Ranajay Saha

Microstructure, Morphology, and Ultrafast Dynamics of a Novel Edible Microemulsion

An edible microemulsion (ME) composed of Tween 80/butyl lactate/isopropyl myristate (IPM)/water has been formulated. Pseudoternary phase diagram of the system contains a large single isotropic region. The phase behavior of the system is also studied at low pH (2.6) and in 0.9% NaCl solution. Conductivity, viscosity, ultrasonic velocity, and compressibility studies find consistent results in the structural transition (from water-in-oil (w/o) to bicontinuous, and from bicontinuous to oil-in-water (o/w)) behavior of the ME. Dynamic light scattering studies reveal the size of the MEs. The absorption and steady state emission spectra of 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl)-4H-pyran (DCM) successfully probe the polarity of the ME at its solvation shell and shows the efficacy of hosting model drug molecules. The rotational anisotropy of the dye has been studied to ascertain the geometrical restriction of the probe molecule. Picosecond-resolved fluorescence spectroscopy applies well to study the relaxation dynamics of water in the solvation shell of the MEs. The study finds strong correlation in the relaxation dynamics of water with the structure of host assembly and offers an edible ME system which could act as a potential drug delivery system and nontoxic nanotemplate for other applications.

Role of solvation dynamics in excited state proton transfer of 1-naphthol in nanoscopic water clusters formed in a hydrophobic solvent.

Excited state proton transfer (ESPT) in biologically relevant organic molecules in aqueous environments following photoexcitation is very crucial as the reorganization of polar solvents (solvation) in the locally excited (LE) state of the organic molecule plays an important role in the overall rate of the ESPT process. A clear evolution of the two photoinduced dynamics in a model ESPT probe 1‐naphthol (NpOH) upon ultrafast photoexcitation is the motive of the present study. Herein, the detailed kinetics of the ESPT reaction of NpOH in water clusters formed in hydrophobic solvent are investigated. Distinct values of time constants associated with proton transfer and solvent relaxation have been achieved through picosecond‐resolved fluorescence measurements. We have also used a model solvation probe Coumarin 500 (C500) to investigate the dynamics of solvation in the same environmental condition. The temperature dependent picosecond‐resolved measurement of ESPT of NpOH and the dynamics of solvation from C500 identify the magnitude of intermolecular hydrogen bonding energy in the water cluster associated with the ultrafast ESPT process.

Slow water dynamics at the surface of macromolecular assemblies of different morphologies

In this contribution we, for the first time, explore the slow dynamical states of confined water molecules in lamellar structures of AOT with various degrees of hydration using a picosecond resolved fluorescence spectroscopic technique using coumarin-500 as the fluorophore. A comparison of slow dynamics between AOT lamellar structures and AOT RMs have been made by preparing RMs that have a diameter the same as the interplanar water layer thickness of lamellar structures and the same number of water molecules in lamellar structures in order to understand the effect of morphology and hydration on the relaxation dynamics of water molecules in these nanoconfining systems. The relaxation time scales obtained in the lamellar systems differ to those of the RM systems and the difference of the timescales has been explained as a interplay between two opposing factors arising out of the morphology and interlayer distance, respectively. The geometrical restriction of the probe at the lamellar interface is determined by measuring time-resolved rotational anisotropy. The hydrogen bond energy of the water molecules residing at the lamellar interface is measured applying the Arrhenius type barrier crossing model.

Light driven ultrafast electron transfer in oxidative redding of Green Fluorescent Proteins

Fluorescent proteins undergoing green to red (G/R) photoconversion have proved to be potential tools for investigating dynamic processes in living cells and for photo-localization nanoscopy. However, the photochemical reaction during light induced G/R photoconversion of fluorescent proteins remains unclear. Here we report the direct observation of ultrafast time-resolved electron transfer (ET) during the photoexcitation of the fluorescent proteins EGFP and mEos2 in presence of electron acceptor, p-benzoquinone (BQ). Our results show that in the excited state, the neutral EGFP chromophore accepts electrons from an anionic electron donor, Glu222, and G/R photoconversion is facilitated by ET to nearby electron acceptors. By contrast, mEos2 fails to produce the red emitting state in the presence of BQ; ET depletes the excited state configuration en route to the red-emitting fluorophore. These results show that ultrafast ET plays a pivotal role in multiple photoconversion mechanisms and provide a method to modulate the G/R photoconversion process.

Protein-cofactor binding and ultrafast electron transfer in riboflavin binding protein under the spatial confinement of nanoscopic reverse micelles.

In this contribution, we study the effect of confinement on the ultrafast electron transfer (ET) dynamics of riboflavin binding protein (RBP) to the bound cofactor riboflavin (Rf, vitamin B2), an important metabolic process, in anionic sodium bis(2‐ethylhexyl) sulfosuccinate reverse micelles (AOT‐RMs) of various hydration levels. Notably, in addition to excluded volume effect, various nonspecific interactions like ionic charge of the confining surface can influence the biochemical reactions in the confined environment of the cell. To this end, we have also studied the ET dynamics of RBP–Rf complex under the confinement of a cationic hexadecyltrimethylammonium bromide (CTAB) RMs with similar water pool size to the anionic AOT‐RMs towards simulating equal restricted volume effect. It has been found that the spatial confinement of RBP in the AOT‐RM of w0 = 10 leads to the loss of its tertiary structure and hence vitamin binding capacity. Although, RBP regains its binding capacity and tertiary structure in AOT‐RMs of w0 ≥20 due to its complete hydration, the ultrafast ET from RBP to Rf merely occurs in such systems. However, to our surprise, the ET process is found to occur in cationic CTAB‐RMs of similar volume restriction. It is found that under the spatial confinement of anionic AOT‐RM, the isoalloxazine ring of Rf is improperly placed in the protein nanospace so that ET between RBP and Rf is not permitted. This anomaly in the binding behaviour of Rf to RBP in AOT‐RMs is believed to be the influence of repulsive potential of the anionic AOT‐RM surface to the protein. Our finding thus suggests that under similar size restriction, both the hydration and surface charge of the confining volume could have major implication in the intraprotein ET dynamics in real cellular environments. Copyright

Modulation of Environmental Dynamics at the Active Site and Activity of an Enzyme under Nanoscopic Confinement: Subtilisin Carlsberg in Anionic AOT Reverse Micelle

Hydration dynamics plays a crucial role in determining the structure, function, dynamics, and stability of an enzyme. These dynamics involve the trapped-water motions within small distance along with the total protein dynamics. However, the exact molecular basis for the induction of enzyme function by water dynamics is still remain unclear. Here, we have studied both enzymatic activity and environmental dynamics at the active site of an enzyme, Subtilisin Carlsberg (SC), under confined environment of the reverse micelle (RM) retaining the structural integrity of the protein. Kinetic measurements show that enzymatic activity increases with increasing the water content of the RM. The picosecond-resolved fluorescence Stokes shift studies indicate faster hydration dynamics at the active site of the enzyme with increasing the water content in the RM (w0 values). Temperature-dependent hydration dynamics studies demonstrate the increased flexibility of the protein at higher temperature under confinement. From temperature-dependent solvation dynamics study, we have also calculated the activation energy that has to be overcome for full orientational freedom to the water molecules from bound to free-state. The results presented here establish a correlation between the enzymatic activity and dynamics of hydration of the encapsulated protein SC in cell-like confined environment within the structural integrity of the enzyme.

Effect of Hydrophobic Interaction on Structure, Dynamics, and Reactivity of Water

The effect of hydrophobic interaction on water is still controversial and requires more detailed experimental and theoretical investigation. The interaction between organic-water molecular complexes might be indicative of the perturbation of hydrogen-bond network in the tetrahedral structure of bulk waters, due to hydrophobic effect. In this contribution, femto/picosecond-resolved solvation dynamics techniques have been adopted to explore the dynamical modification of water clusters in hydrophobic solvent methyl tert-butyl ether (MTBE). The dynamical evolution of water molecules at the surface of micelle-like MTBE has also been studied. Dynamic light scattering techniques have been employed to determine the size of the molecular clusters being formed in respective solvents. Fourier transform infrared (FTIR) spectroscopy well measures the changes in O-H vibration frequency of water induced by MTBE. We have also monitored temperature dependent picosecond-resolved solvation dynamics in order to explore the energetics associated with water solvation in bulk MTBE. Using detailed ab initio calculations at the MP2 level, our study attempts to predict the possible structures, energies, and thermochemical parameters of corresponding MTBE-water molecular complexes in more detail. The chemical reactivity of water further confirms the effect of the hydrophobic interaction on water molecules. The results impart an understanding on hydrophobic interaction imposed by a biomolecule on the structure and reactivity of water, significant for the in vivo cellular condition.

Molecular recognition of a model globular protein apomyoglobin by synthetic receptor cyclodextrin: effect of fluorescence modification of the protein and cavity size of the receptor in the interaction

Labelling of proteins with some extrinsic probe is unavoidable in molecular biology research. Particularly, spectroscopic studies in the optical region require fluorescence modification of native proteins by attaching polycyclic aromatic fluoroprobe with the proteins under investigation. Our present study aims to address the consequence of the attachment of a fluoroprobe at the protein surface in the molecular recognition of the protein by selectively small model receptor. A spectroscopic study involving apomyoglobin (Apo‐Mb) and cyclodextrin (CyD) of various cavity sizes as model globular protein and synthetic receptors, respectively, using steady‐state and picosecond‐resolved techniques, is detailed here. A study involving Förster resonance energy transfer, between intrinsic amino acid tryptophan (donor) and N, N‐dimethyl naphthalene moiety of the extrinsic dansyl probes at the surface of Apo‐Mb, precisely monitor changes in donor acceptor distance as a consequence of interaction of the protein with CyD having different cavity sizes (β and γ variety). Molecular modelling studies on the interaction of tryptophan and dansyl probe with β‐CyD is reported here and found to be consistent with the experimental observations. In order to investigate structural aspects of the interacting protein, we have used circular dichroism spectroscopy. Temperature‐dependent circular dichroism studies explore the change in the secondary structure of Apo‐Mb in association with CyD, before and after fluorescence modification of the protein. Overall, the study well exemplifies approaches to protein recognition by CyD as a synthetic receptor and offers a cautionary note on the use of hydrophobic fluorescent labels for proteins in biochemical studies involving recognition of molecules. Copyright

Ultrafast electron transfer in riboflavin binding protein in macromolecular crowding of nano-sized micelle

In this contribution, we have studied the dynamics of electron transfer (ET) of a flavoprotein to the bound cofactor, an important metabolic process, in a model molecular/macromolecular crowding environments. Vitamin B(2) (riboflavin, Rf) and riboflavin binding protein (RBP) are used as model cofactor and flavoprotein, respectively. An anionic surfactant sodium dodecyl sulfate (SDS) is considered to be model crowding agent. A systematic study on the ET dynamics in various SDS concentration, ranging from below critical micellar concentration (CMC), where the surfactants remain as monomeric form to above CMC, where the surfactants self-assemble to form nanoscopic micelle, explores the dynamics of ET in the model molecular and macromolecular crowding environments. With energy selective excitation in picosecond-resolved studies, we have followed temporal quenching of the tryptophan residue of the protein and Rf in the RBP-Rf complex in various degrees of molecular/macromolecular crowding. The structural integrity of the protein (secondary and tertiary structures) and the vitamin binding capacity of RBP have been investigated using various techniques including UV-Vis, circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) studies in the crowding environments. Our finding suggests that the effect of molecular/macromolecular crowding could have major implication in the intra-protein ET dynamics in cellular environments.

Nanostructure, solvation dynamics, and nanotemplating of plasmonically active SERS substrate in reverse vesicles

Reverse vesicles (RVs) are the organic counterparts to vesicles and are spherical containers in oils consisting of an oily core surrounded by reverse bilayers with water layers present in between. We present here a facile route for forming stable RV from nontoxic surfactants and oil components. The RV formation is characterized by dynamic light scattering and further confirmed by transmission electron microscopic (TEM) techniques. The water channels present in between the bilayers are found to be a potential template for inorganic nanoparticles’ (NPs) synthesis. Both the UV–Vis absorption spectroscopy and the TEM study reveal successful formation of highly clustered silver NPs within the water layers of the RVs. X-ray powder diffraction analyzes the crystalline nature of the NPs. FTIR spectroscopy shows the signature of different kinds of water molecules in between the RV bilayers. The dynamical description of the templating water, dictating the controlled formation of the NPs in the RV, is well revealed in the picosecond-resolved solvation dynamics study of a hydrophilic fluorescence probe 2′-(4-hydroxyphenyl)-5-[5-(4-methylpiperazine-1-yl)-benzimidazo-2-yl-benzimidazole] (H258). The rotational anisotropy study successfully describes geometrical restriction of the probe molecule in the RV. Notably, this study provides the first proof-of-concept data for the ability of the RV to be a template of synthesizing metal NPs. The as-prepared NP clusters are evaluated to be potential surface-enhanced Raman scattering substrate in solution using crystal violet as a model analyte. The present study offers a new RV, which is a prospective nontoxic nanotemplate and is believed to contribute potentially in the emerging NP-vesicle hybrid assembly-based plasmonic applications.Graphical abstractNanotemplating of metal clusters for the efficient SERS detection in liquid phase is reported in a new nontoxic reverse vesicle.