Time-resolved Fluorescence Anisotropy Research Articles

On the Interpretation of subtilisin Carlsberg Time-Resolved Fluorescence Anisotropy Decays: Modeling with Classical Simulations.

In this work, we discuss the challenging time-resolved fluorescence anisotropy of subtilisin Carlsberg (SC), which contains a single Trp residue and is a model fluorescence system. Experimental decay rates and quenching data suggest that the fluorophore should be exposed to water, but the Trp is partially buried in a hydrophobic pocket in the crystallographic structure. In order to study this inconsistency, molecular dynamics simulations were performed to predict the anisotropy decay rates and emission wavelengths of the Trp. We confirmed the inconsistency of the crystallographic structure with the experimentally observed fluorescence data and performed free energy calculations to show that the buried Trp conformation is 2 orders of magnitude (∼3 kcal/mol) more stable than the solvent-exposed one. However, molecular dynamics simulations in which the Trp side chain was restricted to solvent-exposed conformations displayed a maximum Trp emission wavelength shifted toward lower energies and decay rates compatible with the experimentally probed rates. Therefore, if the solvent-exposed conformations are the most important emitters, the experimental anisotropy can be compatibilized with the crystallographic structure. The most likely explanation is that the fluorescence of the most probable conformation in solution, observed in the crystal, is quenched, and this is consistent with the low quantum yield of Trp113 of SC. Additionally, some experiments might have probed denatured or lysed SC structures. SC anisotropy provides an interesting target for the study of fluorescence anisotropy using simulations, which can be used to test and exemplify how modeling can aid the interpretation of experimental data in a system where structure and solution experiments appear to be inconsistent.

Dynamics Gradient of Polymer Chains near a Solid Interface.

The relaxation dynamics of polyisoprene (PI) and nitrile butadiene rubber (NBR) chains at the SiO2 interface were directly probed as a function of distance from the SiO2 surface using time-resolved evanescent wave-induced fluorescence anisotropy, dielectric relaxation spectroscopy, and sum-frequency generation spectroscopy. We found the presence of the dynamics gradient of chains in the interfacial region with the SiO2 surface and tried to assign it to the two kinds of adsorbed chains, namely, loosely and strongly adsorbed, at the interface. The segmental relaxation of chains in the strongly adsorbed layer at the interface could be slower than that of bulk chains by more than 10 orders.

Liquid Structure and Dynamics of Tetraalkylammonium Bromide-Based Deep Eutectic Solvents: Effect of Cation Chain Length.

Deep eutectic solvents (DESs) have emerged in recent years as environmentally sustainable media across several fields. However, knowledge of liquid structure, dynamics, and solute-solvent interactions in many DESs that is essential for exploiting their potential is still lacking. In this work, we make an attempt to obtain some insight into these aspects of a set of less-explored DESs comprising tetraalkylammonium bromide salts and ethylene glycol (EG) by monitoring the fluorescence response of some carefully chosen dipolar (C153 and 4-AP) and nonpolar (9-PA) solutes in these media. Specifically, we have studied the translational and rotational diffusion dynamics of these molecular systems using single-molecule-based fluorescence correlation spectroscopy technique and ensemble-based time-resolved fluorescence anisotropy measurements. These results point to spatial and dynamic heterogeneity of these DESs, which becomes prominent in systems comprising cations with a longer alkyl chain length. This study reveals that diffusion dynamics of the probe molecules is determined not only by the solvent bulk viscosity but also dependent on their microenvironments and solute-solvent interactions experienced in these media.

Solvent dependent relaxation dynamics in lithium ion battery electrolytes: Coupling to medium friction

Solution heterogeneity and dynamics of the industrially relevant electrolyte system, [9.795{xEC + (1 − x)PC} + 0.869LiClO4] with x representing mole fraction, were explored at different loadings of ethylene carbonate (EC) via temperature dependent measurements of time-resolved spectral response and physico-chemical properties. This system was chosen because polarity differed considerably on replacing propylene carbonate (PC) by EC but the solution viscosity remained nearly the same, allowing to investigate the polarity impact on solution inhomogeneity and solute dynamics. Coumarin 153 (C153) and trans‑2‑[4‑(dimethylamino)styryl]‑benzothiazole (trans-DMASBT) were used as fluorescent probes which differ widely in their excited state lifetimes. Driven by the potential for applications of these electrolyte systems in the energy sector, we have investigated them at an optimal composition with (PC + EC)/LiClO4 mole ratio of ~11.3, where direct current (DC) conductivity reaches at the maximum. Signatures of spatio-temporal heterogeneity were detected in steady state fluorescence and time-resolved anisotropy measurements with DMASBT (lifetime ~100–200 ps) but not with C153, suggesting a timescale over which the inhomogeneity density distributions persist in these electrolyte systems. Subsequent Stokes shift dynamics measurements using C153 captured a dynamic shift of ~700–900 cm−1 with an estimated missing portion of ~50%, and biphasic solvation response characterized by sub-100 ps and nanosecond components. Time-resolved fluorescence anisotropy measurements reflect partial viscosity decoupling for the short lifetime probe and bimodal frictional response in these electrolyte systems.

VIPP1 rods engulf membranes containing phosphatidylinositol phosphates

In cyanobacteria and plants, VIPP1 plays crucial roles in the biogenesis and repair of thylakoid membrane protein complexes and in coping with chloroplast membrane stress. In chloroplasts, VIPP1 localizes in distinct patterns at or close to envelope and thylakoid membranes. In vitro, VIPP1 forms higher-order oligomers of >1 MDa that organize into rings and rods. However, it remains unknown how VIPP1 oligomerization is related to function. Using time-resolved fluorescence anisotropy and sucrose density gradient centrifugation, we show here that Chlamydomonas reinhardtii VIPP1 binds strongly to liposomal membranes containing phosphatidylinositol-4-phosphate (PI4P). Cryo-electron tomography reveals that VIPP1 oligomerizes into rods that can engulf liposomal membranes containing PI4P. These findings place VIPP1 into a group of membrane-shaping proteins including epsin and BAR domain proteins. Moreover, they point to a potential role of phosphatidylinositols in directing the shaping of chloroplast membranes.

Behavior of the DPH fluorescence probe in membranes perturbed by drugs.

1,6-Diphenyl-1,3,5-hexatriene (DPH) is one of the most commonly used fluorescent probes to study dynamical and structural properties of lipid bilayers and cellular membranes via measuring steady-state or time-resolved fluorescence anisotropy. In this study, we present a limitation in the use of DPH to predict the order of lipid acyl chains when the lipid bilayer is doped with itraconazole (ITZ), an antifungal drug. Our steady-state fluorescence anisotropy measurements showed a significant decrease in fluorescence anisotropy of DPH embedded in the ITZ-containing membrane, suggesting a substantial increase in membrane fluidity, which indirectly indicates a decrease in the order of the hydrocarbon chains. This result or its interpretation is in disagreement with the fluorescence recovery after photobleaching measurements and molecular dynamics (MD) simulation data. The results of these experiments and calculations indicate an increase in the hydrocarbon chain order. The MD simulations of the bilayer containing both ITZ and DPH provide explanations for these observations. Apparently, in the presence of the drug, the DPH molecules are pushed deeper into the hydrophobic membrane core below the lipid double bonds, and the probe predominately adopts the orientation of the ITZ molecules that is parallel to the membrane surface, instead of orienting parallel to the lipid acyl chains. For this reason, DPH anisotropy provides information related to the less ordered central region of the membrane rather than reporting the properties of the upper segments of the lipid acyl chains.

Specific monovalent cation effect on protein-protein interactions revealed by protein rotational diffusion analysis

The protein-protein interactions induced by cation specific effects were characterized by the rotational diffusion analysis. The rotational diffusion coefficient, Drot, estimated by time-resolved fluorescence anisotropy of fluorescent-labeled lysozyme was reduced responding to monovalent cation chlorides and was able to be approximated by quadratic functions of lysozyme concentration. The resultant first and second terms of lysozyme concentration were observed to be negative and positive according to the species and concentrations of monovalent cations, respectively. These two hydrodynamic interaction parameters demonstrated that the attractive and also repulsive interactions were induced between lysozymes in the order of inverse Hofmeister effect.

Polarized Two-Photon Absorption and Heterogeneous Fluorescence Dynamics in NAD(P)H

Two-photon absorption (2PA) finds widespread application in biological systems, which frequently exhibit heterogeneous fluorescence decay dynamics corresponding to multiple species or environments. By combining polarized 2PA with time-resolved fluorescence intensity and anisotropy decay measurements, we show how the two-photon transition tensors for the components of a heterogeneous population can be separately determined, allowing structural differences between the two fluorescent states of the redox cofactor NAD(P)H to be identified. The results support the view that the two states correspond to alternate configurations of the nicotinamide ring, rather than folded and extended conformations of the entire molecule.

Impact of Topology on the Characteristics of Water inside Cubic Lyotropic Liquid Crystalline Systems.

Water molecules present inside the lipid-based cubic liquid crystalline phases are found to play a major role in wide range of applications, such as protein crystallization, virus detection, delivery of drug and biomolecules, etc. In this regard, it is crucial to elucidate static and dynamic properties of the water molecules in the nanochannels and to explore the effect of geometrical topology on the nature of the water inside the different cubic phases. In the present work, we have incorporated two probes, coumarin-343 (C-343) and coumarin-480 (C-480), in two cubic phases with different symmetries, namely gyroid ( Ia3 d) and double diamond ( Pn3 m) with the same water content (22%), to probe the micropolarity, the microviscosity, and the hydration dynamics at different hydrophobic depths in the mesophases. Steady state results estimate the polarity at the lipid-water interface to be similar to that of ethanol, and the polarity near the more hydrophilic parts of the nanochannel resembles that of ethylene glycol. We have also observed a gradient in the microviscosity inside the LLC nanochannels from time-resolved fluorescence anisotropy studies. The hydration dynamics, which play a key role in the numerous applications of the mesophases, have been probed by the time-dependent Stokes shift method of the two probes, revealing the existence of three kinds of dynamics. The difference in the hydration dynamics inside the two mesophases, where the water molecules confined in the Ia3 d phase exhibit a slower dynamics compared to that in Pn3 m, is the prime importance of this work. The underlying reason for this disparity is majorly associated with the differences in the topology of the two structures including the hydrophobic packing stress, the negative interfacial curvature, and the curvature elastic energy of the lipid-water interface. We believe that this kind of correlation between the structural topology of the different cubic LLC mesophases and nature of the water nanochannel will help to boost the applications of the cubic phases in the future.

Intramolecular Fluorescent Protein Association in a Class of Zinc FRET Sensors Leads to Increased Dynamic Range.

Genetically encoded Förster resonance energy transfer (FRET) sensors enable the visualization of ions, molecules, and processes in live cells. However, despite their widespread use, the molecular states that determine sensor performance are usually poorly understood, which limits efforts to improve them. We used dynamic light scattering (DLS) and time-resolved fluorescence anisotropy to uncover the sensing mechanism of ZifCV1.173, a Zn2+ FRET sensor. We found that the dynamic range (DR) of ZifCV1.173 was dominated by the high FRET efficiency of the Zn2+-free state, in which the donor and acceptor fluorescent proteins were closely associated. Mutating the donor-acceptor interface revealed that the DR of ZifCV1.173 could be increased or decreased by promoting or disrupting the donor-acceptor interaction, respectively. Adapting the same mutations to a related sensor showed the same pattern of DR tuning, supporting our sensing mechanism and suggesting that DLS and time-resolved fluorescence anisotropy might be generally useful in the biophysical characterization of other FRET sensors.

Antiamyloid activity of functionalized cerium oxide nanoparticle on lysozyme fibrillation: Spectroscopic and microscopic investigation

Accumulation of amyloid fibrils of proteins in different organs is a pathological indication for various neurogenerative disorders. Recently nanotechnology is at the centre of interest to tackle with those disorders. In this study we have chosen hen egg white lysozyme as our model protein which is responsible for hereditary systemic Amyloidosis disease. Here we have reported effect of cerium oxide nanoparticle (CeONP), folic acid functionalized CeONP (FA-CeONP) and polyacrylic acid functionalized CeONP (PAA-CeONP) (all of them having size ∼18 nm) on fibrillation of hen egg white lysozyme (Lyz). The amyloid growth is monitored by Thioflavin T (ThT), Congo Red (CR), 8-anilinonapthalene-1-sulfonic acid (ANS) assay, time resolved fluorescence anisotropy, intrinsic fluorescence, circular dichroism, atomic force microscopy and fluorescence microscopy study. Results demonstrate that all nanoparticle (NP) shows antiamyloid activity by broadening lag phase and suppressing growth phase of Lyz amyloid growth, but PAA-CeONP shows maximum inhibitory effect. These bare and functionalized CeONP shows dose dependent antiamyloid activity. We have also demonstrated that such inhibition of amyloid formation is associated with reduction of β-sheet content of protein. The inhibition efficiency is quantified by half maximal inhibition concentration IC50 values. A mechanistic pathway is explained on the basis of electrostatic interaction between NP and Lyz by ζ- potential measurement. This interaction is also analyzed theoretically by docking analysis. Thus our study provides a basis for use of CeONP as a promising candidate in therapeutic application in amyloid related disorders.

Insight into the Excitation-Dependent Fluorescence of Carbon Dots.

High quantum yield, photoluminescence tunability, and sensitivity to the environment are a few distinct trademarks that make carbon nanodots (CDs) interesting for fundamental research, with potential to replace the prevalent inorganic semiconductor quantum dots. Currently, application and fundamental understanding of CDs are constrained because it is difficult to make a quantitative comparison among different types of CDs simply because their photoluminescence properties are directly linked to their size distribution, the surface functionalization, the carbon core structures (graphitic or amorphous) and the number of defects. Herein, we report a facile one-step synthesis of mono-dispersed and highly fluorescent nanometre size CDs from a 'family' of glucose-based sugars. These CDs are stable in aqueous solutions with photoluminescence in the visible range. Our results show several common features in the family of CDs synthesized in that the fluorescence, in the visible region, is due to a weak absorption in the 300-400 nm from a heterogeneous population of fluorophores. Fluorescence quenching experiments suggest the existence of not only surface-exposed fluorophores but more importantly solvent inaccessible fluorophores present within the core of CDs. Interestingly, time-resolved fluorescence anisotropy experiments directly suggest that a fast exchange of excitation energy occurs that results in a homo-FRET based depolarization within 150 ps of excitation.

Ultrafast pump-probe and time-resolved fluorescence anisotropy study of the intramolecular interaction of branched styryl derivatives based on 1,3,5-triazine

A series of branched styryl derivatives based on 1,3,5-triazine were studied by nonlinear optical property measurement, degenerated pump-probe, and time-resolved fluorescence anisotropy methods to elucidate the two-photon absorption (TPA) properties and intramolecular interactions between branches. Significant enhancement of the TPA cross-section was observed in multi-branched derivatives. The anisotropy of multi-branched compounds shows faster decay and small residual values, indicating strong intramolecular interactions between branches, which further confirmed the TPA enhancement mechanism.

Using Time-Resolved Fluorescence Anisotropy of di-4-ANEPPDHQ and F2N12S to Analyze Lipid Packing Dynamics in Model Systems.

The fluorescence probes di-4-ANEPPDHQ and F2N12S have solvochromatic emission spectra and fluorescence lifetimes that are sensitive to order within the environment of lipid membranes. We show in this communication that the time-resolved fluorescence anisotropy of these probes, analyzed either by the wobble-in-a-cone model or by the model-independent order parameter S2, provides complementary information about dynamics and lipid packing in a variety of homogeneous lipid membranes systems.

Understanding the Role of Anionic Lipids in the Interaction of KRas4b with the Membrane Surface

Ras proteins are key drivers of signal transduction where extracellular growth factors control nuclear transcription events involved in cell division, proliferation, and apoptosis. Ras activity is controlled, and signaling mediated by, critical protein-protein interactions with GTPase activating proteins and guanine nucleotide exchange factors. Importantly, these protein complexes are located on the membrane surface where membrane-protein interactions are potentially key features of both the active and inactive forms of Ras. There is incomplete knowledge as to the role the bilayer composition plays in dictating the final orientation of Ras on the surface. We investigated the role of specific lipids in the recruitment of KRas4b to a Nanodisc membrane surface of defined lipid composition. Fluorescence anisotropy experiments revealed that KRas4b has a significant binding preference for Nanodisc bilayers containing PIP2. Molecular Dynamics simulations suggest that the PIP2 specificity arises from long lived salt bridges formed with the positively charged Hyper Variable Region. We show that these long-lived membrane interactions also occur on the globular domain, controlling the orientation of KRas4b at the membrane surface. Two major orientations are observed during all atom simulations. In one case, helix-4 interacts with PIP2 through a bifurcated salt bridge between Arg135 and Lys128 thereby exposing the effector binding interface of the G domain. Mutations of Arg135 and Lys128 to Ala do not dramatically alter the affinity for PIP2 membranes but significantly affects the conformational equilibrium as probed by MD simulations. The topology of KRas4b on the bilayer is experimentally probed by time resolved fluorescence anisotropy and optical waveguide linear dichroism. Our results suggest that both the lipid composition and the positively charged residues on the globular domain play an important role in defining the orientation of Kras4b on the bilayer surface.

Salt Bridges in Ubiquitin Determine the Protein Conformational Flexibility

Ubiquitin family proteins are involved in a wide variety of functions, ranging from proteasomal degradation to signal transduction. Apart from their unique β-grasp fold, with 5 β-strands wrapping around an α-helix, a unique feature of them is a conserved salt bridge connecting the α-helix to a loop between the β3 and β4 strands. Here, we investigated the role of this conserved salt bridge in determining the protein conformational flexibility using ubiquitin as a model system and utilizing time-resolved fluorescence anisotropy and near-UV circular dichroism (CD). Therefore, we inserted a Trp (L43W) at the core near to the conserved salt bridge (K27-D52), as a reporter of protein's microenvironment. CD and fluorescence anisotropy showed that ubiquitin's core is conformationally rigid in the temperature range 5-55 0C. Disrupting the salt bridge by a double mutation (K27A/D52L) increased the conformational flexibility of the protein core at 5 0C, which further increased with temperature up to 55 0C. Interestingly, SUMO2 lacking such a conserved salt bridge, has recently been shown to have similar flexibility in this temperature range.1 These results suggest that the core flexibility of the salt bridge (K27A/D52L) disrupted ubiquitin resembles that of SUMO2. Furthermore, a more severe salt bridge mutation (K27M/D52L) of ubiquitin, replacing Lys with Met to mimic SUMO2, showed that the protein's core is highly flexible even at 5 0C and devoid of any tertiary structure. Our studies on ubiquitin and SUMO2 suggest that the conserved salt bridge of ubiquitin family proteins plays an important role in determining protein conformational flexibility. 1. Shrabasti Bhattacharya and Sri Rama Koti Ainavarapu. Mechanical Softening of a Small Ubiquitin-Related Modifier Protein Due to Temperature Induced Flexibility at the Core. J. Phys. Chem. B (2018), 122, p9128-9136.

Methods toward simplification of time resolved fluorescence anisotropy in proteins labeled with NBD (4-chloro-7-nitrobenzofurazan) adducts

The analysis of time resolved fluorescence anisotropy for NBD tagged proteins is difficult when multiple exponential components arise from heterogeneous amino acid fluorescent adducts. Two approaches were taken toward simplification. First, N terminal selective labeling of tear lipocalin with NBD-Cl was attempted at pH 7.0. While lysines were predominantly labeled at pH 8.0, selective N terminal labeling was attained at neutral pH. Second, fluorescence anisotropic decay analysis was simplified to recover only the rotational correlation time of the protein not the side chain. The boundaries for analysis of anisotropic decays were limited to the longer lifetimes. A modified tail fit enabled fitting the anisotropic decay to a single exponential. The correlation time for tear lipocalin matched published values. Additionally, a method for normalization of acquisition times of vertically (VV) and horizontally (VH) polarized fluorescence emission decays is presented for time-resolved anisotropy. Here it is applied to Picoharp software (Picoquant, Berlin). Picoharp software is programmed with an automatic stop at unequal acquisition times if the fluorescent counts exceeds a default. The method adjusts the intensity decays to the same acquisition time and is applicable to all time-resolved anisotropic decay data collected with time-correlated photon counting.•NBD labeling at pH 7.0 was not selective for N terminus of LCN1.•Constraints for range simplifies fittings of anisotropic decays.•Different acquisition times for decays can be normalized to facilitate fitting in data obtained by Picoharp.

Orientational distribution of DPH in lipid membranes: a comparison of molecular dynamics calculations and experimental time-resolved anisotropy experiments.

Characterization of the membrane phases is a crucial task in cell biology. Cells differ in composition of the lipids and consequently in adopted phases. The phases can be discriminated based upon lipid ordering and molecular diffusion and their identification could be used for characterization of cell membranes. Here we used molecular dynamics (MD) simulations to study the behavior of the fluorescent reporter molecule diphenylhexatriene (DPH) in different lipid phases - liquid disordered (Ld), liquid ordered (Lo), and solid ordered (So) composed of phosphatidylcholines (Ld and So) or a sphingomyelin/cholesterol (SM/Chol) mixture (Lo). To the best of our knowledge, this is the first simulation of DPH in Lo SM/Chol and So DPPC membranes. For the considered membrane compositions DPH is mostly oriented parallel to lipid tails. In the Lo phase we observed a significant fraction of DPH positioned in between membrane leaflets, which agrees with experimental findings, but which has not been observed in previous MD simulations of DPH in phosphatidylcholine membranes. Further, we calculated rotational autocorrelation functions (ROTACF) from our MD simulations in order to model the time-resolved fluorescence anisotropy decay. We observed that order parameters P2 and P4 are sufficient to fully describe the orientation distribution of DPH. We analyzed the ROTACFs by a so-called general model for the time-resolved fluorescence anisotropy [W. van der Meer et al., Biophys. J., 1984, 46, 515] and observed an overestimation of P4. We suggest a rescaling of the recovered P4 yielding an orientation distribution of DPH close to the one observed in our MD simulations.

Time-resolved multirotational dynamics of single solution-phase tau proteins reveals details of conformational variation.

Intrinsically disordered proteins (IDPs) are crucial to many cellular processes and have been linked to neurodegenerative diseases. Single molecules of tau, an IDP associated with Alzheimer's disease, are trapped in solution using a microfluidic device, and a time-resolved fluorescence anisotropy decay is recorded for each molecule. Multiple rotational components are resolved and a novel k-means algorithm is used to sort the molecules into two families of conformations. Differences in rotational dynamics suggest a change in the rigidity and steric hindrance surrounding a sequence (306VQIVYK311) which is central to paired helical filament formation. This single-molecule approach can be applied to other IDPs to resolve heterogeneous populations and underlying differences in conformational dynamics.

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

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.