Molecular design of the γδT cell receptor ectodomain encodes biologically fit ligand recognition in the absence of mechanosensing.

High-acuity αßT cell receptor (TCR) recognition of peptides bound to major histocompatibility complex molecules (pMHCs) requires mechanosensing, a process whereby piconewton (pN) bioforces exert physical load on αßTCR-pMHC bonds to dynamically alter their lifetimes and foster digital sensitivity cellular signaling. While mechanotransduction is operative for both αßTCRs and pre-TCRs within the αßT lineage, its role in γδT cells is unknown. Here, we show that the human DP10.7 γδTCR specific for the sulfoglycolipid sulfatide bound to CD1d only sustains a significant load and undergoes force-induced structural transitions when the binding interface-distal γδ constant domain (C) module is replaced with that of αß. The chimeric γδ-αßTCR also signals more robustly than does the wild-type (WT) γδTCR, as revealed by RNA-sequencing (RNA-seq) analysis of TCR-transduced Rag2-/- thymocytes, consistent with structural, single-molecule, and molecular dynamics studies reflective of γδTCRs as mediating recognition via a more canonical immunoglobulin-like receptor interaction. Absence of robust, force-related catch bonds, as well as γδTCR structural transitions, implies that γδT cells do not use mechanosensing for ligand recognition. This distinction is consonant with the fact that their innate-type ligands, including markers of cellular stress, are expressed at a high copy number relative to the sparse pMHC ligands of αßT cells arrayed on activating target cells. We posit that mechanosensing emerged over ∼200 million years of vertebrate evolution to fulfill indispensable adaptive immune recognition requirements for pMHC in the αßT cell lineage that are unnecessary for the γδT cell lineage mechanism of non-pMHC ligand detection.

Self-Assembly of Amphiphiles into Vesicles and Fibrils: Investigation of Structure and Dynamics Using Spectroscopy and Microscopy Techniques.

Amphiphiles are a class of molecules which are known to assemble into a variety of nanostructures. The understanding and applications of self-assembled systems are based on what has been learned from biology. Among the vast number of self-assemblies, in this article, we have described the formation, characterization, and dynamics of two important biologically inspired assemblies: vesicles and fibrils. Vesicles, which can be classified into several categories depending on the sizes and components, are of great interest due to their potential applications in drug delivery and as nanoscale reactors. The structure and dynamics of vesicles can also mimic the complex geometry of the cell membrane. On the other hand, the self-assembly of proteins, peptides, and even single amino acids leads to a number of degenerative disorders. Thus, a complete understanding of these self-assembled systems is necessary. In this article, we discuss recent work on vesicular aggregates composed of phospholipids, fatty acids, and ionic as well as nonionic surfactants and single amino acid-based fibrils such as phenylalanine and tyrosine. Beside the characterization, we also emphasize the excited-state dynamics inside the aggregates for a proper understanding of the organization, reactivity, and heterogeneity of the aggregates.

A Comparative Study of the Influence of Sugars Sucrose, Trehalose, and Maltose on the Hydration and Diffusion of DMPC Lipid Bilayer at Complete Hydration: Investigation of Structural and Spectroscopic Aspect of Lipid-Sugar Interaction.

It is well-known that sugars protect membrane structures against fusion and leakage. Here, we have investigated the interaction between different sugars (sucrose, trehalose, and maltose) and phospholipid membrane of 1,2-dimyristoyl-sn-glycero-3-phoshpocholine (DMPC) using dynamic light scattering (DLS), transmission electron microscopy (TEM), and other various spectroscopic techniques. DLS measurement reveals that the addition of sugar molecule results a significant increase of the average diameter of DMPC membrane. We have also noticed that in the presence of different sugars the rotational relaxation and solvation time of coumarin 480 (C480) and coumarin 153 (C153) surrounding DMPC membrane increases, suggesting a marked reduction of the hydration behavior at the surface of phospholipid membrane. In addition, we have also investigated the effect of sugar molecules on the lateral mobility of phospholipids. Interestingly, the relative increase in rotational, solvation and lateral diffusion is more prominent for C480 than that of C153 because of their different location in lipid bilayer. It is because of preferential location of comparatively hydrophilic probe C480 in the interfacial region of the lipid bilayer. Sugars intercalate with the phospholipid headgroup through hydrogen bonding and replace smaller sized water molecules from the membrane surface. Therefore, overall, we have monitored a comparative analysis regarding the interaction of different sugar molecules (sucrose, trehalose, and maltose) with the DMPC membrane through DLS, TEM, solvation dynamics, time-resolved anisotropy, and fluorescence correlation spectroscopy (FCS) measurements to explore the structural and spectroscopic aspect of lipid-sugar interaction.

A new strategy to prepare giant vesicles from surface active ionic liquids (SAILs): a study of protein dynamics in a crowded environment using a fluorescence correlation spectroscopic technique.

A simple procedure for the preparation of giant vesicles using surface active ionic liquids (SAILs) has been provided in this paper. SAILs, used to form vesicles, were synthesized by replacing the cationic part of Aerosol OT (AOT) with cations having alkyl chains of different lengths (ammonium and imidazolium cations). The number of carbons in the alkyl chains of the cations was varied from eight to sixteen. From the observed results, the formation of giant vesicles is found to be dependent on the alkyl chain length as well as the organic moieties of the respective cations. These giant vesicles were characterized using fluorescence lifetime imaging microscopy (FLIM). The conformational dynamics of bovine serum albumin (BSA) inside these giant vesicles was determined using fluorescence correlation spectroscopy (FCS) to get an idea about the protein dynamics in a constrained environment. The interaction of the giant vesicles with the protein was confirmed by the change in the diffusion coefficient and the conformational fluctuation time.

Spectroscopy and Fluorescence Lifetime Imaging Microscopy To Probe the Interaction of Bovine Serum Albumin with Graphene Oxide.

The interaction of graphene oxide (GO) with bovine serum albumin (BSA) in aqueous buffer solution has been investigated with various spectroscopic and imaging techniques. At single molecular resolution this interaction has been performed using fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM) techniques. The conformational dynamics of BSA on GO's influence have been explored by FCS and circular dichroism (CD) spectroscopy. For the FCS studies BSA was labeled covalently by a fluorophore, Alexa Fluor 488. On the addition of GO in phosphate buffer of 10 mM at pH 7.4 the diffusion time (τD) and the hydrodynamic radius (Rh) of BSA increase due to adsorption of BSA. Conformational relaxation time components of native BSA drastically vary with the addition of GO, signifying the change of conformational dynamics of BSA after addition of GO. The adsorption isotherm also indicates significant adsorption of BSA on the GO surface. Adsorption of BSA on the GO surface has shown in direct images of atomic force microscopy (AFM) and FLIM. Fluorescence quenching study of BSA with addition of GO also indicates that there is strong interaction between BSA and GO.

How does the surface charge of ionic surfactant and cholesterol forming vesicles control rotational and translational motion of rhodamine 6G perchlorate (R6G ClO4)?

The rotational dynamics and translational diffusion of a hydrophilic organic molecule, rhodamine 6G perchlorate (R6G ClO4) in small unilamellar vesicles formed by two different ionic surfactants, cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), with cholesterol have been investigated using fluorescence spectroscopic methods. Moreover, in this article the formation of vesicle using anionic surfactant, SDS at different cholesterol-to-surfactant molar ratio (expressed by Q value (Q = [cholesterol]/[surfactant])) has also been reported. Visual observation, dynamic light scattering (DLS) study, turbidity measurement, steady state fluorescence anisotropy (r0) measurement, and eventually microscopic images reveal the formation of small unilamellar vesicles in aqueous solution. Also, in this study, an attempt has been made to observe whether the cationic probe molecule, rhodamine 6G (R6G) experiences similar or different microenvironment in cholesterol-SDS and cholesterol-CTAB assemblies with increase in cholesterol concentration. The influence of cholesterol on rotational and translational diffusion of R6G molecules has been investigated by monitoring UV-vis absorption, fluorescence, time-resolved fluorescence anisotropy, and finally fluorescence correlation spectroscopy (FCS) measurements. In cholesterol-SDS assemblies, due to the strong electrostatic attractive interaction between the negatively charged surface of vesicle and cationic R6G molecules, the rotational and diffusion motion of R6G becomes slower. However, in cholesterol-CTAB aggregates, the enhanced hydrophobicity and electrostatic repulsion induces the migration of R6G from vesicle bilayer to aqueous phase. The experimental observations suggest that the surface charge of vesicles has a stronger influence than the hydrophobicity of the vesicle bilayer on the rotational and diffusion motion of R6G molecules.

How does bile salt penetration affect the self-assembled architecture of pluronic P123 micelles?--light scattering and spectroscopic investigations.

The triblock copolymer of the type (PEO)20-(PPO)70-(PEO)20 (P123) forms a mixed supramolecular aggregate with different bile salts, sodium deoxycholate (NaDC) and sodium taurocholate (NaTC), having different hydrophobicity. These mixed micellar systems have been investigated through dynamic light scattering (DLS) and other various spectroscopic techniques. DLS measurements reveal that the bile salts penetrate into the core-corona region of the P123 micelle and further addition of bile salts causes formation of a new supramolecular aggregate. Further CONTIN analysis confirms existence of two types of complexes at higher molar ratios of bile salt-P123 (>1 : 3). Due to the bile salt penetration, the polarity of the core-corona region of bile salt-P123 mixed micelle increases which results in red shift in the absorption and emission spectra of coumarin 153 (C153) and coumarin 480 (C480). The rotational diffusion of the hydrophobic probe C153 and a hydrophilic probe C480 has been investigated in these bile salt-P123 mixed systems and for both the probes a decrease in the average reorientation time has been observed. The reason behind this decrease in the average reorientation time is the increase in both polarity and hydration of the core-corona region in these mixed micelles. Moreover, these bile salt-P123 mixed micelles are characterized by fluorescence correlation spectroscopy (FCS) techniques. As hydrophobic solute 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl)-4H-pyran (DCM) resides in the core region of the bile salt-P123 mixed micelles, the translational diffusion of DCM becomes faster in these mixed micelles compared to that in pure P123 micelle. However, for cationic probe rhodamine 6G perchlorate (R6G), a totally opposite trend in the translational diffusion coefficients has been observed. Both anisotropy and FCS measurements confirm that bile salts affect the core region of the P123 micelle more than the corona region. Besides, all these characterizations confirm that more hydrophobic NaDC interacts in a better way than NaTC with the P123 micelle.

Modulation of the aggregation properties of sodium deoxycholate in presence of hydrophilic imidazolium based ionic liquid: water dynamics study to probe the structural alteration of the aggregates.

In this article, we have investigated the effect of a hydrophilic ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]-BF4), on the aggregation properties of a biological surfactant, sodium deoxycholate (NaDC), in water. In solution, unlike conventional surfactants it shows stepwise aggregation and the effect of the conventional ionic liquid on the aggregation properties is rather interesting. We have observed concentration dependent dual role of the ionic liquid; at their low concentration, the aggregated structure of NaDC reorganizes itself into an elongated rod like structure. However, the aggregated network is disintegrated into small aggregates upon further addition of ionic liquid. TEM (Transmission Electron Microscopy), SEM (Scanning Electron Microscopy) and FLIM (Fluorescence Lifetime Imaging Microscopy) images also confirmed the structural alteration of NaDC upon varying the concentration of the ionic liquid. The proton NMR data indicate that hydrophobic as well as electrostatic interaction is solely responsible for such structural adaptation of NaDC in the presence of an ionic liquid. The host-guest interaction inside the aggregates is monitored using Coumarin-153 (C-153) and the location of C-153 is probed by varying the excitation wavelength from 375 nm to 440 nm and the two binding sites of the aggregates are affected in a different fashion in the presence of ionic liquid. Excitation in the blue region selects the fluorophores which preferably bind to the buried region of the aggregates, whereas 440 nm excitation corresponds to the guest molecules which are exposed to the solvent molecules. The average solvation time of C-153 is increased in the presence of 1.68 wt% [bmim]-BF4 at λexc = 440 nm i.e. the probe molecules relocate themselves to a more restricted region. However, the average solvation time became 2.6 times faster in the presence of 11.2 wt% [bmim]-BF4, which corresponds to a more polar and exposed region. The time resolved anisotropy measurements and polarity determined by pyrene also supported our results in addition to solvation dynamics measurements. In summary, ionic liquids can modulate the host-guest interaction of bile salt aggregates, which can be used as nanocarriers for drug delivery.

Picosecond solvation dynamics--a potential viewer of DMSO-water binary mixtures.

In this work, we have investigated the composition dependent anomalous behavior of dimethyl sulfoxide (DMSO)-water binary mixture by collecting the ultrafast solvent relaxation response around a well known solvation probe Coumarin 480 (C480) by using a femtosecond fluorescence up-conversion spectrometer. Recent molecular dynamics simulations have predicted two anomalous regions of DMSO-water binary mixture. Particularly, these studies encourage us to investigate the anomalies from experimental background. DMSO-water binary mixture has repeatedly given evidences of its dual anomalous nature in front of our systematic investigation through steady-state and time-resolved measurements. We have calculated average solvation times of C480 by two individual well-known methods, among them first one is spectral-reconstruction method and another one is single-wavelength measurement method. The results of both the methods roughly indicate that solvation time of C480 reaches maxima in the mole fraction of DMSO XD = 0.12-0.17 and XD = 0.27-0.35, respectively. Among them, the second region (XD = 0.27-0.35) is very common as most of the thermodynamic properties exhibit deviation in this range. Most probably, the anomalous solvation trend in this region is fully guided by the shear viscosity of the medium. However, the first region is the most interesting one. In this region due to formation of strongly hydrogen bonded 1DMSO:2H2O complexes, hydration around the probe C480 decreases, as a result of which solvation time increases.

Ultrafast FRET to study spontaneous micelle-to-vesicle transitions in an aqueous mixed surface-active ionic-liquid system.

The spontaneous micelle-to-vesicle transition in an aqueous mixture of two surface-active ionic liquids (SAILs), namely, 1-butyl-3-methylimidazolium n-octylsulfate ([C4mim][C8SO4]) and 1-dodecyl-3-methylimidazoium chloride ([C12mim]Cl) is described. In addition to detailed structural characterization obtained by using dynamic light scattering, transmission electron microscopy (TEM), and cryogenic TEM techniques, ultrafast fluorescence resonance energy transfer (FRET) from coumarin 153 (C153) as a donor (D) to rhodamine 6G (R6G) as an acceptor (A) is also used to study micelle-vesicle transitions in the present system. Structural transitions of SAIL micelles ([C4mim][C8SO4] or [C12mim]Cl micelles) to mixed SAIL vesicles resulted in significantly increased D-A distances, and therefore, increased timescale of FRET. In [C4mim][C8SO4] micelles, FRET between C153 and R6G occurs on an ultrafast timescale of 3.3 ps, which corresponds to a D-A distance of about 15 Å. As [C4mim][C8SO4] micelles are transformed into mixed micelles upon the addition of a 0.25 molar fraction of [C12mim]Cl, the timescale of FRET increases to 300 ps, which suggests an increase in the D-A distance to 31 Å. At a 0.5 molar fraction of [C12mim]Cl, unilamellar vesicles are formed in which FRET occurs on multiple timescales of about 250 and 2100 ps, which correspond to D-A distances of 33 and 47 Å. Although in micelles and mixed micelles the obtained D-A distances are well correlated with their radius, in vesicles the obtained D-A distance is within the range of the bilayer thickness.

Effect of encapsulation of curcumin in polymeric nanoparticles: how efficient to control ESIPT process?

This paper demonstrates the photophysics of curcumin inside polymeric nanoparticles (NPs), which are being recently used as targeted drug delivery vehicles. For this purpose, we have prepared three polymeric NPs by ultrasonication method from three well-defined water-insoluble random copolymers. These copolymers having various degrees of hydrophobicity were synthesized via reversible addition-fragmentation transfer (RAFT) method using styrene and three different functional monomers, namely, 2-hydroxyethyl acrylate, 4-formylphenyl acrylate, and 4-vinylbenzyl chloride. The photophysics of the curcumin molecules inside the polymeric NPs have been monitored by applying tools like steady state and time-resolved fluorescence spectroscopy. An increase in fluorescence intensity along with an increase in the lifetime values indicated a perturbation of the excited state intramolecular proton transfer (ESIPT) process of curcumin inside the polymeric NPs.

Spectroscopic investigation of the binding interactions of a membrane potential molecule in various supramolecular confined environments: contrasting behavior of surfactant molecules in relocation or release of the probe between nanocarriers and DNA surface.

The fluorescence and optical properties of membrane potential probes are widely used to measure cellular transmembrane potentials. Hemicyanine dyes are also able to bind to membranes. The spectral properties of these molecules depend upon the charge shift from the donor moiety to the acceptor moiety. Changes in their spectral properties, i.e. absorption and emission maxima or intensities, are helpful in characterizing model membranes, microheterogeneous media, etc. In this article, we have demonstrated the binding interaction of a membrane potential probe, 1-ethyl-2-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-pyridinium perchlorate (LDS 698), with various supramolecular confined environments. The larger dipole moment in the ground state compared to the excited state is a unique feature of hemicyanine dyes. Due to this unique feature, red shifts in the absorption maxima are observed in hydrophobic environments, compared with bulk solvent. On addition of surfactants and CT DNA to an aqueous solution containing LDS 698, significant increase in the emission intensity along with the quantum yield and lifetime indicate partition of the probe molecules into organized assemblies. In the case of the sodium dodecyl sulfate (SDS)-water system, due to interactions between the cationic LDS 698 and the anionic dodecyl sulfate moiety, the fluorescence intensity at ∼666 nm decreases and an additional peak at ∼590 nm appears at premicellar concentration (∼0.20 mM-4.50 mM). But at ∼5.50 mM SDS concentration, the absorbance in the higher wavelength region increases again, indicating encapsulation of the probe in micellar aggregates. This observation indicates that the premicellar aggregation behavior of SDS can also be judged by observing the changes in the UV-vis and fluorescence spectral patterns. The temperature dependent study also indicates that non-radiative deactivation of the dye molecules is highly restricted in the DNA micro-environment, compared with micelles. Besides, we have also investigated the specific interaction of surfactant micelles with DNA. Our observations reveal that, in the presence of CT DNA, LDS 698 interacts exclusively with SDS micelles, but that it preferentially releases from micelles and relocates to DNA surfaces in solutions containing TX-100 micelles.

Interaction of gold nanoclusters with IR light emitting cyanine dyes: a systematic fluorescence quenching study.

This paper describes the intermolecular interactions of gold nanoclusters (Au NCs) with cyanine dyes, namely HITC P, DTTC I, and IR 144. All the cyanine dyes quenched the fluorescence of Au NCs effectively. Steady-state and time-resolved measurements were performed to understand the competition between electron transfer and energy transfer in the Au NCs and cyanine dye system. A significant spectral overlap between the emission spectrum of the Au NCs and the absorption spectrum of cyanine dyes was observed, making both ideal for studying FRET interactions. However, after careful inspection of the steady state spectra and time resolved decays we concluded that photoinduced electron transfer (PET) could be the major pathway to quench the fluorescence intensity of Au NCs. To elucidate the interaction mechanism between Au NCs and cyanine dyes, docking studies were also performed. The docking studies reveal that the quencher molecules, i.e. cyanine dyes, come in close proximity with the 34-cysteine (Cys) in BSA where the Au clusters are located to enable the electron transfer process.

Measuring αß T-Cell Receptor-Mediated Mechanosensing Using Optical Tweezers Combined with Fluorescence Imaging.

T-cell antigen receptors (TCRs) are mechanosensors, which initiate a signaling cascade upon ligand recognition resulting in T-cell differentiation, homeostasis, effector and regulatory functions. An optical trap combined with fluorescence permits direct monitoring of T-cell triggering in response to force application at various concentrations of peptide-bound major histocompatibility complex molecules (pMHC). The technique mimics physiological shear forces applied as cells crawl across antigen-presenting surfaces during immune surveillance. True single molecule studies performed on single cells profile force-bond lifetime, typically seen as a catch bond, and conformational change at the TCR-pMHC bond on the surface of the cell upon force loading. Together, activation and single molecule single cell studies provide chemical and physical triggering thresholds as well as insight into catch bond formation and quaternary structural changes of single TCRs. The present methods detail assay design, preparation, and execution, as well as data analysis. These methods may be applied to a wide range of pMHC-TCR interactions and have potential for adaptation to other receptor-ligand systems.

Single Molecule Force Spectroscopy Reveals Distinctions in Key Biophysical Parameters of αß T-Cell Receptors Compared with Chimeric Antigen Receptors Directed at the Same Ligand.

Chimeric antigen receptor (CAR) T-cell therapies exploit facile antibody-mediated targeting to elicit useful immune responses in patients. This work directly compares binding profiles of CAR and αß T-cell receptors (TCR) with single cell and single molecule optical trap measurements against a shared ligand. DNA-tethered measurements of peptide-major histocompatibility complex (pMHC) ligand interaction in both CAR and TCR exhibit catch bonds with specific peptide agonist peaking at 25 and 14 pN, respectively. While a conformational transition is regularly seen in TCR-pMHC systems, that of CAR-pMHC systems is dissimilar, being infrequent, of lower magnitude, and irreversible. Slip bonds are observed with CD19-specific CAR T-cells and with a monoclonal antibody mapping to the MHC α2 helix but indifferent to the bound peptide. Collectively, these findings suggest that the CAR-pMHC interface underpins the CAR catch bond response to pMHC ligands in contradistinction to slip bonds for CARs targeting canonical ligands.

Anomalous Dynamics in tert-Butyl Alcohol-Water and Trimethylamine N-Oxide-Water Binary Mixtures: A Femtosecond Transient Absorption Study.

In this article, we have investigated the unusual dynamics of tert-butyl alcohol (TBA)-water and trimethylamine N-oxide (TMAO)-water binary mixtures using solvation dynamics as a tool. For this purpose, femtosecond transient absorption spectroscopy has been employed. Although these two molecules are isosteres to each other, a significant difference in water dynamics has been observed. The solvation times in TBA-water binary mixtures are found to be between 1.5 and 15.5 ps. On the contrary, we have observed very fast dynamics in TMAO-water binary mixtures (between 210 and 600 fs). Interestingly, unusual retardation in dynamics is observed at 0.10 mole fraction of TBA and TMAO in both the binary mixtures.

Investigation of Fibril Forming Mechanisms of l-Phenylalanine and l-Tyrosine: Microscopic Insight toward Phenylketonuria and Tyrosinemia Type II.

Phenylketonuria and tyrosinemia type II, the two metabolic disorders, are originated due to the complications in metabolism of phenylalanine (Phe) and tyrosine (Tyr), respectively. Several neurological injuries, involving microcephaly, mental retardation, epilepsy, motor disease, and skin problems etc., are the symptoms of these two diseases. It has been reported that toxic amyloid fibrils are formed at high concentrations of Phe and Tyr. Our study indicates that the fibril forming mechanisms of Phe and Tyr are completely different. In the case of Phe, -NH3+ and -COO- groups of neighboring molecules interact via hydrogen bonding and polar interactions. On the other hand, there is no role of - NH3+ group in the fibril forming mechanism of Tyr. In Tyr fibril, the two hydrogen bonding partners are -OH and -COO- groups. In addition, we have also investigated the effect of three lanthanide cations on the fibrillar assemblies of Phe. It has been observed that the efficiencies of three lanthanides to inhibit the fibrillar assemblies of Phe follow the order Tb3+< Sm3+< Eu3+.

Modulation of the Excited-State Dynamics of 2,2'-Bipyridine-3,3'-diol in Crown Ethers: A Possible Way To Control the Morphology of a Glycine Fibril through Fluorescence Lifetime Imaging Microscopy.

In this article, we have investigated the modulation of excited-state intramolecular double proton transfer (ESIDPT) dynamics of 2,2'-bipyridine-3,3'-diol (BP(OH)2) in two crown ethers (CEs), namely, 18-Crown-6 (18C6) and 15-Crown-5 (15C5). From steady-state UV-visible measurements, we have shown that there is no significant interaction between the dienol tautomeric form of BP(OH)2 and two CEs. However, in the presence of CEs, an additional emission band (∼415 nm) is generated along with the diketo tautomer band (∼465 nm). In time-resolved analysis, we have observed the generation of ∼260 ps rise component in the presence of 18C6. Therefore, by combining the results of steady-state and time-resolved emissions, we have proposed that the water-assisted ESIDPT route of BP(OH)2 generates a hydronium ion (H3O+) in the excited state. 18C6 binds nicely to this H3O+ ion. As a result, retarded ESIDPT dynamics is observed in 18C6. However, as 15C5 cannot bind H3O+ properly, no rise component is found. With the addition of potassium chloride (KCl), the contribution of the rise component decreases due to unavailability of free 18C6 cavity to capture the H3O+ ion generated in the excited state. Addition of calcium chloride (CaCl2) leads to complete removal of the rise component due to the inhibition of the water-assisted ESIDPT route. From wavelength-dependent behavior, we have observed that the rise component is present only at 465 nm in 18C6. We have also shown that the fibrillar morphology of glycine can be successfully probed through fluorescence lifetime imaging microscopy using BP(OH)2 as an imaging agent. Modulation of fibrillar morphology has been found in the presence of two CEs. The interaction of glycine fiber with CEs can be explained by lifetime distribution analysis.

Comparative Fluorescence Resonance Energy-Transfer Study in Pluronic Triblock Copolymer Micelle and Niosome Composed of Biological Component Cholesterol: An Investigation of Effect of Cholesterol and Sucrose on the FRET Parameters.

The formation of pluronic triblock copolymer (F127)-cholesterol-based niosome and its interaction with sugar (sucrose) molecules have been investigated. The morphology of F127-cholesterol -based niosome in the presence of sucrose has been successfully demonstrated using dynamic light scattering (DLS) and transmission electron microscopic (TEM) techniques. The DLS profiles and TEM images clearly suggest that the size of the niosome aggregates increases significantly in the presence of sucrose. In addition to structural characterization, a detailed comparative fluorescence resonance energy transfer (FRET) study has been carried out in these F127-containing aggregates, involving coumarin 153 (C153) as donor (D) and rhodamine 6G (R6G) as an acceptor (A) to monitor the dynamic heterogeneity of the systems. Besides, time-resolved anisotropy and fluorescence correlation spectroscopy measurements have been carried out to monitor the rotational and lateral diffusion motion in these F127-cholesterol-based aggregates using C153 and R6G, respectively. During the course of FRET study, we have observed multiple time constants of FRET inside the F127-cholesterol-based niosomes in contrast with the F127 micelle. This corresponds to the presence of more than one preferential donor-acceptor (D-A) distance in niosomes than in F127 micelle. FRET has also been successfully used to probe the effect of sucrose on the morphology of F127-cholesterol-based niosome. In the presence of sucrose, the time constant of FRET further increases as the D-A distances increase in sucrose-decorated niosome. Finally, the excitation-wavelength-dependent FRET studies have indicated that as the excitation of donor molecules varies from 408 to 440 nm the contribution of the faster rise component of the acceptor enhances considerably, which clearly establishes the dynamics heterogeneity of both systems. Our findings also indicate that FRET is completely intravesicular in nature in these block copolymer-cholesterol-based aggregates.

Unveiling the Mode of Interaction of Berberine Alkaloid in Different Supramolecular Confined Environments: Interplay of Surface Charge between Nano-Confined Charged Layer and DNA.

In this Article, we demonstrate a detailed characterization of binding interaction of berberine chloride (BBCl) with calf-thymus DNA (CT-DNA) in buffer solution as well as in two differently charged reverse micelles (RMs). The photophyscial properties of this alkaloid have been modulated within these microheterogeneous bioassemblies. The mode of binding of this alkaloid with DNA is of debate to date. However, fluorescence spectroscopic measurements, circular dichroism (CD) measurement, and temperature-dependent study unambiguously establish that BBCl partially intercalates into the DNA base pairs. The nonplanarity imposed by partial saturation in their structure causes the nonclassical types of intercalation into DNA. Besides the intercalation, electrostatic interactions also play a significant role in the binding between BBCl and DNA. DNA structure turns into a condensed form after encapsulation into RMs, which is followed by the CD spectra and microscopy study. The probe location and dynamics in the nanopool of the RMs depended on the electrostatic interaction between the charged surfactants and cationic berberine. The structural alteration of CT-DNA from B form to condensed form and the interplay of surface charge between RMs and DNA determine the interaction between the alkaloid and DNA in RMs. Time-resolved study and fluorescence anisotropy measurements successfully provide the binding interaction of BBCl in the nanopool of the RMs in the absence and in the presence of DNA. This study motivates us to judge further the potential applicability of this alkaloid in other biological systems or other biomimicking organized assemblies.