Fluorescent protein with environmentally-sensitive fluorescence lifetime for quantitative pH measurement

被引：0
|
作者：
Simonyan, Tatiana R. [1 ]
Protasova, Elena A. [2 ]
Mamontova, Anastasia, V
Shakhov, Aleksander M. [3 ]
Bodunova, Daria, V
Sidorenko, Svetlana, V [2 ]
Maksimov, Eugene G. [2 ]
Bogdanov, Alexey M. [1 ,4 ]
机构：
[1] Shemyakin Ovchinnikov Inst Bioorgan Chem, Moscow 117997, Russia
[2] MV Lomonosov Moscow State Univ, Fac Biol, Moscow 119992, Russia
[3] NN Semenov Fed Res Ctr Chem Phys, Bio &Nanophoton Lab, Moscow 119991, Moscow, Russia
[4] Izmir Inst Technol, Dept Photon, TR-35430 Izmir, Turkiye
来源：
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS | 2025年 / 766卷
基金：
俄罗斯科学基金会;
关键词：
Fluorescence lifetime; FLIM; pH measurement; Genetically encoded indicators; Live-cell imaging; INTRACELLULAR PH; IMAGING MICROSCOPY; CARBON DOTS; SENSOR; MITOCHONDRIAL; INDICATOR; CELLS; NANOPARTICLES; MECHANISM; VARIANTS;
D O I：
10.1016/j.abb.2025.110350
中图分类号：
Q5 [生物化学]; Q7 [分子生物学];
学科分类号：
071010 ; 081704 ;
摘要：
Intracellular pH is a key factor in cell homeostasis, regulated within specific compartments, and changes in pH can result from or affect biochemical pathways. This study explores a yellow fluorescent protein EYFP-G65T as a core for a time-resolved pH-indicator. Among the tested designs-a circular permutant, a chimeric SypHer3s-like construct, and an unmodified protein-the unmodified EYFP-G65T performed best for live-cell imaging. Upon two-photon excitation, purified EYFP-G65T exhibited a 4.5-fold increase in mean fluorescence lifetime across pH 5.5-7 and a 7-fold change in its major component's lifetime from pH 6.5-8. Using this indicator, we measured pH values ranging from 6 to 8 in various organelles, and mapped pH shifts in mitochondria and the Golgi apparatus in response to stimuli.
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页数：12
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