Time-resolved Fluorescence Anisotropy Measurements Research Articles

Insight into molecular interactions and energetics of surfactant self-assembly in hydrated glucose-urea deep eutectic solvent

Self-assembled structures in highly biocompatible Natural Deep Eutectic Solvent (NADES) are gaining popularity as new drug delivery systems due to their inherent advantages. Although glucose-urea (GU) based NADES satisfies the criterion of an efficient DES in terms of low toxicity, stability at desirable pH range, low viscosity and biocompatible polarity its potential as a medium for molecular self-assembly is yet unexplored. In this work we present first systematic study of self-assembly of pharmaceutically important excipients like sodium deoxycholate (NaDC), cetylpyridinium chloride (CPC) and Brij 35 (B35) in three hydrated GUNADES using isothermal titration calorimetry. The findings are correlated to the microstructure of these solvents as delineated from molecular dynamics simulations. Micro-environment of the micelles in a typical hydrated GU NADES has been studied using time resolved fluorescence anisotropy measurements. Cyto-toxicity of the proposed NADES has been assessed using MTT assay. CMC values of the surfactants increased with increase in glucose-urea concentration in the NADES indicating increased solvophilicity in GUNADES. Fluorescence studies indicate higher polydispersity of sodium deoxycholate in water than GUNADES. Cyto-toxicity study of GU80 shows good biocompatibility.

Time-resolved fluorescence anisotropy with Atto 488-labeled phytochrome Agp1 from Agrobacterium fabrum.

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. Three domains, termed PAS, GAF, and PHY domains, constitute the N-terminal photosensory chromophore module (PCM); the C-terminus is often a histidine kinase module. In the Agrobacterium fabrum phytochrome Agp1, the autophosphorylation activity of the histidine kinase is high in the Pr and low in the Pfr form. Crystal structure analyses of PCMs suggest flexibility around position 308 in the Pr but not in the Pfr form. Here, we performed time-resolved fluorescence anisotropy measurements with different Agp1 mutants, each with a single cysteine residue at various positions. The fluorophore label Atto-488 was attached to each mutant, and time-resolved fluorescence anisotropy was measured in the Pr and Pfr forms. Fluorescence anisotropy curves were fitted with biexponential functions. Differences in the amplitude A2 of the second component between the PCM and the full-length variant indicate a mechanical coupling between position 362 and the histidine kinase. Pr-to-Pfr photoconversion induced no significant changes in the time constant t2 at any position. An intermediate t2 value at position 295, which is located in a compact environment, suggests flexibility around the nearby position 308 in Pr and in Pfr.

Role of Gemini Surfactants with Variable Spacers and SiO2 Nanoparticles in ct-DNA Compaction and Applications toward In Vitro/In Vivo Gene Delivery.

Compaction of calf thymus DNA (ct-DNA) by two cationic gemini surfactants, 12-4-12 and 12-8-12, in the absence and presence of negatively charged SiO2 nanoparticles (NPs) (∼100 nm) has been explored using various techniques. 12-8-12 having a longer hydrophobic spacer induces a greater extent of ct-DNA compaction than 12-4-12, which becomes more efficient with SiO2 NPs. While 50% ct-DNA compaction in the presence of SiO2 NPs occurs at ∼77 nM of 12-8-12 and ∼130 nM of 12-4-12, but a conventional counterpart surfactant, DTAB, does it at its concentration as high as ∼7 μM. Time-resolved fluorescence anisotropy measurements show changes in the rotational dynamics of a fluorescent probe, DAPI, and helix segments in the condensed DNA. Fluorescence lifetime data and ethidium bromide exclusion assays reveal the binding sites of surfactants to ct-DNA. 12-8-12 with SiO2 NPs has shown the highest cell viability (≥90%) and least cell death in the human embryonic kidney (HEK) 293 cell lines in contrast to the cell viability of ≤80% for DTAB. These results show that 12-8-12 with SiO2 NPs has the highest time and dose-dependent cytotoxicity compared to 12-8-12 and 12-4-12 in the murine breast cancer 4T1 cell line. Fluorescence microscopy and flow cytometry are performed for in vitro cellular uptake of YOYO-1-labeled ct-DNA with surfactants and SiO2 NPs using 4T1 cells after 3 and 6 h incubations. The in vivo tumor accumulation studies are carried out using a real-time in vivo imaging system after intravenous injection of the samples into 4T1 tumor-bearing mice. 12-8-12 with SiO2 has delivered the highest amount of ct-DNA in cells and tumors in a time-dependent manner. Thus, the application of a gemini surfactant with a hydrophobic spacer and SiO2 NPs in compacting and delivering ct-DNA to the tumor is proven, warranting its further exploration in nucleic acid therapy for cancer treatment.

Fluorescence of Bimolecular Guanine Quadruplexes: From Femtoseconds to Nanoseconds

The paper deals with the fluorescence of guanine quadruplexes (G4) formed by association of two DNA strands d(GGGGTTTTGGGG) in the presence of K+ cations, noted as OXY/K+ in reference to the protozoon Oxytricha nova, whose telomere contains TTTTGGGG repeats. They were studied by steady-state and time-resolved techniques, time-correlated single photon counting, and fluorescence upconversion. The maximum of the OXY/K+ fluorescence spectrum is located at 334 nm, and the quantum yield is 5.8 × 10-4. About 75% of the photons are emitted before 100 ps and stem from ππ* states, possibly with a small contribution of charge transfer. Time-resolved fluorescence anisotropy measurements indicate that ultrafast (<330 fs) excitation transfer, due to internal conversion among exciton states, is more efficient in OXY/K+ compared to previously studied G4 structures. This is attributed to the arrangement of the peripheral thymines in two diagonal loops with restricted mobility, facilitating the interaction among them and with guanines. Thymines should also be responsible for a weak intensity excimer/exciplex emission band, peaking at 445 nm. Finally, the longest living fluorescence component (∼2.1 ns) is observed at the blue side of the spectrum. So far, high-energy long-lived emitting states had been reported only for double-stranded structures but not for G4.

Dynamics of transition dipole orientation in representative fluorescent proteins

Molecules of fluorescent proteins behave like antennas: they exhibit distinct directionality of light absorption and emission. In our previous work we determined the mean orientations of the ‘molecular antennas’ (transition dipole moments) within the atomic framework of fluorescent protein molecules. In the presented work we use molecular dynamics simulations, as well as time resolved fluorescence anisotropy measurements, in order to investigate the dynamics of transition dipole moment orientation in representative fluorescent proteins.

Short-Range Backbone Dihedral Rotations Modulate Internal Friction in Intrinsically Disordered Proteins.

Protein folding and dynamics are governed by an intricate interplay of thermal and viscosity-mediated effects. The solvent viscosity contributes to the frictional drag in protein dynamics. In addition to this viscosity-dependent effect, there is also an intriguing viscosity-independent component that represents the intrinsic resistance of the polypeptide chain to changing its conformation. This solvent-independent component is termed internal friction. A longstanding question is what is the fundamental molecular origin of internal friction in highly solvated and rapidly fluctuating intrinsically disordered proteins (IDPs) devoid of any persistent intrachain interactions? Here, we present a unique case to directly demonstrate that sequence-specific backbone dihedral barriers control local internal friction in an archetypal IDP, namely, α-synuclein. We performed site-directed fluorescence depolarization kinetics using picosecond time-resolved fluorescence anisotropy measurements to directly observe the directional decorrelation arising due to short-range backbone torsional fluctuations in the dihedral space. A linear viscosity-dependent model of the dihedral relaxation time yielded a finite zero-viscosity intercept that corresponds to internal friction. Our site-specific dynamic readouts were able to detect localized sequence-specific frictional components that are otherwise skewed in viscosity-dependent long-range chain fluctuations. Our results revealed the presence of low internal friction in nonproline sequence segments. In contrast, a proline introduces torsional stiffness in the segment exhibiting high internal friction that can be compensated by a conformationally flexible glycine. Such an intriguing interplay of local dihedral dynamics can modulate sequence-dependent internal friction in a wide range of IDPs involved in a myriad of important events including folding, binding, assembly, and phase transitions.

Effect of Lithium-Ion on the Structural Organization of Monocationic and Dicationic Ionic Liquids.

In recent times, ionic liquid-based (ILs) electrolytic system has emerged as suitable alternative to the conventional organic solvent-based electrolytic system. However, since, anion of ILs is known to form aggregates in the presence of lithium-ions (Li+), and this can influence the transport properties of Li+ ion in a significant manner, it is, therefore, important to understand how lithium-ions influence the structure and dynamics of ILs. With this objective, in the present study, intermolecular interaction, structural organization, and dynamics of monocationic ILs (MILs) and dicationic IL (DIL) have been studied in the absence and presence of lithium salt. Specifically, for this purpose, two MILs, 1-methyl-3-propylimidazolium bis(trifluoromethylsulfonyl)imide ([C3C1im][NTf2]), 1-hexyl-3-propylimidazolium bis(trifluoromethylsulfonyl)imide ([C6C1im][NTf2]), and a DIL, 1,6-bis(3-methylimidazolium-1-yl)hexane bis(trifluoromethylsulfonyl)amide ([C6(mim)2][NTf2]2) have been chosen in such a way that either the alkyl chain of MILs becomes equal or half of the spacer chain length of DIL. To understand the effect of the addition of lithium-ion on the structural organization of MILs and DIL, steady-state absorption and fluorescence spectroscopies, time-resolved fluorescence anisotropy and nuclear magnetic resonance (NMR) techniques have been used. Structural organization in the apolar and polar domains of ILs has been probed by following the rotational diffusion of suitably chosen solute in the concerned media through time-resolved fluorescence anisotropy (TRFA) measurements. TRFA studies have revealed that with the addition of Li+ ion, coordination between the Li+ ions and anions of MILs and DILs takes place in the ionic region leading to a change in the structural organization of the apolar regions of the respective medium. In fact, upon adding lithium-ions, a reduction in the packing of alkyl chains has also been observed for the MILs. However, not much change in the structural organization of the apolar region of the DIL has been observed when Li+ ion is added to it. In the presence of Li+ ions, a similar trend in the change of structural organization of polar regions for both MILs and DIL has been observed. Further, measurements of the self-diffusion coefficient through NMR have also supported the observation that Li+ ion also perturbs the nanostructural organization of the MIL in a significant manner than that it does for the DIL. The behavior of DIL in the presence of Li+ ion, as revealed by the present study, has been rationalized by considering the folded arrangement of DIL in the fluid-structure. Essentially, all of these investigations have suggested that the addition of lithium-ion significantly alters the microscopic behavior of MILs in comparison to that of DIL. The outcome of this study is expected to be helpful in realizing the potentials of these media as electrolytes in battery applications.

Phenylalanine Interacts with Oleic Acid-Based Vesicle Membrane. Understanding the Molecular Role of Fibril–Vesicle Interaction under the Context of Phenylketonuria

In the present contribution, on the basis of a spectroscopic and microscopic investigation, the characterization and photophysics of various assemblies of oleic acid/oleate solution at three pH values, namely, 8.28, 9.72, and 11.77, were explored. The variation in the dynamic response of aqua molecules in and around the assemblies has been interrogated by a picoseconds solvation dynamics experiment using a time-correlated single-photon counting setup employing coumarin-153 as a probe. On the one hand, the time-resolved fluorescence anisotropy measurement along with the fluorescence correlation spectroscopy experiment was executed to extract information regarding the comparison of the extent of the internal restricted confinement of these assemblies. On the other hand, an effort to investigate the cross-interaction between the self-assembled architectures of l-phenylalanine (l-Phe), responsible for phenylketonuria (PKU) disorder, and the oleic acid at the vesicle-forming pH established that the l-Phe fibrillar morphologies strongly alter the dynamic properties of the vesicle membrane formed by the oleic acid. Specifically, the interaction of the l-Phe assemblies with the oleic acid vesicle membrane is found to introduce the flexibility of the vesicle membrane and alter the hydration properties of the membrane. To track the fibril-induced alterations of the oleic acid vesicle properties, various spectroscopic and microscopic investigations were performed. The mutual reconciliation of the experimental outputs, therefore, portrays the state of the art, which accounts for the fibril-induced alterations of the properties of the oleic acid vesicle membrane, the mimicking setup of the cellular membrane, thereby informing us that alterations of such a property of the membrane should be taken into active consideration during the rational development of therapeutic modulators against disorders like PKU.

Role of Ligand-Binding and Non-Active Site Residues in the Dynamics, Conformational Integrity, and Catalysis of the Disordered Enzyme Monomeric Chorismate Mutase

The dominant view in enzymology is that an increase in structural disorderedness tends to decrease catalytic efficiency. Surprisingly, engineered monomeric chorismate mutase (mCM), an enzyme with properties of molten globule (intrinsically disordered), efficiently catalyses the conversion of chorismate to prephenate. Therefore, here, we investigate the underlying structural plasticity mechanisms that facilitate this catalysis. We generated rationally designed mutations of mCM and used a battery of spectroscopic tests including Forster resonance energy transfer (FRET) and time-resolved fluorescence anisotropy measurements to probe what influences catalysis. We found that the integrity of the secondary structure, and ligand-induced global compaction (disorder-to-order transition) play a crucial role in catalysis. Intriguingly, we find that even minor perturbation in regions distant from the active site can significantly impact the enzyme efficiency by changing the conformation, segmental dynamics, ligand-induced global compaction, and stability.

Exciton energy transfer and bi-exciton annihilation in the emitting layers of thermally activated delayed fluorescence-based OLEDs

Time-resolved fluorescence anisotropy measurements and molecular dynamics simulations are used to study the energy transfer among dopant molecules in the emitting layer of organic light emitting diodes.

Effects of fatty acid from deep-sea microorganisms on lipid bilayer membrane fluidity under high pressure: comparison of branched-chain and polyunsaturated fatty acid

In this study, the effects of unsaturated and saturated branchedchain fatty acids in biomembranes of microorganisms living under high temperature and high pressure on the fluidity of biomembranes were investigated by time-resolved fluorescence anisotropy measurements. First, the relationship between the order parameter S and the rotational diffusion coefficient Dw, which can be calculated from the fluorescence anisotropy measurements, and the motion of lipid molecules was investigated using lipids with three different structures, and it was found that the former was related to the spacing of lipid molecules and the latter to the motion of lipid molecules. Next, we investigated the S and Dw values of lipid bilayer membrane containing the saturated branched-chain fatty acid 12-methyltridecanoic acid (12-MTA) and the polyunsaturated fatty acid (PUFA) cis-4,7,10,13,16,19-docosahexaenoic acid (DHA). The results showed that 12-MTA increased the S value and decreased the Dw value. On the other hand, DHA tended to reduce the S value and increase the Dw value, albeit slightly. These results mean that 12-MTA narrows the molecular spacing of lipids and inhibits lipid molecular movement, while DHA tends to widen the molecular spacing of lipids and promote lipid molecular movement.

Observation of Intramolecular Interaction in Fluorescent Star-Shaped Polymers: Evidence for Energy Hopping between Branch Chains.

Time-resolved fluorescence anisotropy measurements were performed on three-branched star-shaped polymers, based on precisely synthesized poly(9,9-di-n-octyl-fluorene vinylene)s containing C6F5 end groups. The star-shaped polymers showed identical fluorescence spectra, fluorescence lifetimes, and quantum yields to those of the reference single-chain oligomer. However, a rapid fluorescence anisotropy decay was observed in two kinds of star-shaped polymers, while such decay was not seen in the corresponding single-chain oligomer. On the basis of the analysis using an incoherent hopping model, the observed rapid anisotropy decay is attributable to energy hopping processes between branches within a single polymer species, and its rate was deduced to be ca.100 ps depending upon the core part.

Interaction of a hydrophilic molecule with bovine serum albumin: A combined multi-spectroscopic, microscopic and isothermal calorimetric study in the presence of graphene oxide

One of the most widely studied plasma protein is bovine serum albumin (BSA), and it plays a crucial role in the binding and transportation of many exogenous and endogenous drugs. Coumarin derivatives play a crucial role as an analgesic, anticancer, and anticoagulant agent. Here in this report, we have studied the binding interaction between a 7-amino coumarin derivative, namely 7-(N, N′-diethylamino)coumarin-3-carboxylic acid (7-DCCA) with BSA and also have found out the effect of graphene oxide (GO) on the binding interaction. The binding interaction was probed by employing various spectroscopic, microscopic, isothermal titration calorimetric (ITC), and by using molecular docking studies. The change in absorption and emission maxima position of BSA and 7-DCCA in the absence and presence of GO along with the change in absorbance and emission intensity when we titrated by 7-DCCA or BSA respectively indicates the strong complexation between 7-DCCA and BSA. From the fluorescence resonance energy transfer study, the average distance between tryptophan 213 of the BSA (donor), and the 7-DCCA (acceptor) is found ~50 Ǻ. From excited-state lifetime and fluorescence lifetime imaging (FLIM) studies, we observed that the average lifetime of 7-DCCA is much higher in the presence of BSA compared to buffer medium and in the presence of BSA a heterogeneous environment was created around 7-DCCA. Again in the presence of GO, the formation of a complex of 7-DCCA with a low concentration of BSA shows greater binding interaction as compared to that in the absence of GO. Time-resolved fluorescence anisotropy measurement shows that the addition of GO provides a restricted environment surrounding the 7-DCCA molecule in the presence of both low and high concentrations of BSA. CD study demonstrates the same extent of reduction of α-helical content of BSA protein on binding with 7-DCCA in the absence and presence of GO. ITC study revealed that the interaction between 7-DCCA and BSA is favoured by negative enthalpy change and positive entropy change. Also, from the ITC study, we find out that GO doesn't inflict significant change in the overall global binding interaction pattern, rather it inflicts changes in the mechanistic pathways of binding between 7-DCCA and BSA. Step-scan FTIR studies show the modification of amide I and amide II bonds with time when complexation occurs between BSA and 7-DCCA in the absence and presence of GO. The docking result reveals that the deprotonated form of 7-DCCA has positioned at a distance of ~3.7 nm from Trp 213. Thus these interactions play a central role in the development of coumarin derivative based small molecules to be used for both academic and industrial purposes.

Simplification in the Acquisition and Analysis of Fluorescence Decays Acquired with Polarized Emission for Time-Resolved Fluorescence Anisotropy Measurements.

This study introduces a global fluorescence decay analysis that substantially simplifies the acquisition and analysis of time-resolved fluorescence decays acquired with a vertically polarized excitation and vertically (IVV(t)) and horizontally (IVH(t)) polarized emission for time-resolved fluorescence anisotropy (TRFA) measurements. TRFA measurements were conducted whereby the IVV(t) and IVH(t) fluorescence decays of a series of oligoquinolines labeled at one end with an oligo(phenylenevinylene) dye (OPV-Qn with n = 4, 7, 17, 24, 33) were acquired according to the standard protocol that is currently accepted in the scientific literature which involves toggling the emission polarizer before fitting linear combinations of the IVV(t) and IVH(t) decays or acquiring the IVV(t) and IVH(t) decays with static polarizers before fitting them globally. The rotational time (ϕ) and initial anisotropy (r0) retrieved from these analyses were identical within experimental error regardless of whether the decays were acquired with toggling or static polarizers and fitted according to the standard protocol or globally. These experimental results were further supported by retrieving the parameters used to generate mono-, bi-, and tri-exponential TRFAs from the global analysis of simulated IVV(t) and IVH(t) fluorescence decays which were found to match perfectly the values that were inputted. Together, these experiments and simulations demonstrated that the parameters describing any type of TRFA can be extracted directly from the analysis of the IVV(t) and IVH(t) fluorescence decays acquired with a standard time-resolved fluorometer, a substantial simplification compared to the protocols currently in place to determine the TRFA.

Controlling balance between homo-FRET and hetero-FRET within hetero-chromophoric systems by tuning nature of solvent

Using fluorescence lifetime and time-resolved fluorescence anisotropy measurements, here we report how the nature of the solvent controls Förster resonance energy transfer (FRET) in a system containing two chromophores, disodium fluorescein and rhodamine-6G, and their aggregates. For water, significant energy transfer between unlike chromophores (i.e. hetero-FRET between fluorescein and rhodamine) is observed with increasing concentration which reduces a bit at higher concentration. In contrast, for glycerol, homo-FRET overwhelms hetero-FRET at higher concentration; a mixed behavior is noted for water-glycerol mixture. Also, the heterogeneity of the system leads to observation of ‘dip-and-rise’ type temporal fluorescence anisotropy traces. The absence of hetero-FRET at high concentration is explained based on spectral broadening which are further fine-tuned by solvent viscosity. Accompanied density functional theory calculations reveal enhanced stability of rhodamine-6G homo-dimers over other types of dimers which comply with the experimental findings. Thus, the results strengthen our understanding of explicit role of solvent in controlling mechanism of energy transfer within multi-chromophoric aggregates.

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.

Fluorescence Depolarization Kinetics to Study the Conformational Preference, Structural Plasticity, Binding, and Assembly of Intrinsically Disordered Proteins.

Fluorescence depolarization kinetics measured by the time-resolved fluorescence anisotropy decay serves as a sensitive and powerful methodology to study the conformational dynamics of macromolecules. This methodology allows us to delineate the different modes of biomolecular motional dynamics including the local, segmental, and global rotational dynamics on the timescale ranging from picoseconds to nanoseconds. In this chapter, we describe the principles and applications of this methodology to obtain unique molecular insights into the intrinsically disordered proteins (IDPs). Fluorescence depolarization kinetics, when performed in a site-specific manner, can offer a reliable tool to monitor the intrinsic backbone torsional dynamics of expanded IDPs and is capable of discerning the conformational preference of IDPs. Additionally, the time-resolved fluorescence anisotropy measurements allow us to investigate the mechanism of binding and assembly of a wide range of IDPs that are involved in crucial function and disease.

Revealing Conformational Variants of Solution-Phase Intrinsically Disordered Tau Protein at the Single-Molecule Level.

Intrinsically disordered proteins, such as tau protein, adopt a variety of conformations in solution, complicating solution-phase structural studies. We employed an anti-Brownian electrokinetic (ABEL) trap to prolong measurements of single tau proteins in solution. Once trapped, we recorded the fluorescence anisotropy to investigate the diversity of conformations sampled by the single molecules. A distribution of anisotropy values obtained from trapped tau protein is conspicuously bimodal while those obtained by trapping a globular protein or individual fluorophores are not. Time-resolved fluorescence anisotropy measurements were used to provide an explanation of the bimodal distribution as originating from a shift in the compaction of the two different families of conformations.

Rotational Dynamics of a Probe in Rubbery Polymers Characterized by Time‐Resolved Fluorescence Anisotropy Measurement

AbstractThe rotational relaxation time (τrot) of a fluorescent molecule, coumarin 153 (C153), dispersed in different rubbery polymers is characterized by time‐resolved fluorescence anisotropy measurement, and an attempt is made to quantitatively combine it with the segmental relaxation time (τseg) of the corresponding matrix polymer obtained by dielectric relaxation spectroscopy. This study here demonstrates that τseg extrapolated to higher temperatures using the Vogel–Fulcher–Tammann law can be superimposed on τrot, resulting in a single curve. This behavior is common for polymers with different glass transition temperatures such as polyisoprene and acrylonitrile/butadiene copolymer, implying that the rotational dynamics of C153 is a useful tool for the characterization of polymer dynamics.