Open Access
International Journal of Molecular Sciences, volume 23, issue 21, pages 12907
A Single Fluorescent Protein-Based Indicator with a Time-Resolved Fluorescence Readout for Precise pH Measurements in the Alkaline Range
Simonyan Tatiana R
1, 2
,
Protasova Elena A
2, 3
,
Mamontova Anastasia S.
1, 2
,
SHAKHOV A. M.
2, 4
,
Maksimov Eugene G.
2, 3
,
Bogdanov Alexey M.
2
Publication type: Journal Article
Publication date: 2022-10-26
Quartile SCImago
Q1
Quartile WOS
Q1
Impact factor: 5.6
ISSN: 16616596, 14220067
PubMed ID:
36361706
Catalysis
Organic Chemistry
Inorganic Chemistry
Physical and Theoretical Chemistry
Computer Science Applications
Spectroscopy
Molecular Biology
General Medicine
Abstract
The real-time monitoring of the intracellular pH in live cells with high precision represents an important methodological challenge. Although genetically encoded fluorescent indicators can be considered as a probe of choice for such measurements, they are hindered mostly by the inability to determine an absolute pH value and/or a narrow dynamic range of the signal, making them inefficient for recording the small pH changes that typically occur within cellular organelles. Here, we study the pH sensitivity of a green-fluorescence-protein (GFP)-based emitter (EGFP-Y145L/S205V) with the alkaline-shifted chromophore’s pKa and demonstrate that, in the pH range of 7.5–9.0, its fluorescence lifetime changes by a factor of ~3.5 in a quasi-linear manner in mammalian cells. Considering the relatively strong lifetime response in a narrow pH range, we proposed the mitochondria, which are known to have a weakly alkaline milieu, as a target for live-cell pH measurements. Using fluorescence lifetime imaging microscopy (FLIM) to visualize the HEK293T cells expressing mitochondrially targeted EGFP-Y145L/S205V, we succeeded in determining the absolute pH value of the mitochondria and recorded the ETC-uncoupler-stimulated pH shift with a precision of 0.1 unit. We thus show that a single GFP with alkaline-shifted pKa can act as a high-precision indicator that can be used in a specific pH range.
Citations by journals
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The Analyst
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The Analyst
1 publication, 100%
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Citations by publishers
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Royal Society of Chemistry (RSC)
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Royal Society of Chemistry (RSC)
1 publication, 100%
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Simonyan T. R. et al. A Single Fluorescent Protein-Based Indicator with a Time-Resolved Fluorescence Readout for Precise pH Measurements in the Alkaline Range // International Journal of Molecular Sciences. 2022. Vol. 23. No. 21. p. 12907.
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Simonyan T. R., Protasova E. A., Mamontova A. S., SHAKHOV A. M., Lukyanov K. A., Maksimov E. G., Bogdanov A. M. A Single Fluorescent Protein-Based Indicator with a Time-Resolved Fluorescence Readout for Precise pH Measurements in the Alkaline Range // International Journal of Molecular Sciences. 2022. Vol. 23. No. 21. p. 12907.
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TY - JOUR
DO - 10.3390/ijms232112907
UR - https://doi.org/10.3390%2Fijms232112907
TI - A Single Fluorescent Protein-Based Indicator with a Time-Resolved Fluorescence Readout for Precise pH Measurements in the Alkaline Range
T2 - International Journal of Molecular Sciences
AU - Simonyan, Tatiana R
AU - Protasova, Elena A
AU - Mamontova, Anastasia S.
AU - SHAKHOV, A. M.
AU - Maksimov, Eugene G.
AU - Bogdanov, Alexey M.
AU - Lukyanov, Konstantin A.
PY - 2022
DA - 2022/10/26 00:00:00
PB - Multidisciplinary Digital Publishing Institute (MDPI)
SP - 12907
IS - 21
VL - 23
PMID - 36361706
SN - 1661-6596
SN - 1422-0067
ER -
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@article{2022_Simonyan,
author = {Tatiana R Simonyan and Elena A Protasova and Anastasia S. Mamontova and A. M. SHAKHOV and Eugene G. Maksimov and Alexey M. Bogdanov and Konstantin A. Lukyanov},
title = {A Single Fluorescent Protein-Based Indicator with a Time-Resolved Fluorescence Readout for Precise pH Measurements in the Alkaline Range},
journal = {International Journal of Molecular Sciences},
year = {2022},
volume = {23},
publisher = {Multidisciplinary Digital Publishing Institute (MDPI)},
month = {oct},
url = {https://doi.org/10.3390%2Fijms232112907},
number = {21},
pages = {12907},
doi = {10.3390/ijms232112907}
}
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MLA
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Simonyan, Tatiana R., et al. “A Single Fluorescent Protein-Based Indicator with a Time-Resolved Fluorescence Readout for Precise pH Measurements in the Alkaline Range.” International Journal of Molecular Sciences, vol. 23, no. 21, Oct. 2022, p. 12907. https://doi.org/10.3390%2Fijms232112907.