Fluorescence Dynamics of a FRET Probe Designed for Crowding Studies

The Journal of Physical Chemistry. B
Megan CurrieAhmed A Heikal

Abstract

Living cells are crowded with macromolecules and organelles. As a result, there is an urgent need for molecular sensors for quantitative, site-specific assessment of the macromolecular crowding effects on a myriad of biochemical processes toward quantitative cell biology and biophysics. Here we investigate the excited-state dynamics and translational diffusion of a novel FRET sensor (mCerulean-linker-mCitrine) in a buffer (PBS, pH 7.4) at room temperature. Complementary experiments were carried out on free CFP, YFP, and the cleaved FRET probe as controls. The wavelength-dependent fluorescence lifetime measurements of the donor and acceptor in the FRET probe, using the time-correlated single-photon counting technique, indicate an energy transfer efficiency of 6.8 ± 0.9% in PBS, with distinct excited-state dynamics from the recombinant CFP and YFP. The estimated mCerulean-mCitrine distance in this FRET probe is 7.7 ± 0.2 nm. The energy transfer efficiency increases (11.5 ± 0.9%) as the concentration of Ficoll-70 increases over the range of 0-300 g/L with an estimated mCerulean-mCitrine distance of 6.1 ± 0.2 nm. Complementary time-resolved anisotropy measurements suggest that the rotational diffusion of hetero-FRET in PBS is sensi...Continue Reading

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Citations

Sep 12, 2018·Physical Chemistry Chemical Physics : PCCP·Hong Bok LeeAhmed A Heikal
Oct 22, 2018·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Anh CongAhmed A Heikal
Oct 13, 2017·The Journal of Physical Chemistry Letters·Mizuki TakeuchiTakakazu Nakabayashi
Apr 4, 2019·The Journal of Physical Chemistry. B·Joshua D SlocumRalph Jimenez
Jul 26, 2018·Journal of the American Chemical Society·Shahar SukenikMartin Gruebele
Jan 10, 2019·Analytical Chemistry·Shuntaro TakahashiNaoki Sugimoto

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