. Chertkova, Konstantin A. Lukyanov, Alexander G. Petrenko, Igor E. Deyev

Research output: Contribution to journalArticlepeer-review

15 Citations (Scopus)

Abstract

Fluorescent protein Dendra2 is a monomeric GFP-like protein that belongs to the group of Kaede-like photoconvertible fluorescent proteins with irreversible photoconversion from a green- to red-emitting state when exposed to violet-blue light. In an acidic environment, photoconverted Dendra2 turns green due to protonation of the phenolic group of the chromophore with pKa of about 7.5. Thus, photoconverted form of Dendra2 can be potentially used as a ratiometric pH-sensor in the physiological pH range. However, incomplete photoconversion makes ratiometric measurements irreproducible when using standard filter sets. Here, we describe the method to detect fluorescence of only photoconverted Dendra2 form, but not nonconverted green Dendra2. We show that the 350 nm excitation light induces solely the fluorescence of photoconverted protein. By measuring the red to green fluorescence ratio, we determined intracellular pH in live CHO and HEK 293 cells. Thus, Dendra2 can be used as a novel ratiometric genetically encoded pH sensor with emission maxima in the green-red spectral region, which is suitable for application in live cells.

Original languageEnglish
Pages (from-to)1518-1521
Number of pages4
JournalBiochemical and Biophysical Research Communications
Volume493
Issue number4
DOIs
Publication statusPublished - 2 Dec 2017
Externally publishedYes

Keywords

  • Dendra2
  • Fluorescent proteins
  • GFP
  • Intracellular pH
  • pH
  • pH sensor

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