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

Abstract

Living cells are complex, crowded, and dynamic and continually respond to environmental and intracellular stimuli. They also have heterogeneous ionic strength with compartmentalized variations in both intracellular concentrations and types of ions. These challenges would benefit from the development of quantitative, noninvasive approaches for mapping the heterogeneous ionic strength fluctuations in living cells. Here, we investigated a class of recently developed ionic strength sensors that consists of mCerulean3 (a cyan fluorescent protein) and mCitrine (a yellow fluorescent protein) tethered via a linker made of two charged α-helices and a flexible loop. The two helices are designed to bear opposite charges, which is hypothesized to increase the ionic screening and therefore a larger intermolecular distance. In these protein constructs, mCerulean3 and mCitrine act as a donor–acceptor pair undergoing Förster resonance energy transfer (FRET) that is dependent on both the linker amino acids and the environmental ionic strength. Using time-resolved fluorescence of the donor (mCerulean3), we determined the sensitivity of the energy transfer efficiencies and the donor–acceptor distances of these sensors at variable concentrations of the Hofmeister series of salts (KCl, LiCl, NaCl, NaBr, NaI, Na2SO4). As controls, similar measurements were carried out on the FRET-incapable, enzymatically cleaved counterparts of these sensors as well as a construct designed with two electrostatically neutral α-helices (E6G2). Our results show that the energy transfer efficiencies of these sensors are sensitive to both the linker amino acid sequence and the environmental ionic strength, whereas the sensitivity of these sensors to the identity of the dissolved ions of the Hofmeister series of salts seems limited. We also developed a theoretical framework to explain the observed trends as a function of the ionic strength in terms of the Debye screening of the electrostatic interaction between the two charged α-helices in the linker region. These controlled solution studies represent an important step toward the development of rationally designed FRET-based environmental sensors while offering different models for calculating the energy transfer efficiency using time-resolved fluorescence that is compatible with future in vivo studies.

Time-Resolved Fluorescence Anisotropy and Molecular Dynamics Analysis of a Novel GFP Homo-FRET Dimer

“…Thus, the combination of time-resolved fluorescence anisotropy and MD simulations allows us to gain detailed insight into the behavior of the GFP dimer. We propose this construct as a homo-FRET standard to be used for comparison with homo-FRET-based biosensors ( 103 ) and potentially as a reference when studying dimerization processes in cells via FRET between GFP.…”

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).…”

“…The energy transfer efficiency among a given donor-acceptor pair is dependent on the distance that separates the donor and acceptor molecule, the orientation parameter [κ ( Jareserijman and Jovin, 2006 )] of their relative dipole moments, and the spectral overlap between the donor’s emission and the acceptor’s absorption ( Piston and Kremers, 2007 ; Gadella, 2009 ; Choi et al, 2012 ; Currie et al, 2017 ). 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 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.…”