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Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor.
Biochem Biophys Res Commun 2017; 493(4):1518-1521BB

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.

Authors+Show Affiliations

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia. Electronic address: alpah@mail.ru.Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

28986251

Citation

Pakhomov, Alexey A., et al. "Fluorescent Protein Dendra2 as a Ratiometric Genetically Encoded PH-sensor." Biochemical and Biophysical Research Communications, vol. 493, no. 4, 2017, pp. 1518-1521.
Pakhomov AA, Martynov VI, Orsa AN, et al. Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor. Biochem Biophys Res Commun. 2017;493(4):1518-1521.
Pakhomov, A. A., Martynov, V. I., Orsa, A. N., Bondarenko, A. A., Chertkova, R. V., Lukyanov, K. A., ... Deyev, I. E. (2017). Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor. Biochemical and Biophysical Research Communications, 493(4), pp. 1518-1521. doi:10.1016/j.bbrc.2017.09.170.
Pakhomov AA, et al. Fluorescent Protein Dendra2 as a Ratiometric Genetically Encoded PH-sensor. Biochem Biophys Res Commun. 2017 12 2;493(4):1518-1521. PubMed PMID: 28986251.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor. AU - Pakhomov,Alexey A, AU - Martynov,Vladimir I, AU - Orsa,Alexander N, AU - Bondarenko,Alena A, AU - Chertkova,Rita V, AU - Lukyanov,Konstantin A, AU - Petrenko,Alexander G, AU - Deyev,Igor E, Y1 - 2017/10/03/ PY - 2017/09/19/received PY - 2017/09/30/accepted PY - 2017/10/8/pubmed PY - 2017/10/31/medline PY - 2017/10/8/entrez KW - Dendra2 KW - Fluorescent proteins KW - GFP KW - Intracellular pH KW - pH KW - pH sensor SP - 1518 EP - 1521 JF - Biochemical and biophysical research communications JO - Biochem. Biophys. Res. Commun. VL - 493 IS - 4 N2 - 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. SN - 1090-2104 UR - https://www.unboundmedicine.com/medline/citation/28986251/Fluorescent_protein_Dendra2_as_a_ratiometric_genetically_encoded_pH_sensor_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0006-291X(17)31960-5 DB - PRIME DP - Unbound Medicine ER -