Fluorescence Correlation Spectroscopy Research Articles

The molecular basis for the synergistic aggregation of tau by alpha-synuclein.

Age-related neurodegenerative disorders like Alzheimer's disease (AD) and Parkinson's disease (PD) take an overwhelming toll on individuals and society. Traditionally, aggregation of a single protein (tau in AD and α-synuclein, or αS, in PD) was thought to be responsible for causing these different pathologies. However, there is increasing evidence that the pathologies of these two diseases overlap, and the individual proteins primarily associated with each disease promote each other's aggregation. Both tau and αS are intrinsically disordered proteins, lacking stable secondary structures under physiological conditions. Here, we complement ensemble aggregation studies with fluorescence correlation spectroscopy to investigate the interactions between tau and αS. Specifically, we measure interactions between full-length tau and constructs corresponding to its proline rich region (PRR) and microtubule binding domain (K18) with monomeric and aggregated forms of αS. We find only weak interactions between any of the tau variants and monomeric αS. Binding to αS aggregates, however, is enhanced relative to monomer, particularly by the PRR. The presence of the C-terminal tail of αS further amplifies this interaction. Interestingly, neither full-length nor truncated αS seeding has a significant impact on tau aggregation under the conditions of our experiments. The findings of our study draw attention to a central role of the PRR in mediating protein-protein interactions and underscores the complexity of the homotypic and heterotypic interactions underlying pathological protein aggregation.

Using single-molecule fluorescence techniques to understand the enzymatic cycle of sex-biased RNA helicases DDX3X and DDX3Y.

Sex differences are pervasive in human health and disease with the most striking differences lying in the sex chromosomes, which encode a group of sex-specific protein homologs. DDX3X and DDX3Y are a pair of sexually dimorphic non-processive ATP-dependent RNA helicases. We previously showed that DDX3Y forms larger, less dynamic phase separated condensates compared to DDX3X, possibly due to the lower ATPase activity of DDX3Y and different dynamic interactions with RNA. We used single-molecule FRET of immobilized, fluorescently labeled RNA duplexes and reported that DDX3X and DDX3Y show different binding dynamics and partial unwinding activities (Shen and Yanas et al., Molecular Cell 2022). To further investigate the enzymatic activities of DDX3, we used multiparameter confocal spectroscopy to obtain unwinding, time-resolved anisotropy, diffusion coefficients, and structural changes by FRET of freely diffusing RNA duplexes upon addition of DDX3, with and without ATP. Duplex RNA with a 5’ overhang was labeled with a Cy3 and Alexa647 FRET pair at the chain termini. The proportion of duplex RNA was quantified and used as a readout for helicase activity, and DDX3X shows higher helicase activity than DDX3Y. With ns time-resolved fluorescence anisotropy, we found that the shorter, non-overhanging strand RNA is released with helicase activity, whereas protein remains bound to the longer RNA strand. Diffusion constants from fluorescence correlation spectroscopy revealed formation of enzymatically active protein clusters at low DDX concentrations. Removal of N- and C-terminal intrinsically disordered regions suppressed formation of clusters and reduced enzymatic activity. We hypothesize that these clusters act as “helicase hubs,” possibly tiny nuclei of liquid granules, which may be translational centers in the cell, leading to efficient translation initiation.

Cellular Uptake of Bevacizumab in Cervical and Breast Cancer Cells Revealed by Single-Molecule Spectroscopy

Bevacizumab is a biological drug that is now extensively studied in clinics against various types of cancer. Although bevacizumab's action is preferably extracellular, there are reports suggesting its internalization into cancer cells, consequently decreasing its therapeutic potential. Here we are solving this issue by applying fluorescence correlation spectroscopy in living cells. We tracked single molecules of fluorescent bevacizumab in MDA-MB-231 and HeLa cells and proved that mobility measurements bring significant added value to standard imaging techniques. We confirmed the presence of the drug in intracellular vesicles. Additionally, we explicitly excluded the presence of a free cytosolic fraction of bevacizumab in both studied cell types. Extracellular and intracellular concentrations of the drug were measured, giving a partition coefficient on the order of 10-5, comparable with the spontaneous uptake of biologically inert nanoparticles. Our work presents how techniques and models developed for physics can answer biological questions.

Quantitative characterization of membrane-protein reversible association using FCS

Functionally meaningful reversible protein-membrane interactions mediate many biological events. Fluorescence correlation spectroscopy (FCS) is increasingly used to quantitatively study the non-reversible binding of proteins to membranes using lipid vesicles in solution. However, the lack of a complete description of the phase and statistical equilibria in the case of reversible protein-membrane partitioning has hampered the application of FCS to quantify the partition coefficient (Kx). In this work, we further extend the theory that describes membrane-protein partitioning to account for spontaneous protein-membrane dissociation and reassociation to the same or a different lipid vesicle. We derive the probability distribution of proteins on lipid vesicles for reversible binding and demonstrate that FCS is a suitable technique for accurate Kx quantification of membrane-protein reversible association. We also establish the limits to Kx determination by FCS studying the Cramer-Rao bound on the variance of the retrieved parameters. We validate the mathematical formulation against reaction-diffusion simulations to study phase and statistical equilibria and compare the Kx obtained from a computational FCS titration experiment with the experimental ground truth. Finally, we demonstrate the application of our methodology studying the association of anti-HIV broadly neutralizing antibody (10E8-3R) to the membrane.

Photophysical Rate Constants and Oxygen Dependence for Si and Ge Rhodamine Zwitterions.

The family of group XIV rhodamine zwitterions are fluorescence probes with carbon, silicon, germanium, or tin substituted in the 10-position of the xanthene ring. Because of their inherent near-infrared fluorescence, photostability and high quantum yields in aqueous solutions, the Si and Ge containing fluorophores in this class have become increasingly important for fluorescent labeling of proteins and biological molecules. This study fully characterizes photophysical rates derived from a model consisting of a singlet ground state, the lowest singlet excited state, and the lowest triplet excited state for two exemplar group XIV rhodamine zwitterions, one containing Si and the other Ge. Within a simple Jablonski diagram, all radiative and non-radiative rates, including intersystem crossing and triplet depopulation rates, were measured as a function of oxygen concentration. It was shown that the triplet depopulation rates are intrinsically fast in comparison with traditional xanthene containing fluorophores, probably due to the increased spin-obit coupling from the Si and Ge substitution in the xanthene ring. Dissolved oxygen increases both the intersystem crossing and triplet depopulation rates. Stern-Volmer analysis was conducted to estimate rates of quenching by oxygen. The experimental data was used to estimate the initial rates for reactive oxygen production by Si and Ge containing fluorophores in aqueous solutions containing different concentrations of dissolved O2. These estimates showed a significantly slower initial rate of reactive oxygen production in comparison with rhodamine 6G. This goes a long way to explaining their inherent photostability. Spectroscopic experiments were also conducted in 77 K viscous aqueous glasses where it was observed that the fluorescence spectra remained unchanged, and the quantum yields increased from 0.53 to 0.84 and from 0.52 to 0.89 for the Si and Ge containing fluorophores respectively; no phosphorescence was observed. All intersystem crossing and triplet depopulation rates were measured using fluorescence correlation spectroscopy (FCS) and analyzed using a new method that extrapolated the power dependence of the FCS curves to optical saturation. This method was verified using published data.

Combined Fluorescence Fluctuation and Spectrofluorometric Measurements Reveal a Red-Shifted, Near-IR Emissive Photo-Isomerized Form of Cyanine 5.

Cyanine fluorophores are extensively used in fluorescence spectroscopy and imaging. Upon continuous excitation, especially at excitation conditions used in single-molecule and super-resolution experiments, photo-isomerized states of cyanines easily reach population probabilities of around 50%. Still, effects of photo-isomerization are largely ignored in such experiments. Here, we studied the photo-isomerization of the pentamethine cyanine 5 (Cy5) by two similar, yet complementary means to follow fluorophore blinking dynamics: fluorescence correlation spectroscopy (FCS) and transient-state (TRAST) excitation-modulation spectroscopy. Additionally, we combined TRAST and spectrofluorimetry (spectral-TRAST), whereby the emission spectra of Cy5 were recorded upon different rectangular pulse-train excitations. We also developed a framework for analyzing transitions between multiple emissive states in FCS and TRAST experiments, how the brightness of the different states is weighted, and what initial conditions that apply. Our FCS, TRAST, and spectral-TRAST experiments showed significant differences in dark-state relaxation amplitudes for different spectral detection ranges, which we attribute to an additional red-shifted, emissive photo-isomerized state of Cy5, not previously considered in FCS and single-molecule experiments. The photo-isomerization kinetics of this state indicate that it is formed under moderate excitation conditions, and its population and emission may thus deserve also more general consideration in fluorescence imaging and spectroscopy experiments.

Shrinking gate fluorescence correlation spectroscopy yields equilibrium constants and separates photophysics from structural dynamics

Fluorescence correlation spectroscopy is a versatile tool for studying fast conformational changes of biomolecules especially when combined with Förster resonance energy transfer (FRET). Despite the many methods available for identifying structural dynamics in FRET experiments, the determination of the forward and backward transition rate constants and thereby also the equilibrium constant is difficult when two intensity levels are involved. Here, we combine intensity correlation analysis with fluorescence lifetime information by including only a subset of photons in the autocorrelation analysis based on their arrival time with respect to the excitation pulse (microtime). By fitting the correlation amplitude as a function of microtime gate, the transition rate constants from two fluorescence-intensity level systems and the corresponding equilibrium constants are obtained. This shrinking-gate fluorescence correlation spectroscopy (sg-FCS) approach is demonstrated using simulations and with a DNA origami-based model system in experiments on immobilized and freely diffusing molecules. We further show that sg-FCS can distinguish photophysics from dynamic intensity changes even if a dark quencher, in this case graphene, is involved. Finally, we unravel the mechanism of a FRET-based membrane charge sensor indicating the broad potential of the method. With sg-FCS, we present an algorithm that does not require prior knowledge and is therefore easily implemented when an autocorrelation analysis is carried out on time-correlated single-photon data.

Trans-cis isomerization kinetics of cyanine dyes reports on the folding states of exogeneous RNA G-quadruplexes in live cells.

Guanine (G)-rich nucleic acids are prone to assemble into four-stranded structures, so-called G-quadruplexes. Abnormal GGGGCC repeat elongations, and in particular their folding states, are associated with amyotrophic lateral sclerosis and frontotemporal dementia. Due to methodological constraints however, most studies of G quadruplex structures are restricted to in vitro conditions. Evidence of how GGGGCC repeats form into G-quadruplexes in vivo is sparse. We devised a readout strategy, exploiting the sensitivity of trans-cis isomerization of cyanine dyes to local viscosity and sterical constraints. Thereby, folding states of cyanine-labeled RNA, and in particular G-quadruplexes, can be identified in a sensitive manner. The isomerization kinetics, monitored via fluorescence blinking generated upon transitions between a fluorescent trans isomer and a non-fluorescent cis isomer, was first characterized for RNA with GGGGCC repeats in aqueous solution using fluorescence correlation spectroscopy and transient state (TRAST) monitoring. With TRAST, monitoring the isomerization kinetics from how the average fluorescence intensity varies with laser excitation modulation characteristics, we could then detect folding states of fluorescently tagged RNA introduced into live cells.

Agonist-controlled competition of RAR and VDR nuclear receptors for heterodimerization with RXR is manifested in their DNA binding

We found previously that nuclear receptors (NRs) compete for heterodimerization with their common partner, retinoid X receptor (RXR), in a ligand-dependent manner. To investigate potential competition in their DNA binding, we monitored the mobility of retinoic acid receptor (RAR) and vitamin D receptor (VDR) in live cells by fluorescence correlation spectroscopy. First, specific agonist treatment and RXR coexpression additively increased RAR DNA binding, while both agonist and RXR were required for increased VDR DNA binding, indicating weaker DNA binding of the VDR/RXR dimer. Second, coexpression of RAR, VDR, and RXR resulted in competition for DNA binding. Without ligand, VDR reduced the DNA-bound fraction of RAR and vice versa, i.e., a fraction of RXR molecules was occupied by the competing partner. The DNA-bound fraction of either RAR or VDR was enhanced by its own and diminished by the competing NR's agonist. When treated with both ligands, the DNA-bound fraction of RAR increased as much as due to its own agonist, whereas that of VDR increased less. RXR agonist also increased DNA binding of RAR at the expense of VDR. In summary, competition between RAR and VDR for RXR is also manifested in their DNA binding in an agonist-dependent manner: RAR dominates over VDR in the absence of agonist or with both agonists present. Thus, side effects of NR-ligand-based (retinoids, thiazolidinediones) therapies may be ameliorated by other NR ligands and be at least partly explained by reduced DNA binding due to competition. Our results also complement the model of NR action by involving competition both for RXR and for DNA sites.

Quantifying membrane binding and diffusion with fluorescence correlation spectroscopy diffusion laws

Many transient processes in cells arise from the binding of cytosolic proteins to membranes. Quantifying this membrane binding and its associated diffusion in the living cell is therefore of primary importance. Dynamic photonic microscopies, e.g., single/multiple particle tracking, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy (FCS), enable non-invasive measurement of molecular mobility in living cells and their plasma membranes. However, FCS with a single beam waist is of limited applicability with complex, non-Brownian, motions. Recently, the development of FCS diffusion laws methods has given access to the characterization of these complex motions, although none of them is applicable to the membrane binding case at the moment. In this study, we combined computer simulations and FCS experiments to propose an FCS diffusion law for membrane binding. First, we generated computer simulations of spot-variation FCS (svFCS) measurements for a membrane binding process combined to 2D and 3D diffusion at the membrane and in the bulk/cytosol, respectively. Then, using these simulations as a learning set, we derived an empirical diffusion law with three free parameters: the apparent binding constant KD, the diffusion coefficient on the membrane D2D, and the diffusion coefficient in the cytosol, D3D. Finally, we monitored, using svFCS, the dynamics of retroviral Gag proteins and associated mutants during their binding to supported lipid bilayers of different lipid composition or at plasma membranes of living cells, and we quantified KD and D2D in these conditions using our empirical diffusion law. Based on these experiments and numerical simulations, we conclude that this new approach enables correct estimation of membrane partitioning and membrane diffusion properties (KD and D2D) for peripheral membrane molecules.

Influence of chain length and branching on poly(ADP-ribose)-protein interactions.

Hundreds of proteins interact with poly(ADP-ribose) (PAR) via multiple PAR interaction motifs, thereby regulating their physico-chemical properties, sub-cellular localizations, enzymatic activities, or protein stability. Here, we present a targeted approach based on fluorescence correlation spectroscopy (FCS) to characterize potential structure-specific interactions of PAR molecules of defined chain length and branching with three prime PAR-binding proteins, the tumor suppressor protein p53, histone H1, and the histone chaperone APLF. Our study reveals complex and structure-specific PAR-protein interactions. Quantitative Kd values were determined and binding affinities for all three proteins were shown to be in the nanomolar range. We report PAR chain length dependent binding of p53 and H1, yet chain length independent binding of APLF. For all three PAR binders, we found a preference for linear over hyperbranched PAR. Importantly, protein- and PAR-structure-specific binding modes were revealed. Thus, while the H1-PAR interaction occurred largely on a bi-molecular 1:1 basis, p53-and potentially also APLF-can form complex multivalent PAR-protein structures. In conclusion, our study gives detailed and quantitative insight into PAR-protein interactions in a solution-based setting at near physiological buffer conditions. The results support the notion of protein and PAR-structure-specific binding modes that have evolved to fit the purpose of the respective biochemical functions and biological contexts.

Phase-separating pyrenoid proteins form complexes in the dilute phase

While most studies of biomolecular phase separation have focused on the condensed phase, relatively little is known about the dilute phase. Theory suggests that stable complexes form in the dilute phase of two-component phase-separating systems, impacting phase separation; however, these complexes have not been interrogated experimentally. We show that such complexes indeed exist, using an in vitro reconstitution system of a phase-separated organelle, the algal pyrenoid, consisting of purified proteins Rubisco and EPYC1. Applying fluorescence correlation spectroscopy (FCS) to measure diffusion coefficients, we found that complexes form in the dilute phase with or without condensates present. The majority of these complexes contain exactly one Rubisco molecule. Additionally, we developed a simple analytical model which recapitulates experimental findings and provides molecular insights into the dilute phase organization. Thus, our results demonstrate the existence of protein complexes in the dilute phase, which could play important roles in the stability, dynamics, and regulation of condensates.

Ultraviolet Nanophotonics Enables Autofluorescence Correlation Spectroscopy on Label-Free Proteins with a Single Tryptophan.

Using the ultraviolet autofluorescence of tryptophan amino acids offers fascinating perspectives to study single proteins without the drawbacks of fluorescence labeling. However, the low autofluorescence signals have so far limited the UV detection to large proteins containing several tens of tryptophan residues. This limit is not compatible with the vast majority of proteins which contain only a few tryptophans. Here we push the sensitivity of label-free ultraviolet fluorescence correlation spectroscopy (UV-FCS) down to the single tryptophan level. Our results show how the combination of nanophotonic plasmonic antennas, antioxidants, and background reduction techniques can improve the signal-to-background ratio by over an order of magnitude and enable UV-FCS on thermonuclease proteins with a single tryptophan residue. This sensitivity breakthrough unlocks the applicability of UV-FCS technique to a broad library of label-free proteins.

A quantitative map of nuclear pore assembly reveals two distinct mechanisms

Understanding how the nuclear pore complex (NPC) is assembled is of fundamental importance to grasp the mechanisms behind its essential function and understand its role during the evolution of eukaryotes1–4. There are at least two NPC assembly pathways—one during the exit from mitosis and one during nuclear growth in interphase—but we currently lack a quantitative map of these events. Here we use fluorescence correlation spectroscopy calibrated live imaging of endogenously fluorescently tagged nucleoporins to map the changes in the composition and stoichiometry of seven major modules of the human NPC during its assembly in single dividing cells. This systematic quantitative map reveals that the two assembly pathways have distinct molecular mechanisms, in which the order of addition of two large structural components, the central ring complex and nuclear filaments are inverted. The dynamic stoichiometry data was integrated to create a spatiotemporal model of the NPC assembly pathway and predict the structures of postmitotic NPC assembly intermediates.

Single-molecule quantification of the oligomeric state of ZIP transporters in mammalian cells with fluorescence correlation spectroscopy.

The SLC39 family of transporters, otherwise known as ZIPs for Zrt and Irt-like Proteins, function to increase cytosolic levels of transition metals. ZIP transporters have been identified at all phylogenetic levels and are members of the SoLute Carrier (SLC) superfamily. There are fourteen ZIP transporters encoded in the human genome. ZIP transmembrane proteins are expressed in the plasma membrane or membranes of intracellular organelles and have unique expression profiles across cell types. While direct structural efforts including x-ray crystallography, NMR and ab initio approaches have been effective tools in elucidating the structure of ZIPs, direct elucidation of the oligomeric state of these proteins is essential in understanding how wild type ZIP proteins function and whether mutations alter the oligomeric state of ZIPs. Unfortunately, several tools to quantify oligomeric states of proteins require overexpression of proteins which can lead to artifacts in experimental results. In contrast, fluorescence correlation spectroscopy (FCS) is a single-molecule technique which can be used to quantify the oligomeric state of transmembrane proteins. FCS takes advantage of the observation that the molecular brightness of a cluster of fluorescent molecules is directly proportional to the number of fluorescent molecules within the protein complex. This chapter describes how to implement FCS, focused on ZIP transporters, to quantify the oligomeric state of transmembrane in vivo. Included within this chapter are procedures to design constructs for experiments, transfection of mammalian cells as well as data acquisition and analysis. Taken together, FCS is a powerful mechanism to investigate the oligomeric state of proteins embedded within membranes of cells.

Fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy for deciphering the morphological evolution of supramolecular self-assembly.

The properties and functions of non-covalent interaction-driven fluorescent supramolecular self-assembly depend greatly on their evolution dynamics. Electron microscopy, atomic force microscopy, and confocal laser scanning microscopy have been used to elucidate the formation of molecular self-assembly. However, some pertinent issues, such as the drying or freezing of the sample for electron microscopy, the influence of the interactions between the tip and the sample in atomic force microscopy imaging, and the low spatial resolution of confocal laser scanning microscopy images, often impede the real-time analysis and exploration of the dynamics of molecular self-assembly processes. In this context, fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy have recently been explored to unravel the physical picture of the in situ growth dynamics and stimuli-induced morphological transformation of luminescent self-assembled structures. The current highlight article demonstrates the need for fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy to acquire precise information on the dynamics and morphological evolution of fluorescent self-assembled architectures using a few remarkable recent studies. In addition to the current status and challenges, the future directions for the further exploration of dynamic self-assembly processes towards developing next-generation functional materials have been delineated.

In vivo measurement of autophagic flux by fluorescence correlation spectroscopy.

Autophagy is a fundamental and phylogenetically conserved self-degradation process and plays a very important role in the selective degradation of deleterious proteins, organelles, and other macromolecules. Although flow cytometry and fluorescence imaging techniques have been used to assess autophagic flux, we remain less able to in vivo monitor autophagic flux in a highly sensitive, robust, and well-quantified manner. Here, we reported a new method for real-time and quantitatively monitoring autophagosomes and assessing autophagic flux in living cells based on fluorescence correlation spectroscopy (FCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) fused with an enhanced green fluorescent protein (EGFP-LC3B) was used as a biomarker to label autophagosomes in living cells, and FCS was used to monitor EGFP-LC3B labeled autophagosomes by using the characteristic diffusion time (τD) value and brightness per particle (BPP) value. By analyzing the distribution frequency of the τD values in living cells stably expressing EGFP-LC3B, mutant EGFP-LC3B (EGFP-LC3BΔG) and enhanced green fluorescent protein (EGFP), we found that the τD value greater than 10 ms was attributed to the signal of EGFP-LC3B labeled autophagosomes. So, we proposed a parameter PAP as an indicator to assess the basal autophagic activity and induced autophagic flux. This new method was able to evaluate autophagy inducers, early-stage autophagy inhibitors, and late-stage autophagy inhibitors. Compared with current methods, our method shows high spatiotemporal resolution and very high sensitivity for autophagosomes in low EGFP-LC3B expressing cells and will become an attractive and alternative method for biological and medical studies, some drug screening, and disease treatment.

Protein-Protein Interactions in Abiotic Stress Signaling: An Overview of Biochemical and Biophysical Methods of Characterization.

The identification and characterization of bona fide abiotic stress signaling proteins can occur at different levels of the complete in vivo signaling cascade or network. Knowledge of a particular abiotic stress signaling protein could theoretically lead to the characterization of complete networks through the analysis of unknown proteins that interact with the previously known protein. Such signaling proteins of interest can indeed be experimentally used as bait proteins to catch interacting prey proteins, provided that the association of bait proteins and prey proteins should yield a biochemical or biophysical signal that can be detected. To this end, several biochemical and biophysical techniques are available to provide experimental evidence for specific protein-protein interactions, such as co-immunoprecipitation, bimolecular fluorescence complementation, tandem affinity purification coupled to mass spectrometry, yeast two hybrid, protein microarrays, Förster resonance energy transfer, or fluorescence correlation spectroscopy. This array of methods can be implemented to establish the biochemical reality of putative protein-protein interactions between two proteins of interest or to identify previously unknown partners related to an initially known protein of interest. The ultimate validity of these methods however depends on the in vitro/in vivo nature of the approach and on the heterologous/homologous context of the analysis. This chapter will review the application and success of some classical methods of protein-protein interaction analysis in the field of plant abiotic stress signaling.

Membrane interactions of apoptotic inhibitor Bcl-xL: What can be learned using fluorescence spectroscopy

Permeabilization of the mitochondrial outer membrane–a point of no return in apoptotic regulation–is tightly controlled by proteins of the Bcl-2 family. Apoptotic inhibitor Bcl-xL is an important member of this family, responsible for blocking the permeabilization, and is also a promising target for anti-cancer drugs. Bcl-xL exists in the following conformations, each believed to play a role in the inhibition of apoptosis: (i) a soluble folded conformation, (ii) a membrane-anchored (by its C-terminal α8 helix) form, which retains the same fold as in solution and (iii) refolded membrane-inserted conformations, for which no structural data are available. In this review, we present the summary of the application of various methods of fluorescence spectroscopy for studying membrane interaction of Bcl-xL, and specifically the formation of the refolded inserted conformation. We discuss the application of environment-sensitive probes, Förster resonance energy transfer, fluorescence correlation spectroscopy, and fluorescent quenching for structural, thermodynamic, and functional characterization of protein-lipid interactions, which can benefit studies of other members of Bcl-2 (e.g., Bax, BAK, Bid). The conformational switching between various conformations of Bcl-xL depends on the presence of divalent cations, pH and lipid composition. This insertion-refolding transition also results in the release of the BH4 regulatory domain from the folded structure of Bcl-xL, which is relevant to the lipid-regulated conversion between canonical and non-canonical modes of apoptotic inhibition.

Assessing the impact of choline chloride and benzyltrimethylammonium chloride-based deep eutectic solvents on the structure and conformational dynamics of bovine serum albumin: a combined steady-state, time-resolved fluorescence and fluorescence correlation spectroscopic study.

Although deep eutectic solvents (DESs) are regarded as useful substitutes for both ionic liquids and common organic solvents for storage and applications of biomolecules, it is still unclear whether all DESs or only specific types of DESs will be suitable for the said purpose. In view of this, the current study aims to report on the structure and conformational dynamics of BSA in the presence of two DESs, namely ethaline (choline chloride:ethylene glycol) and BMEG (benzyltrimethyl ammonium chloride:ethylene glycol), having the same hydrogen bond donor but with a distinct hydrogen bond acceptor, so that how small changes in one constituent of a DES alter the protein-DES interaction at the molecular level can be understood. The protein-DES interaction is investigated by exploiting both ensemble-averaged measurements like steady-state and time-resolved fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and single-molecule sensitive techniques based on fluorescence correlation spectroscopy (FCS). Interestingly, the results obtained from these studies have demonstrated that while a very small quantity of BMEG completely unfolds the native structure of the protein, it remains in a partially unfolded state even at very high ethaline content. More interestingly, it has been found that at very high concentrations of BMEG, the unfolded protein undergoes enhanced protein-protein interaction resulting in the aggregation of BSA. All of the results obtained from these investigations have essentially suggested that both protein-DES interaction and interspecies interaction among the constituent of DESs play a crucial role in governing the overall stability and conformational dynamics of the protein in DESs.