Time-resolved Fluorescence Anisotropy Research Articles

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.

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

Förster resonance energy transfer (FRET) is a powerful tool to investigate the interaction between proteins in living cells. Fluorescence proteins, such as the green fluorescent protein (GFP) and its derivatives, are coexpressed in cells linked to proteins of interest. Time-resolved fluorescence anisotropy is a popular tool to study homo-FRET of fluorescent proteins as an indicator of dimerization, in which its signature consists of a very short component at the beginning of the anisotropy decay. In this work, we present an approach to study GFP homo-FRET via a combination of time-resolved fluorescence anisotropy, the stretched exponential decay model, and molecular dynamics simulations. We characterize a new, to our knowledge, FRET standard formed by two enhanced GFPs (eGFPs) and a flexible linker of 15 aminoacids (eGFP15eGFP) with this protocol, which is validated by using an eGFP monomer as a reference. An excellent agreement is found between the FRET efficiency calculated from the fit of the eGFP15eGFP fluorescence anisotropy decays with a stretched exponential decay model (〈EFRETexp〉 = 0.25 ± 0.05) and those calculated from the molecular dynamics simulations (〈EFRETMD〉 = 0.18 ± 0.14). The relative dipole orientation between the GFPs is best described by the orientation factors 〈κ2〉 = 0.17 ± 0.16 and 〈|κ|〉 = 0.35 ± 0.20, contextualized within a static framework in which the linker hinders the free rotation of the fluorophores and excludes certain configurations. The combination of time- and polarization-resolved fluorescence spectroscopy with molecular dynamics simulations is shown to be a powerful tool for the study and interpretation of homo-FRET.

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.

Structure and dynamics of poly(methacrylic acid) and its interpolymer complex probed by covalently bound rhodamine-123

The dynamics and structural characteristics of polymethacrylic acid bound rhodamine-123 (PMAA–R123) and its interpolymer complex formed through hydrogen bonding between the monomeric units with poly(vinylpyrrolidone) were investigated using single molecular fluorescence studies. The time resolved fluorescence anisotropy decay of PMAA-R123 under acidic pH exhibits an associated anisotropy decay behavior characteristic of two different environments experienced by the fluorophore with one shorter and another longer rotational correlation time. The anisotropy decay retains normal bi-exponential behavior under neutral pH. Fluorescence correlation spectroscopic investigation reveals that the attached fluorophore undergoes hydrolysis under basic condition which results in the release of the fluorophore from the polymer backbone. Shrinkage in the hydrodynamic radius of PMAA is observed on addition of the complementary polymer PVP which is attributed to the formation compact solubilized nanoparticle like aggregates. The size of particle further decreases on the addition of NaCl. The detailed results show that these complexes have potential for use as drug-delivery system under physiological conditions.

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.

Residence and diffusion of a dynamically prototropic hydration probe in AOT reverse micelles

We investigated the hydration dynamics in the nanosized water pool of an Aerosol-OT (AOT) reverse micelle (RM) using a prototropic (proton-donating) fluorescent dye, N-methyl-7-hydroxyquinolinium (NM7HQ+), as the probe. NM7HQ+ is a photoacid, and the deprotonation of its excited-state cationic form (C*) gives rise to a neutral keto species (K*). In the present study, the residence sites of C* and K* in water pools of various sizes were determined by monitoring their time-resolved fluorescence anisotropy. Additionally, the evolutions of the C* and K* bands in the time-resolved fluorescence spectra were analyzed by fitting the spectra to the sum of two lognormal peak functions. The time-dependent spectral shifts of the species with different formal charges revealed the characteristic hydration behavior at the interface of the anionic AOT headgroups and the bound water region, as well as the rotational and translational diffusion of the probe within the RM.

Partial Viscosity Decoupling of Solute Solvation, Rotation, and Translation Dynamics in Lauric Acid/Menthol Deep Eutectic Solvent: Modulation of Dynamic Heterogeneity with Length Scale.

Deep eutectic solvents (DESs) are new-generation media that can be fine-tuned to have desired properties circumventing economic and environmental issues. Typically, these are ionic, and only recently, nonionic DESs, having interesting properties, are being explored. In this report, we examined the structure and dynamics of a nonionic lauric acid/menthol (LA/Men) DES through steady-state emission, solvation dynamics, time-resolved fluorescence anisotropy, and translational diffusion dynamics. The zero shift in the emission spectra of coumarin 153 (a solvatochromic dye) as a function of the excitation wavelength suggests that LA/Men DES is spatially homogenous. Decoupling (p = 0.63) of the average solvation time, ⟨τs⟩, from medium viscosity suggests the presence of mild dynamic heterogeneity in the system. Rotational time, ⟨τr⟩, which reflects the nature of the first solvation shell, shows little decoupling (p = 0.81), suggesting it to be fairly dynamically homogeneous at a shorter length scale. An Arrhenius-type analysis also proves that rotation is mainly controlled by medium viscosity. Translational diffusion time, ⟨τD⟩, which provides information at a larger length scale, is strongly decoupled from medium viscosity (p = 0.29). This indicates that at a larger length scale, the DES is quite dynamically heterogeneous. The slow component of solvation time, which is believed to originate at a larger length scale, correlates well with the translational diffusion timescale having similar activation energies. This suggests that their origin is same. Expectedly, for the long component of solvation time, the decoupling is quite strong (p = 0.30). Overall, our result demonstrates the structure and dynamics of the nonionic LA/Men DES, and the existence of length scale-dependent heterogeneity has been proposed.

Modulation of excited-state photodynamics of ESIPT probe 1′-hydroxy-2′-acetonaphthone (HAN) on interaction with bovine serum albumin

The 1′-hydroxy-2′-acetonaphthone (HAN) is a well-known excited-state intramolecular proton-transfer (ESIPT) dye that has been used as a useful fluorescence probe to understand various homogenous and microheterogenous media. However, interaction of HAN with proteins is still largely less explored. The present study is aimed to investigate the interaction of HAN with the most abundant animal circulatory protein, bovine serum albumin (BSA), using multi-spectroscopic techniques and molecular docking studies. The photophysical properties of HAN undergo significant modulation upon its interaction with BSA protein. The confinement and reduced polarity experienced by bound HAN into BSA binding pocket causes a large fluorescence enhancement. The binding constant for HAN-BSA complex is evaluated to be ∼4.3 × 104 M−1. Time-resolved fluorescence measurements reveal the exclusive presence of only keto (K*) form and twisted keto rotamer (KR*) form of the dye in the excited state inside the BSA binding pocket, though in the ground state the dye exists entirely in its enol (E) form. Results suggest a quantitative ESIPT reaction for HAN even inside the BSA pocket. Time resolved fluorescence anisotropy studies indicate very tight binding of HAN into the BSA pocket such that independent rotational diffusion of the dye is completely ceased. Molecular docking studies provide best docking conformation and involved interaction forces, suggesting the preferred binding of HAN to the site IIA (Sudlow's site I) of the BSA protein. Observed photophysical results provide valuable insights of HAN–BSA binding interaction, revealing for the first time that the intriguing excited-state equilibrium between K* and KR* forms of HAN is largely shifted towards KR* form, as the dye binds to the BSA pocket. To the best of our knowledge such an intriguing modulation in the excited state prototautomeric conversion process has not been reported before for the studied dye in the presence of any other host. Results of the present study can be useful in understanding the pharmacology of structurally similar dyes/drugs, having relevance to drug delivery, pharmaceutical sciences, sensors, and so on.

Physicochemical properties of L-alpha dipalmitoyl phosphatidylcholine large unilamellar vesicles: Effect of hydrophobic block (PLA/PCL) of amphipathic diblock copolymers

In the present work, we show how amphipathic diblock copolymers affect the physicochemical properties of the lipid bilayer of DPPC liposome. Diblock copolymers proposed for this study are focused in the difference between PLA and PCL hydrophobic block, because PLA and PCL differ in their glass transition temperature, where a higher ratio of PLA, lowers the flexibility of the diblock copolymer. On the contrary, a greater proportion of PCL makes the diblock copolymer more flexible. This flexibility difference between hydrophobic block would affect the physicochemical properties of lipid bilayer of DPPC. The difference of rigidity or flexibility of hydrophobic block and their interaction with DPPC large unilamellar vesicles (LUVs) was evaluated at low and high copolymers concentration. The copolymer concentrations used were chosen based on their respective cmc. We measure (a) Thermotropic behavior from GP of Laurdan and fluorescence anisotropy of DPH; (b) Relation between wavelength excitation and generalized polarization of Laurdan; (c) Time-resolved fluorescence anisotropy of DPH; (d) Water outflow through the lipid bilayer and (e) calcein release from DPPC LUVs. Furthermore, large unilamellar vesicles in the absence and in the presence of different copolymers were characterized by size and zeta-potential. The results show that the diblock copolymer at high PLA/PCL ratio, that is, greater rigidity of hydrophobic block produces an increase of the phase transition temperature (Tm). For DPPC LUVs, Tm increase 3.5 °C at low and about 4.5 °C at high copolymers concentration, sensed by Laurdan and DPH fluorescent probes, although the DPPC/copolymers molar ratio for Cop4 is higher than Cop3, Cop2 and Cop1. In addition, we observed a decrease in the polarity of microenvironments in the bilayer and an increase in the order of the acyl chains in the bilayer to a high proportion of PLA. Furthermore, the presence of diblock copolymer with high proportion of PLA, decreases water outflow from DPPC liposome and water efflux is slower; leading to a decrease in calcein release from DPPC liposomes. Our results clearly show that the greater the stiffness of the hydrophobic block, greater degree of packaging of the lipid bilayer, greater the order of the acyl chains, and greater retention of water and calcein inside the liposome. Therefore, the presence of AB-type diblock copolymers with a more rigid hydrophobic block, stabilizes the lipid bilayer and would allow a more controlled release of water, and encapsulated molecules inside of the DPPC liposome.

A spectroscopic deciphering of the differential interaction behavior of alkaloid drugs with native B-DNA and protonated DNA

The present study demonstrates a detailed spectroscopic investigation on the differential extent and modes of interaction of two alkaloid drugs, Sanguinarine (SG) and Berberine (BR), with biologically significant protonated and B-forms of herring sperm DNA in aqueous buffer medium of pH 3.5 and 7.4 respectively. The steady-state spectroscopic results substantiate the binding interaction of either drug molecule with protonated as well as B-form of DNA. The drug:DNA binding strength obtained employing the Benesi-Hildebrand equation reveals a significantly higher binding affinity of BR with B-DNA at pH 7.4 compared to that with protonated DNA at pH 3.5. However, the binding constant obtained for interaction of iminium form of SG with protonated DNA is found to be an order of magnitude higher compared to that in case of interaction with B-DNA. Moreover, the steady-state emission results validate the role of preferential electrostatic interaction between the cationic forms of the drugs (cationic BR and the iminium form of SG) and the negatively charged phosphate backbone of B-DNA under physiological conditions at pH 7.4 which has been further corroborated by monitoring the effect of a strong electrolyte on drug:DNA interaction under the various experimental conditions. The circular dichroic (CD) spectroscopic studies reveal the probable intercalative binding of the drug molecules within DNA at either pH. The time-resolved fluorescence decay measurements along with the time-resolved fluorescence anisotropy studies further substantiate the binding interaction of the drugs with protonated as well as B-form of herring sperm DNA.

Time-Resolved Fluorescence Anisotropy of a Molecular Rotor Resolves Microscopic Viscosity Parameters in Complex Environments.

Understanding viscosity in complex environments remains a largely unanswered question despite its importance in determining reaction rates in vivo. Here, time-resolved fluorescence anisotropy imaging (TR-FAIM) is combined with fluorescent molecular rotors (FMRs) to simultaneously determine two non-equivalent viscosity-related parameters in complex heterogeneous environments. The parameters, FMR rotational correlation time and lifetime, are extracted from fluorescence anisotropy decays, which in heterogeneous environments show dip-and-rise behavior due to multiple dye populations. Decays of this kind are found both in artificially constructed adiposomes and in live cell lipid droplet organelles. Molecular dynamics simulations are used to assign each population to nano-environments within the lipid systems. The less viscous population corresponds to the state showing an average 25° tilt to the lipid membrane normal, and the more viscous population to the state showing an average 55° tilt. This combined experimental and simulation approach enables a comprehensive description of the FMR probe behavior within viscous nano-environments in complex, biological systems.

Effects of titanium dioxide and zinc oxide nano-materials on lipid order in model membranes

Engineered nano-materials (ENM) have been reported to affect lipid membrane permeability in cell models, but a mechanistic understanding of how these materials interact with biological membranes has not been described. To assess mechanisms of permeability, liposomes composed of DOPC, DOPS, or POPC, with or without cholesterol, were used as model membranes for measuring ENM-induced changes to lipid order to improve our understanding of ENM effects on membrane permeability. Liposomes were treated with either titanium dioxide (TiO2) or zinc oxide (ZnO) ENM, and changes to lipid order were measured by time-resolved fluorescence anisotropy of a lipophilic probe, Di-4-ANEPPDHQ. Both ENM increased lipid order in two lipid models differing in headgroup charge. TiO2 increased lipid order of POPC liposomes (neutral charge), while ZnO acted primarily on DOPS liposomes (negative charge). Addition of cholesterol to these models significantly increased lipid order while in some cases attenuated ENM-induced changes to lipid order. To assess the ability of ENM to induce membrane permeability, liposomes composed of the above lipids were assayed for membrane permeability by calcein leakage in response to ENM. Both ENM caused a dose-dependent increase in permeability in all liposome models tested, and the addition of cholesterol to the liposome models neither blocked nor reduced calcein leakage. Together, these experiments show that ENM increased permeability of small molecules (calcein) from model liposomes, and that the magnitude of the effect of ENM on lipid order depended on ENM surface charge, lipid head group charge and the presence of cholesterol in the membrane.

Transient Deprotonation of the Chromophore Affects Protein Dynamics Proximal and Distal to the Linear Tetrapyrrole Chromophore in Phytochrome Cph1.

Phytochromes are biological red/far-red light sensors found in many organisms. Prototypical phytochromes, including Cph1 from the cyanobacterium Synechocystis 6803, act as photochemical switches that interconvert between stable red (Pr)- and metastable far-red (Pfr)-absorbing states induced by photoisomerization of the bilin chromophore. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY) and the C-terminal transmitter (output) module, usually a histidine kinase, as in the case of Cph1. The chromophore deprotonates transiently during the Pr → Pfr photoconversion in association with extensive global structural changes required for signal transmission. Here, we performed equilibrium studies in the Pr state, involving pH titration of the linear tetrapyrrole chromophore in different Cph1 constructs, and measurement of pH-dependent structural changes at various positions in the protein using picosecond time-resolved fluorescence anisotropy. The fluorescent reporter group was attached at positions 371 (PHY domain), 305 (GAF domain), and 120 (PAS domain), as well as at sites in the PAS-GAF bidomain. We show direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains. Our results suggest that chromophore deprotonation is closely associated with a higher protein mobility (conformational space) both in proximal and in distal protein sites, implying a causal relationship that might be important for the global large protein arrangements and thus intramolecular signal transduction.

Using a Model Lysosome Membrane to Study Nanomaterial-Membrane Interactions

Some engineered nanomaterials (ENM), phagocytosed by cells and transported to lysosomes can cause the release of hydrolytic enzymes into the cytosol of the cell, resulting in an inflammatory response and cytotoxicity. We designed model lipid systems to study interactions between ENM and lipid membranes to assess ENM-lipid interaction. Liposomes (100 nm) were generated from lipids found in lysosomes: bis(monooleoylglycero)phosphate (BMP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), in a ratio of 2:3:3. Tris-buffered saline (TBS, pH 7.4) and acetate-buffered saline (ABS, pH 4.5) was used to mimic the pH of early and late lysosomes. Liposome structure was evaluated using transmission electron microscopy; all preparations were ∼100 nm in diameter and unilamellar. Liposomes were exposure to two types of titanium dioxide (TiO2) ENM, nanospheres (TNS) and nanobelts (TNB), or zinc oxide (ZnO) nanospheres for 2 hours at 25, 100 or 200 µg/ml. Time-resolved fluorescence anisotropy was performed using the fluorescence probe di-4-ANEPPDHQ and a custom-built time-resolved fluorometer to measure changes in membrane fluidity (reported in cone angle). Liposomes (BMP:DOPC:DOPE) exposed to TNS (100 µg/ml) showed no difference in cone angle compared to control in either buffer. However, TNB exposure produced significant decreases in cone angle in TBS at 200 µg/ml (36.42±0.31° to 32.84±0.33°) and in ABS at both 100 and 200 µg/ml (34.92±0.52° to 34.30±0.83° and 30.88±0.12°). Even though the surface area of TNS was greater than TNB the lack of effect supported that shape was important. ZnO ENM exposed liposomes composed of BMP:DOPC:DOPE showed no significant change in cone angle (100 µg/ml). However, BMP:DOPC:DOPS liposome had a significant decrease in cone angle (37.60±0.35° to 34.12±0.30°), indicating lipid headgroup can influence interactions. Funding: NIH R01ES023209, 1F32ES027324, P20GM103546, P30GM103338 and J Murdock Charitable Trust.

A microenvironment-sensitive coumarin-labeled peptide for the assessment of lipid-peptide interactions

Fluorescent-labeled peptides were synthesized as antimicrobial peptide models for evaluating the peptide interaction with eukaryotic and bacterial lipid membrane models and their dynamics in lipid membrane by fluorescence techniques. In this work, we discuss the behavior of two peptides of 15 residues, C-PEP and PEP (C-PEP being labeled with a coumarin 343) synthesized employing the solid-phase methodology (SPPS). The interaction of these peptides with lipid membranes of different phospholipid composition was studied by steady-state and time-resolved fluorescence spectroscopy, steady-state and time-resolved fluorescence anisotropy and circular dichroism. Both peptides were random coil in an aqueous medium and change their conformation to an α-helix when they were incorporated into DOPC lipid bilayer. In this lipid membrane, the fluorophore-peptide (C-PEP) displayed membrane interaction with a slightly higher partition within the lipid bilayer in comparison with PEP; nevertheless, the secondary structure adopted within the lipid membrane was not significantly modified by the conjugation with the coumarin moiety. In DPPC:DOPC (50:50) liposomes both peptides were less partitioned, as the incorporation was hindered because the lipid membrane is less fluid. And in the liposomes made of POPE:POPG (70:30), a bacterial model lipid membrane, a larger partition constant was observed with the adoption of structures of polyproline II (PPII)-type. The minor conformational disruption as a result of the conjugation with the coumarin moiety would allow modeling the peptide dynamic interaction with membranes using fluorescence techniques.

Probing the Hydrogen Bond Involving Acridone Trapped in a Hydrophobic Biological Nanocavity: Integrated Spectroscopic and Docking Analyses.

Spectroscopic analyses reveal that acridone (AD) penetrates through the structure and enters the hydrophobic cavity of the protein β-lactoglobulin (βLG). Although the protein contains two tryptophan (Trp) residues, AD interacts with only one (Trp-19), which is authenticated by the appearance of a single isoemissive point in TRANES. Alteration in the secondary structure of the protein while AD pierces through βLG is evident from the circular dichroism spectroscopic study. The ground-state interaction between AD and βLG is proven from the UV-vis spectroscopic study and the static nature of quenching of intrinsic fluorescence of the protein by the ligand. The steady-state fluorescence study in varied temperatures indicates the involvement of hydrogen bonding in the ligand-protein interaction. Further, the time-resolved fluorescence anisotropy study gives a hint of the presence of a hydrogen bond in AD-βLG interaction, which possibly involves the rotamers of Trp-19. In fact, the idea of involvement of rotamers of Trp-19 is obtained from the increase in fluorescence lifetime of βLG in the presence of AD. The docking study agrees to the involvement of hydrogen bonding in AD-βLG interaction. The direct evidence of hydrogen bonding between Trp and AD is obtained from the laser flash photolysis studies where the signature of formation of ADH• and Trp• through hydrogen abstraction between Trp and AD, loosely bound through hydrogen bonding, gets prominence. Thus, binding of AD to βLG involves hydrogen bonding in a hydrophobic pocket of the protein.

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.

Energy migration captures membrane-induced oligomerization of the prion protein

Excitation energy migration via homo-Förster resonance energy transfer (homo-FRET) can serve as an intermolecular proximity ruler within complex biomolecular assemblies. Here we present a unique case to demonstrate that energy migration can be a novel and sensitive readout to capture the membrane-mediated misfolding and oligomerization of the human prion protein (PrP), which is known to undergo an aberrant conformational conversion from an α-helical form into a self-propagating aggregated β-rich state causing deadly transmissible neurodegenerative diseases. Using site-specific energy migration studies by monitoring steady-state and time-resolved fluorescence anisotropy of fluorescently-tagged PrP, we elucidate the molecular details of lipid membrane-induced oligomers. We show that the intrinsically disordered N-terminal segment is critical for lipid-induced conformational sequestration of PrP into higher-order, β-rich oligomeric species that exhibit membrane permeabilization. Our results revealed that the N-terminal regions constitute the central core of the oligomeric architecture, whereas the distal C-terminal ends participate in peripheral association with the lipid membrane. Our study will find applications in the sensitive detection and in the structural characterization of membrane-induced protein misfolding and aggregation in a variety of deadly amyloid diseases.

Entrapment in micellar assemblies switches the excimer population of potential therapeutic luminophore azapodophyllotoxin.

Azapodophyllotoxin is a new class of anti-tumor agent with brilliant therapeutic activity and understanding its physicochemical nature in bio-mimetic microenvironments may provide substantial importance in context of its intercellular localization, efficacy as well as delivery. The present work epitomizes environment-sensitive fluorescence modulation of a prodigy, 4-(2-Hydroxyethyl)-10-phenyl-3,4,6,7,8,10- hexahydro-1H-cyclopenta[g]furo[3,4-b]quinoline-1-one (HPFQ) from the class of anti-cancer agent Azapodophyllotoxin, in differently charged model bio-mimetic micellar microenvironment of cationic CTAB, anionic SDS and neutral Triton X-100 using UV-visible absorption, steady state fluorescence, time-resolved fluorescence and fluorescence anisotropy studies. As a distinct phenomenon, anticancer HPFQ exhibits prolific fluorescence in solvents of varying polarity, originating from a mixed contribution of locally excited, charge transfer and excimer emission. A dramatic modulation in the photophysics of HPFQ has been observed in two types of surfactant consortiums: pre-micellar and post-micellar at physiological and anoxic pH. On photo-excitation, anti-cancer HPFQ exists in monomer-excimer equilibrium with varying ratios in different polarity regions. The marked enhancement in fluorescence intensity of HPFQ in post-micellar region of the surfactant under study probably arises due to regeneration of the monomer from its excimer. This reoccurrence reduces the possibility of Förster resonance energy transfer (FRET) from monomer to excimer, which essentially increases the desired emission intensity. Localization of HPFQ in micellar systems highly depends on polarity gradient inside the micelle, electrostatic, hydrophobic and intermolecular hydrogen bonding interactions. Further corroboration with the polarity sensitive experiments in dioxane-water mixture indicates towards spatial localization of the probe molecule in the stern layer of cationic CTAB, sheer surface of neutral TX100 and outer Gouy-Chapman layer in anionic SDS micelles. A molecular binary logic gate correlates the dominance of micellization over the polarity factor, which enhances the fluorescence response of HPFQ. The enhancement of the emissive potential of anti-cancer HPFQ in biomimetic environments by switching its excimer population may have an immense importance to achieve the status of a dual therapeutic and imaging agent altogether in progressive biomedical research.

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.