Crowding Effects on Energy-Transfer Efficiencies of Hetero-FRET Probes As Measured Using Time-Resolved Fluorescence Anisotropy

Abstract

Macromolecular crowding is prevalent in all living cells due to the presence of large biomolecules and organelles. Cellular crowding is heterogeneous and is known to influence biomolecular transport, biochemical reactions, and protein folding. Emerging evidence suggests that some cell pathologies may be correlated with compartmentalized crowding. As a result, there is a need for robust biosensors that are sensitive to crowding as well as quantitative, noninvasive fluorescence methods that are compatible with living cells studies. Here, we have developed a model that describes the rotational dynamics of hetero-Förster resonance energy transfer (FRET) biosensors as a means to determine the energy-transfer efficiency and donor–acceptor distance. The model was tested on wavelength-dependent time-resolved fluorescence anisotropy of hetero-FRET probes (mCerulean3–linker–mCitrine) with variable linkers in both crowded (Ficoll-70) and viscous (glycerol) solutions at room temperature. Our results indicate that the energy-transfer efficiencies of these FRET probes increase as the linker becomes shorter and more flexible in pure buffer at room temperature. In addition, the FRET probes favor compact structures with enhanced energy-transfer efficiencies and a shorter donor–acceptor distance in the heterogeneous, polymer-crowded environment due to steric hindrance. In contrast, the extended conformation of these FRET probes is more favorable in viscous, homogeneous environments with a reduced energy-transfer efficiency compared to those in pure buffer, which we attribute to reduced structural fluctuations of the mCerulean3–mCitrine FRET pair in the glycerol-enriched buffer. Our results represent an important step toward the application of quantitative and noninvasive time-resolved fluorescence anisotropy of hetero-FRET probes to investigate compartmentalized macromolecular crowding and protein–protein interactions in living cells as well as in controlled environments.

FRET Analysis of Ionic Strength Sensors in the Hofmeister Series of Salt Solutions Using Fluorescence Lifetime Measurements

“…When excited at 425 nm, the time-resolved fluorescence, F ( t ), of the donor (475/50 nm) in the intact RD, KE, and RE constructs is satisfactorily described as a biexponential decay model such that In this model, we assume that α 1 and α 2 are amplitude fractions (α 1 + α 2 = 1) of two distinct subpopulations; one undergoes FRET (at a rate of k ET + k fl D and amplitude α 1 ) and the other population decays only via fluorescence (i.e., no FRET) at a rate of k fl D and amplitude α 2 . The biexponential decay model was satisfactory following the Akaike information criterion .…”

Quantification of FRET-induced angular displacement by monitoring sensitized acceptor anisotropy using a dim fluorescent donor

“…The well-separated absorption/emission bands of eGFP and mRFP1 ensured minimal donor bleed-through, which inevitably resulted in smaller spectral overlap, and, hence, less efficient FRET. The group of Heikal developed mCerulean-mCitrine-based biosensors to study macromolecular crowding in cells 44 , 45 . They used time-resolved anisotropy to analyze the rotational dynamics of the probes, but eliminating donor background and subsequent quantification of angular displacement were not in the scope of their research.…”

Molecular Brightness Approach for FRET Analysis of Donor-Linker-Acceptor Constructs at the Single Molecule Level: A Concept

“…This model system was selected due to its low energy transfer efficiency (7.1 ± 0.5)% as measured using time-resolved fluorescence ( Aplin et al, 2020 ) to test the sensitivity of the proposed FCS approach. For the control experiments on the donor alone, the linker region of the FRET probe (mTurquoise2.1–link–mCitrine) was digested using the serine protease, proteinase K ( Leopold et al, 2019 ; Schwarz et al, 2019 ; Miller et al, 2020 ; Aplin et al, 2021 ) ( Figure 1B ). The absorption and emission spectra of crTC2.1 construct in a buffer are also shown ( Figure 1C ), where the excitation wavelength of the donor (405 nm) and its fluorescence detection can be visualized (arrows).…”

“…In addition, FRET has been used successfully to study intermolecular interactions and their dynamics in a myriad of biological systems, both in vitro and in vivo ( Lakowicz, 2006 ; Piston and Kremers, 2007 ; Zhang et al, 2013 ; Shrestha et al, 2015 ; Okamoto and Sako, 2017 ; Schwarz et al, 2019 ; Miller et al, 2020 ). FRET applications in scientific research include molecule-molecule interactions ( Margineanu et al, 2016 ), conformational changes of biomolecules ( Ha et al, 1996 ), and environmental sensing ( Leopold et al, 2019 ; Schwarz et al, 2019 ; Miller et al, 2020 ).…”

“…In these constructs, the donor (mCerluean3) and acceptor (mCitrine) act as a FRET pair that are tethered together by either neutral (crowding) or two oppositely charged α-helices (ionic strength) in the linker region ( Liu et al, 2017b ). These environmental sensors have been investigated using ensemble averaging techniques such as time-resolved fluorescence ( Currie et al, 2017 ; Schwarz et al, 2019 ; Miller et al, 2020 ) and time-resolved anisotropy ( Currie et al, 2017 ; Leopold et al, 2019 ; Aplin et al, 2021 ) measurements. However, time-resolved fluorescence methods require expensive and specialized equipment as well as sophisticated users for experimental design and data analysis.…”