Steady-state Anisotropy Research Articles

Effect of phosphatidylcholine regioisomerism on lateral segregation of milk sphingomyelin in bilayer membranes

Milk fat globule membrane (MFGM) promotes the lateral phase separation of milk lipids and stabilizes the fat globules in milk. The composition and structures of lipids have a significant impact on physicochemical properties of MFGM, which in turn influences the digestion and absorption of milk lipids. Phospholipids (PL), sphingolipids, and cholesterol are the major lipid constituents of MFGM. While the effects of the head-group and structure of the fatty acids (FAs) on membrane properties are commonly studied, little is known on the impact of PL regioisomerism. The present study investigated the impact of phosphatidylcholine (PC) regioisomerism on lateral segregation of milk-sphingomyelin (milk-SM) as well as the influence on the interaction of milk-SM with ceramide and cholesterol in simulated membrane systems. The regioisomer pairs of four molecular species PC 16:0/18:1n-9, PC 16:0/18:2n-6, PC 16:0/18:3n-3, and PC 16:0/20:4n-6 were included in this study. The lateral segregation was determined using lifetime analysis of trans-parinaric acid (tPA) fluorescence. Thermostability of the domains was detected using steady-state anisotropy of tPA. Our results demonstrated a clear impact of PC regioisomerism on membrane properties. PC regioisomers having the unsaturated FAs at the sn-2 position enhanced the lateral segregation of milk-SM with and without the presence of ceramide and cholesterol compared to the regioiosmers having 16:0 at the sn-2 position. Furthermore, the characteristics i. e. the acyl chain length and degree of unsaturatin of sn-2 FA of the PCs had a major impact on the milk-SM gel phase and the intermolecular forces between milk-SM and ceramide/cholesterol. This work is the first investigation showing the effect of PL regioisomerism on milk-SM domains, which might have significant influence on functional properties of MFGM.

Exploring the Role of Neuropeptide PACAP in Cytoskeletal Function Using Spectroscopic Methods.

The behavior and presence of actin-regulating proteins are characteristic of various clinical diseases. Changes in these proteins significantly impact the cytoskeletal and regenerative processes underlying pathological changes. Pituitary adenylate cyclase-activating polypeptide (PACAP), a cytoprotective neuropeptide abundant in the nervous system and endocrine organs, plays a key role in neuron differentiation and migration by influencing actin. This study aims to elucidate the role of PACAP as an actin-regulating polypeptide, its effect on actin filament formation, and the underlying regulatory mechanisms. We examined PACAP27, PACAP38, and PACAP6-38, measuring their binding to actin monomers via fluorescence spectroscopy and steady-state anisotropy. Functional polymerization tests were used to track changes in fluorescent intensity over time. Unlike PACAP27, PACAP38 and PACAP6-38 significantly reduced the fluorescence emission of Alexa488-labeled actin monomers and increased their anisotropy, showing nearly identical dissociation equilibrium constants. PACAP27 showed weak binding to globular actin (G-actin), while PACAP38 and PACAP6-38 exhibited robust interactions. PACAP27 did not affect actin polymerization, but PACAP38 and PACAP6-38 accelerated actin incorporation kinetics. Fluorescence quenching experiments confirmed structural changes upon PACAP binding; however, all studied PACAP fragments exhibited the same effect. Our findings indicate that PACAP38 and PACAP6-38 strongly bind to G-actin and significantly influence actin polymerization. Further studies are needed to fully understand the biological significance of these interactions.

Photophysical Characterization and In Vitro Evaluation of α-Mangostin-Loaded HDL Mimetic Nano-Complex in LN-229 Glioblastoma Spheroid Model.

Cytotoxic activity has been reported for the xanthone α-mangostin (AMN) against Glioblastoma multiforme (GBM), an aggressive malignant brain cancer with a poor prognosis. Recognizing that AMN's high degree of hydrophobicity is likely to limit its systemic administration, we formulated AMN using reconstituted high-density lipoprotein (rHDL) nanoparticles. The photophysical characteristics of the formulation, including fluorescence lifetime and steady-state anisotropy, indicated that AMN was successfully incorporated into the rHDL nanoparticles. To our knowledge, this is the first report on the fluorescent characteristics of AMN with an HDL-based drug carrier. Cytotoxicity studies in a 2D culture and 3D spheroid model of LN-229 GBM cells and normal human astrocytes showed an enhanced therapeutic index with the rHDL-AMN formulation compared to the unincorporated AMN and Temozolomide, a standard GBM chemotherapy agent. Furthermore, treatment with the rHDL-AMN facilitated a dose-dependent upregulation of autophagy and reactive oxygen species generation to a greater extent in LN-229 cells compared to astrocytes, indicating the reduced off-target toxicity of this novel formulation. These studies indicate the potential therapeutic benefits to GBM patients via selective targeting using the rHDL-AMN formulation.

Spectroscopic behavior of bufotenine and bufotenine N-oxide: Solvent and pH effects and interaction with biomembrane models

Bufotenine is a fluorescent analog of Dimethyltryptamine (DMT) that has been widely studied due to its psychedelic properties and biological activity. However, little is known about its spectroscopic properties in different media. Thus, we present in this work, for the first time, the spectroscopic behavior of bufotenine and bufotenine N-oxide by means of their fluorescence properties. Both molecules exhibit changes in optical absorption and emission spectra with variations in pH of the medium and in different solvents. Assays in the presence of biomembranes models, like micelles and liposomes, were also performed. In surfactants titration experiments, the spectral shift observed in fluorescence shows the interaction of both molecules with pre-micellar structures and with micelles. Steady state anisotropy measurements show that both bufotenine and bufotenine N-oxide, in the studied concentration range, interact with liposomes without causing changes in the fluidity of the lipid bilayer. These results can be useful in studies that aim at searching for new compounds, inspired by bufotenine and bufotenine N-oxide, with relevant pharmacological activities and also in studies that use these molecules as markers of psychiatric disorders.

Exploring the Structural Importance of the C3=C4 Double Bond in Plant Alkaloids Harmine and Harmaline on Their Binding Interactions with Hemoglobin.

Harmine and harmaline are two structurally similar heterocyclic β-carboline plant alkaloids with various therapeutic properties, having a slight structural difference in the C3=C4 double bond. In the present study, we have reported the nature of the interaction between hemoglobin (Hb) with harmine and harmaline by employing several multispectroscopic, calorimetric, and molecular docking approaches. Fluorescence spectroscopic studies have shown stronger interaction of harmine with Hb compared to that of almost structurally similar harmaline. Steady-state anisotropy experiments further show that the motional restriction of harmine in the presence of Hb is substantially higher than that of the harmaline-Hb complex. Circular dichroism (CD) study demonstrates no conformational change of Hb in the presence of both alkaloids, but CD study in 1-cm cuvette path length also demonstrates stronger affinity of harmine toward Hb compared to harmaline. From the thermal melting study, it has been found that both harmine and harmaline slightly affect the stability of Hb. From isothermal titration calorimetry (ITC), we have found that the binding process is exothermic and enthalpy driven. Molecular docking studies indicated that both harmine and harmaline prefer identical binding sites in Hb. This study helps us to understand that slight structural differences in harmine and harmaline can alter the interaction properties significantly, and this key information may help in the drug discovery processes.

Unveiling the interaction modes of Imiquimod with DNA: Biophysical and computational studies

Herein the manuscript, we report the interaction of an important drug, Imiquimod (IMQ), with Calf Thymus Deoxyribonucleic Acid (ct-DNA). Despite of being used IMQ in multiple medicinal applications such as immune response modifier, antitumor and antiviral drug its interaction with DNA is not studied yet. To understand their interaction and characterize the binding mechanism between groove and intercalation modes, we used various spectroscopic tools. Fluorescence quenching, time-resolved emission, steady state anisotropy technique, ethidium bromide (EtBr), and 4′,6-diamidino-2-phenylindole (DAPI) displacement assays provided us biophysical aspects of IMQ-DNA interactions. All the above studies revealed the binding mode as intercalation mechanism. Circular Dichroism (CD) also well corroborated the intercalation of IMQ into DNA. Thermodynamic parameters of these bindinginteractions were interpreted with the help of temperature dependent spectroscopic studies characterizing the interaction as enthalpy driven. Further, the binding interactions is explored with the help of molecular docking studies. Based on experimental and computational studies it was concluded that the drug binds to DNA via intercalation mode in AT-rich DNA sequences. These biophysical investigations not only improve our comprehension of IMQ's therapeutic effectiveness but also clarifies the sequence dependent binding processes underlying comparable drug-DNA interactions.

Deciphering conformational changes in human serum albumin induced by bile salts using spectroscopic and molecular modeling approaches

Bile salts are physiologically-important natural amphiphilic biosurfactants synthesized in the liver and play a vital role in the solubilization and digestion of dietary lipids, cholesterol, and other fat-soluble compounds in the body. Bile salts also exhibit pharmacological and biological uses as carriers for transporting scarce water-soluble drugs and other chemicals owing to their unique emulsifying, solubilizing capacities and micelle forming ability. In this context, we present an endeavor towards the possibilities underlying the interaction and binding between the most abundant plasma protein namely, human serum albumin (HSA) and bile salts to demonstrate an intriguing interplay of hydrophobic, electrostatic and hydrogen bonding effects using steady state absorption, fluorescence emission, anisotropy, time-resolved emission, and molecular modelling approaches. The outcome illuminates how amphiphilic interfaces of bile salts under various physico-chemical conditions trigger the conformational changes and binding affinities of native and molten-globule forms of HSA. To elucidate non-covalent interactions during HSA-bile salt supramolecular host–guest complex formation, changes in intrinsic (tryptophan) and extrinsic (ANS) fluorescence have been investigated. The results reveal upon binding of bile salts in subdomain IIA of HSA, the protein undergoes conformational changes mediated primarily by hydrophobic interactions. Furthermore, time-resolved fluorescence measurements provide important structural and dynamical insights into the protein-bile salt supramolecular complexes. Additionally, molecular docking studies on these complexes clearly reveal spontaneous binding of bile salts into subdomain IIA of HSA while suggesting that the binding affinity decreases with the decreasing order of hydrophobicity of the bile salts (NaDC > NaC > NaTC) (ΔGdock = −29.64 kJ mol−1, −26.15 kJ mol−1, −14.35 kJ mol−1), respectively. This study exclusively highlights the molecular mechanism of conformational perturbation in the native (pH 7) and molten-globule (pH 3) forms of HSA, induced by bile salts. We believe that the results reported herein will be helpful in the design and formulation of protein-bile salt-based pharmacological carriers suitable for drug delivery.

Nature of Linear Spectral Properties and Fast Relaxations in the Excited States and Two-Photon Absorption Efficiency of 3-Thiazolyl and 3-Phenyltiazolyl Coumarin Derivatives.

Coumarin-based fluorescent agents play an important role in the manifold fundamental scientific and technological areas and need to be carefully studied. In this research, linear photophysics, photochemistry, fast vibronic relaxations, and two-photon absorption (2PA) of the coumarin derivatives, methyl 4-[2-(7-methoxy-2-oxo-chromen-3-yl)thiazol-4-yl]butanoate (1) and methyl 4-[4-[2-(7-methoxy-2-oxo-chromen-3-yl)thiazol-4-yl]phenoxy]butanoate (2), were comprehensively analyzed using stationary and time-resolved spectroscopic techniques, along with quantum-chemical calculations. The steady-state one-photon absorption, fluorescence emission, and excitation anisotropy spectra, as well as 3D fluorescence maps of 3-hetarylcoumarins 1 and 2 were obtained at room temperature in solvents of different polarities. The nature of relatively large Stokes shifts (∼4000-6000 cm-1), specific solvatochromic behavior, weak electronic π → π* transitions, and adherence to Kasha's rule were revealed. The photochemical stability of 1 and 2 was explored quantitatively, and values of photodecomposition quantum yields, on the order of ∼10-4, were determined. A femtosecond transient absorption pump-probe technique was used for the investigation of fast vibronic relaxation and excited-state absorption processes in 1 and 2, while the possibility of efficient optical gain was shown for 1 in acetonitrile. The degenerate 2PA spectra of 1 and 2 were measured by an open aperture z-scan method, and the maximum 2PA cross-sections of ∼300 GM were obtained. The electronic nature of the hetaryl coumarins was analyzed by quantum-chemical calculations using DFT/TD-DFT level of theory and was found to be in good agreement with experimental data.

Rotational dynamics of 7-(dimethyl amino)-4-methoxy-1- methyl-1, 8-napthyridin-2(1H)-one in DMSO and acetonitrile solvents

Steady-State anisotropy and time-resolved fluorescence decay measurements have been carried out by varying temperatures in DMSO and acetonitrile solvents to study the rotational reorientation dynamics of 7-(dimethylamino)-4-methoxy-1-methyl-1,8-napthyridin-2(1H)-one(Quinolon-390). Experimentally determined rotational re-orientation time values in DMSO are found to be higher than acetonitrile, which implies that Qu-390 experiences more friction in DMSO than acetonitrile. To understand this friction, the theoretically measured rotational reorientation times using Stokes-Einstein’s-Debye (SED), Geirer-Wirtz (GW), and Dote-Kivelson and Schwartz (DKS) models are compared with experimental ones. Theoretically, estimated Qu-390 rotational reorientation times in DMSO and acetonitrile underestimate the experimental values which emphasize that mechanical friction contribution is less and dielectric friction contribution is more. Interestingly the experimental value and stick theoretical value 164 Ps at around 320 K is the same in DMSO.

Fluorescence Anisotropy as a Self-Referencing Readout for Ion-Selective Sensing and Imaging Using Homo-FRET between Chromoionophores.

Fluorescence anisotropy has been widely used in developing biosensors and immunoassays, by virtue of the self-reference and environment-sensitive properties. However, fluorescence anisotropic chemical sensors on inorganic ions are limited by the total anisotropy change. To this end, we demonstrate here fluorescence anisotropic ion-selective optodes based on the homo-FRET (Förster resonance energy transfer) of the crowded chromoionophores. The conventional fluorescence on-off mode is transformed into the anisotropic mode. Variation of the target ion concentration changes the inter-chromoionophore distance in the organic sensing phase, leading to different extents of homo-FRET and steady-state anisotropy. A theoretical model is developed by coupling homo-FRET and anisotropy. Anisotropic detections of pH, K+, and Na+ are demonstrated as examples based on the different ionophores for H+, K+, and Na+, respectively. Further, fluorescence imaging of the nano-optodes, plasticized poly(vinyl chloride) sensing films, and live cells are demonstrated using a homemade fluorescence anisotropic imaging platform. The results form the basis of an ion-selective analytical method operating in the fluorescence anisotropic mode, which could potentially be applied to other fluorescence on-off probes based on homo-FRET.

Photophysical Behavior of Plant Flavonols Galangin, Kaempferol, Quercetin, and Myricetin in Homogeneous Media and the DMPC Model Membrane: Unveiling the Influence of the B-Ring Hydroxylation of Flavonols.

Flavonols have been studied extensively because of their interesting biological activities and excited-state intramolecular proton transfer (ESIPT) behavior. Galangin, kaempferol, quercetin, and myricetin are structurally related flavonols that differ only in the number of B-ring hydroxyl substituents. In this work, we have carried out a detailed study on the photophysical behavior of these structurally related flavonols in various solvents and a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) small unilamellar vesicles (SUVs) model membrane. We observed that these flavonols exist in different forms in the ground and excited states depending on the nature of the solvent. The weak intrinsic fluorescence of these flavonols gets enhanced in hydrogen-bond-accepting and alcoholic solvents. The phototautomer fluorescence intensity of these flavonols increases significantly in the DMPC membrane compared to water, suggesting ESIPT activation via binding interaction between flavonols and the membrane. According to our findings, both the number of B-ring hydroxy groups and membrane fluidity affect the flavonol binding with the membrane. The steady-state fluorescence intensity, steady-state anisotropy, fluorescence lifetime, and fluorescence anisotropy decay of flavonols were sensitive towards the temperature-induced DMPC membrane phase change. A quenching study has been performed to investigate the location and distribution of flavonols in the DMPC SUVs. Moreover, the antioxidant potential of flavonols in DMPC SUVs has been examined using the DPPH scavenging method. Our results reveal that the B-ring hydroxy groups significantly affect the photophysics, binding affinity, location, distribution, and DPPH scavenging activity of polyhydroxy-flavonols in the DMPC SUVs.

Monitoring the Anisotropy and Fluidity of the HDL Monolayer as Surrogates of HDL Functionality.

The fluidity of the biological lipid layers modulates processes involved in cardiovascular disease. High-density lipoprotein (HDL) monolayer fluidity is considered as a surrogate of HDL functionality. In particular, the more fluid the HDL monolayer is, the greater the cholesterol efflux (ChE) is observed. Fluidity depends on cholesterol and on the saturation and length of the fatty acids present in lipid layers. Specifically, low cholesterol and short-chain and/or low-saturated fatty acids content in the lipid layers increases fluidity. Lipid peroxidation is also involved in regulating the monolayers' fluidity. HDL oxidation decreases its fluidity and ChE capacity. Accordingly, the presence of antioxidants in biological membranes and in HDL increases fluidity. The fluidity is assessed in polarization studies that measures the steady-state anisotropy (r) using fluorescent probes (such as 1,6-diphenyl-1,3,5-hexatriene; DPH) that mimic the molecular movements of the sample analyzed. Since r refers to the rigidity and fluidity refers to the viscosity of lipid layers, the fluidity index is the inverse value of r (i.e., 1/r). This chapter describes a method for measuring HDL monolayer fluidity and r. The reproducibility of this method was excellent as the intra-assay coefficients of variation (CV) were <2.5 (20 replicates on the same day) and the interassay CV were <5% (60 replicates measured on 3 different days; 20 replicates/day). The method therefore represents a reproducible and useful tool to evaluate HDL functionality as an emerging cardiovascular risk factor.

Interactions and Dynamics in Aqueous Solutions of pH-Responsive Polymers: A Combined Fluorescence and Dielectric Relaxation Study.

Interaction and dynamics of aqueous solutions of pH-responsive smart polymers are investigated via steady-state, time-resolved fluorescence emission spectroscopy with the help of external local reporter coumarin 153 (C153), while MHz to GHz dielectric relaxation spectroscopic (DRS) measurement reports the intrinsic medium relaxation features. A series of pH-responsive random copolymers (DPL-DP60) comprising of a pH-responsive moiety 2-((leucinyl)oxy)ethyl methacrylate (l-Leu-HEMA) and hydrophobic methyl methacrylate (MMA) are synthesized and characterized. A balance between the pH-responsive (l-Leu-HEMA) and the hydrophobic (MMA) content dictates the phase transition pH, which is found to be ∼5-7 for these aqueous copolymer solutions (1 mg/mL). Dynamic light scattering measurements in aqueous solutions of these polymers reflect a small particle size (∼2-8 nm) at solution pH below their individual phase transition pH, while a large particle size (∼140-340 nm) forms beyond their phase transition pH. No signature of a phase transition pH-driven abrupt change in static and dynamic properties of aqueous polymer solutions has been registered from pH-dependent dielectric relaxation as well as solute (C153)-centric fluorescence measurements. A significant impact of varying the l-Leu-HEMA/MMA segment ratio on steady-state fluorescence emission and rotational anisotropy decay of the fluorophore solute (C153) has been observed. MHz to GHz DRS in aqueous solutions of these pH-responsive polymers reflects bulk water-like dielectric features.

Therapeutic lithium alters polar head-group region of lipid bilayer and prevents lipid peroxidation in forebrain cortex of sleep-deprived rats

Lithium is regarded as a unique therapeutic agent for the management of bipolar disorder (BD). In efforts to explain the favourable effects of lithium in BD, a wide range of mechanisms was suggested. Among those, the effect of clinically relevant concentrations of lithium on the plasma membrane was extensively studied. However, the biophysical properties of brain membranes isolated from experimental animals exposed to acute, short-term and chronic lithium have not been performed to-date. In this study, we compared the biophysical parameters and level of lipid peroxidation in membranes isolated from forebrain cortex (FBC) of therapeutic lithium-treated and/or sleep-deprived rats. Lithium interaction with FBC membranes was characterized by appropriate fluorescent probes. DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulphonate) were used for characterization of the hydrophobic lipid core and Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) for the membrane-water interface. Lipid peroxidation was determined by immunoblot analysis of 4-HNE-(4-hydroxynonenal)-protein adducts. The organization of polar head-group region of FBC membranes, measured by Laurdan generalized polarization, was substantially altered by sleep deprivation and augmented by lithium treatment. Hydrophobic membrane interior characterized by steady-state anisotropy of DPH and TMA-DPH fluorescence was unchanged. Chronic lithium had a protective effect against peroxidative damage of membrane lipids in FBC. In summary, lithium administration at a therapeutic level and/or sleep deprivation as an animal model of mania resulted in changes in rat FBC membrane properties.

Fluorescence depolarization dynamics of ionic strength sensors using time-resolved anisotropy

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

Covalent immobilization and characterization of Rhizopus oryzae lipase on core-shell cobalt ferrite nanoparticles for biodiesel production

Rhizopus oryzae lipase (ROL) was immobilized on the surface of silica coated amino modified CoFe2O4 nanoparticles and applied for biodiesel production. The results indicated more affinity of the ROL toward its substrate upon immobilization, as revealed by a lower Km value for the immobilized ROL compared to its free counterpart. Intrinsic fluorescence spectroscopy indicated a lower intensity for ROL immobilized on CoFe2O4 nanoparticles. Besides, immobilized ROL steady state anisotropy measurements presented lower values, which implied assembly of ROL molecules on magnetic nanoparticles upon immobilization as well as their restricted rotation upon covalent attachment. Thermal stability analysis revealed improved activity at higher temperatures for the immobilized enzyme compared to its free counterpart. Accordingly, Pace analysis to determine protein thermal stability revealed preservation of the protein conformation in the presence of increasing temperatures upon immobilization on nanoparticles. Finally, ROL immobilized on CoFe2O4 nanoparticles exhibited improved efficiency of biodiesel production in agreement with thermal activity profile. Therefore, the authors suggest application of the lipase molecules immobilized on CoFe2O4 nanoparticles for more efficient biodiesel production.

Comparative binding analysis of noscapine and piperine with tRNA: A structural perturbation and energetic study.

In this study, we have exploring the binding mechanisms of the two anticancer alkaloid noscapine (NOS) and piperine (PIP) with tRNA using different spectroscopy and computational method. Absorbance and emission spectra revealed that both the drugs show strong binding with tRNA, where NOS intercalate between the base pairs of tRNA and PIP binds in the groove of tRNA. Competitive binding study and steady state anisotropy further confirms the intercalative mode of binding between NOS and tRNA and groove binding in PIP-tRNA complex. The observed thermodynamic parameters suggested that NOS-tRNA complex formation is endothermic and entropy driven, however it was exothermic, and enthalpy driven in case of PIP-tRNA complex. CD and time resolved fluorescence studies show the structural perturbations and conformational change in tRNA structure with NOS as well as PIP. Molecular docking studies are comparable with experimental results and further confirmed that the hydrophobic interactions involved in the NOS-tRNA binding, whereas hydrogen binding and van der Waals interactions play important role in the PIP-tRNA complex formation. This study can be useful to understand the potential binding and resultant tRNA damage by alkaloids and deigned new target specific anticancer drug.

Determining the Stoichiometry of Small Protein Oligomers Using Steady-State Fluorescence Anisotropy

A large fraction of soluble and membrane-bound proteins exists as non-covalent dimers, trimers, and higher-order oligomers. The experimental determination of the oligomeric state or stoichiometry of proteins remains a nontrivial challenge. In one approach, the protein of interest is genetically fused to green fluorescent protein (GFP). If a fusion protein assembles into a non-covalent oligomeric complex, exciting their GFP moiety with polarized fluorescent light elicits homotypic Förster resonance energy transfer (homo-FRET), in which the emitted radiation is partially depolarized. Fluorescence depolarization is associated with a decrease in fluorescence anisotropy that can be exploited to calculate the oligomeric state. In a classical approach, several parameters obtained through time-resolved and steady-state anisotropy measurements are required for determining the stoichiometry of the oligomers. Here, we examined novel approaches in which time-resolved measurements of reference proteins provide the parameters that can be used to interpret the less expensive steady-state anisotropy data of candidates. In one approach, we find that using average homo-FRET rates (kFRET), average fluorescence lifetimes (τ), and average anisotropies of those fluorophores that are indirectly excited by homo-FRET (rET) do not compromise the accuracy of calculated stoichiometries. In the other approach, fractional photobleaching of reference oligomers provides a novel parameter a whose dependence on stoichiometry allows one to quantitatively interpret the increase of fluorescence anisotropy seen after photobleaching the candidates. These methods can at least reliably distinguish monomers from dimers and trimers.

A Multiparametric Fluorescence Probe to Understand the Physicochemical Properties of Small Unilamellar Lipid Vesicles in Poly(ethylene glycol)-Water Medium.

FDAPT (2-formyl-5-(4'-N,N-dimethylaminophenyl)thiophene) efficiently senses the minimum alteration of lipid bilayer microenvironment with all six different fluorescence parameters namely emission wavelength, fluorescence intensity, steady-state anisotropy, and their corresponding time-dependent parameters (Sahu et al., J. Phys. Chem. B 2018, 122, 7308-7318). In the present work, the effect of poly(ethylene glycol) on the small unilamellar vesicle is demonstrated with the emission behavior of the FDAPT probe. A medium and a high molecular weight PEG were chosen to perturb the lipid vesicles. The alteration of the bilayer polarity, water content inside bilayer, lipid packing density in the perturbed vesicles reflect significant changes in different fluorescence parameters of FDAPT probe. The effect of PEG on the unilamellar vesicle was rationalized with the alteration of the emission behavior, fluorescence lifetime, steady-state anisotropy and anisotropy decay of the probe. The simple and convenient fluorescence measurements provide new insights into the effect of PEG on the packing density, water volume, micro polarity, and microviscosity of the small unilamellar vesicle. The physiological understanding was extended to rationalize the cryoprotecting behavior of PEG.

Spectroscopic Studies of a Phosphonium Ionic Liquid in Supercritical CO2

Fluorescence spectroscopy was used to study a solution comprised of coumarin 153 (C153)+ trihexyltetradecylphosphonium bis(trifluoromethylsulfonyl)imide ([P6,6,6,14]+ [Tf2N]−)+ supercritical CO2 (scCO2). We compare the spectroscopy of C153 in neat scCO2 to that of C153/scCO2 with the addition of ionic liquid (IL). Excitation and emission peak frequencies of C153 in scCO2 and in IL/scCO2 diverged at reduced densities (ρr = ρ/ρc) below the CO2 critical density. At low fluid density, spectral changes in the IL/scCO2 solutions showed evidence that C153 experiences a very different microenvironment—one that is unlike neat scCO2. The data show that the presence of IL clearly influences the C153 excitation and emission profiles. Excitation was broadened and red shifted by &gt;2000 cm−1 and the presence of an additional low-energy emission component that was red shifted by ~3000 cm−1 was clearly visible and not observed in neat scCO2. The solution heterogeneity was controlled by changing the scCO2 density and at high fluid density, both the excitation and emission spectra were more similar to those in neat scCO2. Steady-state anisotropy also showed that at low fluid density, the C153 emission was significantly polarized. Aggregation of C153 has been reported in the literature and this led us to hypothesize the possibility that C153 dimer (aggregation) formation may be occurring in scCO2. Another possible explanation is that dye–IL aggregates may dissolve into the scCO2 phase due to C153 acting as a “co-solvent” for the IL. Time-resolved intensity decay measurements yielded only slightly non-exponential decays with accompanying time constants of ~3–4 ns that were significantly shorter than the 5–6 ns time constants in neat scCO2, which are suggestive of C153–IL interactions. However, these data did not conclusively support dimer formation. Pre-exponential factors of the time constants showed that almost all of the emission was due to monomeric C153.