We characterize a novel macromolecular crowding biosensor using multiple modalities of ultrafast time-resolved two-photon fluorescence for FRET, rotational dynamics, and thermodynamic equilibrium analyses in controlled Ficoll-70 solutions.

Recently, we have investigated the sensitivity of an mEGFP-linker-mScarlet-I construct (GE2.3) in response to macromolecular crowding using ensemble time-resolved two-photon (2P) fluorescence measurements [Mersch et al., Phys. Chem. Chem. Phys. 2024, 26(5), 3927-3940] as a point of reference for developing a single-molecule approach for Förster resonance energy transfer (FRET). Here, we investigate the fluorescence fluctuations, FRET, molecular brightness, and translational diffusion of GE2.3 as a model system using fluorescence correlation spectroscopy (FCS), at the single molecule level, as a function of the excitation and detection wavelengths of the donor (mEGFP) and the acceptor (mScarlet-I). We hypothesize that the molecular brightness (number of fluorescence photons per molecule) of the donor of GE2.3, in the presence and absence of the acceptor, would be distinct due to FRET at the single-molecule level. To test this hypothesis, we used wavelength-dependent FCS to quantify the molecular brightness of intact and enzymatically cleaved GE2.3 as a function of Ficoll-70 (a crowding agent, 0-300 g L-1) at room temperature. Our results indicate that the molecular brightness of intact GE2.3 in a buffer is smaller than that of the cleaved counterpart under 488-nm excitation of the donor, which is attributed to FRET. In contrast, the molecular brightness of both cleaved and intact GE2.3 seems to be the same under the 561-nm excitation of the acceptor due to the absence of FRET. Our results also show that the FRET efficiency of GE2.3 increases as the concentration of Ficoll increases up to 200 g L-1, which agrees with our previous time-resolved 2P-fluorescence measurements. Fluctuation autocorrelation analysis shows that the translational diffusion of intact and cleaved GE2.3 sensors deviates from the Stokes-Einstein model in Ficoll crowded solutions. Additionally, we highlight the multiscale translational and rotational diffusion coefficients of GE2.3 in terms of the average distance between neighboring Ficoll molecules, over the same concentration range, to elucidate the spatio-temporal scaling aspect of FRET and protein-protein interactions. These single-molecule studies would be beneficial for future studies in living cells, where very low GE2.3 expression levels will be required as compared with ensemble, time-resolved 2P-fluorescence measurements.

Microenvironmental ionic strength in living cells plays important roles in protein folding, protein-protein interactions and aggregation, intrinsically disordered protein structures, enzymatic activity, and electrostatic interactions. The cellular ionic strength also varies in response to the extracellular environment, osmosis, and metabolic activities. Here we investigate the sensitivity of an mCerulean3-linker-mCitrine construct (RD) to the ionic strength of potassium chloride solutions, at the single-molecule level, using fluorescence correlation spectroscopy (FCS).

Living cells are dynamic and heterogeneous microenvironments, in which macromolecular crowding impacts transport, intermolecular interactions, and kinetics. Here, we characterize a protein biosensor that has been designed to detect changes in macromolecular crowding using Förster energy transfer (FRET). We investigate the fluorescence fluctuations, molecular brightness, and translational diffusion of the mEGFP-linker-mScarlet-I construct (GE2.3) using fluorescence correlation spectroscopy (FCS) of the donor (mEGFP) in the presence and absence of the acceptor (mScarlet-I) in response to environmental crowding.

Macromolecular crowding and ionic strength in living cells influence a myriad of biochemical processes essential to cell function and survival. For example, macromolecular crowding is known to affect diffusion, biochemical reaction kinetics, protein folding, and protein-protein interactions. In addition, enzymatic activities, protein folding, and cellular osmosis are also sensitive to environmental ionic strength. Recently, genetically encoded mCerulean3-linker-mCitrine constructs have been developed and characterized using time-resolved fluorescence measurements as a function of the amino acid sequence of the linker region as well as the environmental crowding and ionic strength. Here, we investigate the thermodynamic equilibrium of structural conformations of mCerulean3-linker-mCitrine constructs in response to the environmental macromolecular crowding and ionic strength. We have developed a theoretical framework for thermodynamic equilibrium of the structural conformations of these environmental sensors. In addition, we tested these theoretical models for thermodynamic analysis of these donor-linker-acceptor sensors using time-resolved fluorescence measurements as a function of the amino acid sequence of the linker region. Employing ultrafast time-resolved fluorescence measurements for gaining thermodynamic energetics would be helpful for Förster Resonance Energy Transfer (FRET) studies of protein-protein interactions in both living cells and controlled environments.

Laser-induced two-photon (2P) fluorescence microscopy is routinely used as a noninvasive, quantitative methodology for a wide range of cell and tissue studies due to its enhanced penetration depth, reduced laser scattering, and minimum extended photobleaching. Macromolecular crowding affects many cellular processes such as diffusion, biochemical reaction kinetics, protein-protein interactions, and protein folding. Here, we carried out time-resolved 2P fluorescence measurements of a novel mEGFP-linker-mScarlet-I macromolecular crowding construct (GE2.3) to characterize its environmental sensitivity in biomimetic crowded solutions (Ficoll-70, 0-300 g/L) using Förster resonance energy transfer (FRET) analysis. For these studies, the excited-state 2P-fluorescence dynamics of the donor (mEGFP) were investigated in the presence (intact) and absence (enzymatically cleaved) of the acceptor (mScarlet-I). In addition, time-resolved 2P-fluorescence measurements of intact GE2.3 were used to determine the corresponding equilibrium constant and the Gibbs free energy associated with the structural conformation equilibrium in response to environmental crowding. We further carried out time-resolved 2P-fluorescence depolarization anisotropy to examine both the macromolecular crowding and the linker flexibility effects on GE2.3 rotational dynamics within the context of Stokes-Einstein model as compared with theoretical predictions based on its molecular weight. Our results further the development of a rational engineering design of bioenvironmental sensors. Additionally, these results in well-defined environments will inform our future in vivo studies of genetically encoded GE2.3 towards the mapping of crowded intracellular environment under different physiological conditions. Importantly, this GE2.3 sensor allows for minimal interference of the intrinsic cellular autofluorescence with the FRET analysis and crowding studies.

Different modalities of time-resolved fluorescence lifetime techniques are typically used for Förster resonance energy transfer (FRET) studies in vivo and in solution and have been used to measure macromolecular processes and interactions. Here, we examine the sensitivity of the estimated FRET efficiency and donor-acceptor distance to different modalities of time-resolved two-photon (2P) fluorescence measurements, experimental design, and data analysis. As a model, we used the biosensor (mTurquoise-linker-mCitrine), where mTurquoise-mCitrine serves as the FRET pair tethered with a flexible linker.

Macromolecular crowding influences many biological processes in living cells such as protein folding, protein-protein interactions, translational diffusion, and biochemical reaction kinetics. In order to investigate the correlation between the heterogenous, dynamic macromolecular crowding and cell physiology, site-specific biosensors and noninvasive, quantitative methodology are critically needed. Here, we investigate the effects of donor identity (mCerulean3, mTurqoise2.1, and mTurqoise2.0) on the environmental sensitivity of genetically encoded sensors (donor-linker-mCitrine) using time-resolved two-photon fluorescence measurements.

The heterogeneous cellular environment influences a myriad of biological processes. For example, macromolecular crowding affects biochemical reactions, protein-protein interactions, and protein folding. Additionally, the structure-function relationship of biomolecules and enzymatic activities are sensitive to the surrounding ionic strength. In this contribution, we highlight our recent studies on a family of donor–linker– acceptor constructs, which were designed for mapping the macromolecular crowding and ionic strength in living cells. Integrated ultrafast laser spectroscopy methods have been employed to quantify the Förster resonance energy transfer (FRET) and the donor-acceptor distance as a measure of the sensitivity of these constructs to environmental changes. The donor-acceptor FRET pairs are intrinsically fluorescent cyan and yellow proteins, respectively, that can be genetically encoded in living cells. The sensitivity of these constructs to environmental biomimetic crowding and ionic strength was investigated as a function of the sequence and charge of the linker regions, as well as the identity of the donor protein. Integrating noninvasive, quantitative laser-induced fluorescence methods with FRET, as a molecular ruler, provides a powerful tool for cellular studies towards mapping out macromolecular crowding and ionic strength in living cells. Our results are key for the development of rational design strategies for engineering enhanced noninvasive biosensors with better environmental sensitivities. The same sensors were used as a model system for developing new experimental approaches for protein-protein interaction and FRET studies. Importantly, these diagnostic molecular and analytical tools set the stage for understanding the correlation between these environmental factors and cellular functions.

Compartmentalized, dynamic ionic strength within living cells influences numerous biochemical mechanisms such as catalytic function, protein folding, osmotic pressure, and energy production. Recent development of genetically encoded donor-linker-acceptor biosensors that undergo Förster resonance energy transfer (FRET) offers a promising methodology towards noninvasive, site-specific, quantitative, and sensitive mapping of in vivo ionic strength. Here, we investigate the effects of amino acid sequence in the flexible linker on the FRET efficiency and hence the sensitivity to ionic strength of varying KCl concentration.

The heterogeneity of the cellular ionic strength is critical for in vivo biological functions. We have previously characterized hetero-FRET biosensors that are sensitive to the environmental ionic strength using integrated fluorescence methods. This family of biosensors consists of the FRET pair, mCerulean3 (donor) and mCitrine (acceptor), that is linked through a flexible hinge of two charged alpha-helices (one that is basic and the other that is acidic). We have demonstrated that as the ionic strength increases, energy transfer decreases due to electrostatic screening that leads to an increase in the donor-acceptor distance. In this contribution, we are investigating a new ionic strength biosensor that has a single basic alpha-helix, K6, as a means to elucidate the underlying mechanism for its ionic strength sensitivity, as well as developing rational design strategies for environmental sensing. As a control, we carried out similar measurements on the parent E6 sensor that has an electrostatically neutral alpha-helix in the linker region. These studies are carried out using integrated fluorescence methods such as time-resolved fluorescence and polarization anisotropy as a function of the environmental ionic strength using different Hofmeister salt solutions.

Eukaryotic cells exploit dynamic and compartmentalized ionic strength to impact a myriad of biological functions such as enzyme activities, protein-protein interactions, and catalytic functions. Herein, we investigated the fluorescence depolarization dynamics of recently developed ionic strength biosensors (mCerulean3-linker-mCitrine) in Hofmeister salt (KCl, NaCl, NaI, and Na2SO4) solutions. The mCerulean3-mCitrine acts as a Förster resonance energy transfer (FRET) pair, tethered together by two oppositely charged α-helices in the linker region. We developed a time-resolved fluorescence depolarization anisotropy approach for FRET analyses, in which the donor (mCerulean3) is excited by 425-nm laser pulses, followed by fluorescence depolarization analysis of the acceptor (mCitrine) in KE (lysine-glutamate), arginine-aspartate, and arginine-glutamate ionic strength sensors with variable amino acid sequences. Similar experiments were carried out on the cleaved sensors as well as an E6G2 construct, which has neutral α-helices in the linker region, as a control. Our results show distinct dynamics of the intact and cleaved sensors. Importantly, the FRET efficiency decreases and the donor-acceptor distance increases as the environmental ionic strength increases. Our chemical equilibrium analyses of the collapsed-to-stretched conformational state transition of KE reveal that the corresponding equilibrium constant and standard Gibbs free energy changes are ionic strength dependent. We also tested the existing theoretical models for FRET analyses using steady-state anisotropy, which reveal that the angle between the dipole moments of the donor and acceptor in the KE sensor are sensitive to the ionic strength. These results help establish the time-resolved depolarization dynamics of these genetically encoded donor-acceptor pairs as a quantitative means for FRET analysis, which complement traditional methods such as time-resolved fluorescence for future in vivo studies.

In this report, we have developed a simple approach using single-detector fluorescence autocorrelation spectroscopy (FCS) to investigate the Förster resonance energy transfer (FRET) of genetically encoded, freely diffusing crTC2.1 (mTurquoise2.1–linker–mCitrine) at the single molecule level. We hypothesize that the molecular brightness of the freely diffusing donor (mTurquoise2.1) in the presence of the acceptor (mCitrine) is lower than that of the donor alone due to FRET. To test this hypothesis, the fluorescence fluctuation signal and number of molecules of freely diffusing construct were measured using FCS to calculate the molecular brightness of the donor, excited at 405 nm and detected at 475/50 nm, in the presence and absence of the acceptor. Our results indicate that the molecular brightness of cleaved crTC2.1 in a buffer is larger than that of the intact counterpart under 405-nm excitation. The energy transfer efficiency at the single molecule level is larger and more spread in values as compared with the ensemble-averaging time-resolved fluorescence measurements. In contrast, the molecular brightness of the intact crTC2.1, under 488 nm excitation of the acceptor (531/40 nm detection), is the same or slightly larger than that of the cleaved counterpart. These FCS-FRET measurements on freely diffusing donor-acceptor pairs are independent of the precise time constants associated with autocorrelation curves due to the presence of potential photophysical processes. Ultimately, when used in living cells, the proposed approach would only require a low expression level of these genetically encoded constructs, helping to limit potential interference with the cell machinery.