Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor

Journal Article · Sat Dec 02 04:00:00 EST 2017 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/J.BBRC.2017.09.170· OSTI ID:22719147

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

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. - Highlights: • Dendra2 is a monomeric green-to-red photoconvertible fluorescent protein. • Photoconverted form of Dendra2 fluoresces green at low pH and red at high pH. • It can be used as a ratiometric pH sensor using photoconverted Dendra2-specific excitation peak. • Dendra2 is suitable for pH measurements in physiological range in live cells.

• OSTI ID: 22719147
• Journal Information: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 4 Vol. 493; ISSN 0006-291X; ISSN BBRCA9
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
EXCITATION
FLUORESCENCE
PH VALUE
PHENOLS
PROTEINS
SENSORS