Rajib Kumar Mitra

Temperature-Dependent Simultaneous Ligand Binding in Human Serum Albumin

Human serum albumin (HSA) is a soluble protein in our circulatory system, which is known to bind a variety of drugs and ligands. Since Sudlows pioneering works on the ligand-binding sites, a major effort of the biophysical/biochemical research has been directed to characterize the structural, functional, and dynamical properties of this protein. Structural studies on HSA have revealed distinct temperature-induced folded states. Despite knowing about the ligand-binding properties and residues important for the binding, less is understood about the temperature-dependent molecular recognition of the protein. Here, we have prepared thermally induced unfolded states of the protein and characterized those by circular dichroism (CD) and differential thermal analysis (DTA) techniques. The change in the globular structure of the protein as a consequence of thermal unfolding has also been characterized by dynamic light scattering (DLS) measurements. We have used two fluorescent ligands (4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl) 4H-pyran) (DCM; hydrophobic; neutral) and Nile blue (NB; cationic) of different natures to characterize the ligand-binding properties of the protein in the native and thermally unfolded states. The possible binding sites of the ligands have been characterized by competitive binding with other drug molecules having definite binding sites in HSA. Picosecond-resolved Förster resonance energy transfer (FRET) studies along with steady-state and polarization-gated spectroscopies on the ligands in the protein reveal the dynamics of the binding sites at various temperatures. From the FRET studies, an attempt has been made to characterize the simultaneous binding of the two ligands in various temperature-dependent folded states of HSA.

Do hydration dynamics follow the structural perturbation during thermal denaturation of a protein: a terahertz absorption study.

We investigate the thermal denaturation of human serum albumin and the associated solvation using terahertz (THz) spectroscopy in aqueous buffer solution. Far- and near-ultraviolet circular dichroism spectroscopy reveal that the protein undergoes a native (N) to extended (E) state transition at temperature ≤55°C with a marginal change in the secondary and tertiary structure. At 70°C, the protein transforms into an unfolded (U) state with significant irreversible disruption of its structures. We measure the concentration- and temperature-dependent THz absorption coefficient (α) of the protein solution using a p-Ge THz difference spectrometer (2.1-2.8 THz frequency range), thereby probing the collective protein-water network dynamics. When the solvated protein is heated up to 55°C and cooled down again, a reversible change in THz absorption is observed. When increasing the temperature up to 70°C, we find a dramatic irreversible change of THz absorption. The increase in THz absorption compared to bulk water is attributed to a blue shift in the spectrum of the solvated protein compared to bulk water. This is supported by measurements of THz absorption coefficients using THz time-domain spectroscopy (0.1-1.2 THz frequency range). We also use picosecond-resolved fluorescence spectroscopy of the tryptophan 214 moiety of human serum albumin. All experimental observations can be explained by a change in the hydration dynamics of the solvated protein due to the additional exposure of hydrophobic residues upon unfolding.

Modulation of Dynamics and Reactivity of Water in Reverse Micelles of Mixed Surfactants

In this contribution, we attempt to correlate the change in water dynamics in a reverse micellar (RM) core caused by the modification of the interface by mixing an anionic surfactant, sodium bis(2-ethylhexyl) sulfosuccinate (AOT), and a nonionic surfactant, tetraethylene glycol monododecyl ether (Brij-30), at different proportions, and its consequent effect on the reactivity of water, measured by monitoring the solvolysis reaction of benzoyl chloride (BzCl). The dimension of the RM droplets at different mixing ratios of AOT and Brij-30 (X(Brij-30)) has been measured using dynamic light scattering (DLS) technique. The physical properties of the RM water have been determined using Fourier transform infrared spectroscopy (FTIR) and compressibility studies, which show that with increasing X(Brij-30), the water properties tend toward that of bulk-like water. The solvation dynamics, probed by coumarin 500 dye, gets faster with X(Brij-30). The rotational anisotropy studies along with a wobbling-in-cone analysis show that the probe experiences less restriction at higher X(Brij-30). The kinetics of the water-mediated solvolysis also gets faster with X(Brij-30). The increased rate of solvolysis has been correlated with the accelerated solvation dynamics, which is another consequence of surfactant headgroup-water interaction.

Interactions of Nile blue with micelles, reverse micelles and a genomic DNA.

In this contribution we report studies on the nature of binding of a small ligand/drug Nile blue (NB) with sodium dodecyl sulfate (SDS) micelles, bis-(2-ethylehexyl) sulfosuccinate (AOT)/isooctane reverse micelles (RM) and a genomic DNA extracted from Salmon sperm. With detailed steady state and picosecond resolved optical spectroscopic techniques, we examined the fluorescence quenching of the ligand upon complexation with the SDS monomers and DNA. Polarization analyzed picosecond-resolved fluorescence measurements reveal geometrical restriction on the probe in SDS micelles, AOT-RM and DNA. Steady state and time resolved studies on the probe in nanocages of AOT RM with various degrees of hydration (w0) reveal the existence of NB as two distinct species namely, neutral and cationic. This study confirms that the emission of NB in aqueous micelles and DNA solution is due to the cationic form of the drug. Our experiments clearly identified non-specific electrostatic and intercalative modes of interaction of the probe with the DNA at lower and higher DNA concentrations respectively. The nature of binding of NB in presence of the DNA and SDS micelles reveals that the binding affinity of the probe is higher with the micelles than with the DNA. The complex rigidity of NB with DNA and its fluorescence quenching with DNA elucidate a strong recognition mechanism between NB and DNA.

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 hydration on the functionality of a proteolytic enzyme α-chymotrypsin under crowded environment

Enzymes and other bio-macromolecules are not only sensitive to physical parameters such as pH, temperature and solute composition but also to water activity. A universally instructive way to vary water activity is the addition of osmotically active but otherwise inert solvents which also mimic the condition of an intercellular milieu. In the present contribution, the role of hydration on the functionality of a proteolytic enzyme α-chymotrypsin (CHT) is investigated by modulating the water activity with the addition of polyethylene glycols (PEG with an average molecular weight of 400). The addition of PEG increases the affinity of the enzyme to its substrate, however, followed by a decrease in the turnover number (k(cat)). Energetic calculations show that entrance path for the substrate is favoured, whereas the exit channel is restricted with increasing concentration of the crowding agent. This decrease is attributed to the thinning of the hydration shell of the enzyme due to the loss of critical water residues from the hydration surface of the enzyme as evidenced from volumetric and compressibility measurements. The overall secondary and tertiary structures of CHT determined from far-UV and near-UV circular dichroism (CD) measurements show no considerable change in the studied osmotic stress range. From kinetic and equilibrium data, we calculate 115 ± 30 numbers of water molecules to be altered during the enzymatic catalysis of CHT. Spectroscopic observation of water relaxation and rotational dynamics of ANS-CHT complex at various concentrations of the osmoting agent also support the dehydration of the hydration layer. Such dehydration/hydration processes during turnover imply a significant contribution of solvation to the energetics of the conformational changes.

The influence of charge on the structure and dynamics of water encapsulated in reverse micelles

Hydrogen-bonded structure and relaxation dynamics of water entrapped inside reverse micelles (RMs) composed of surfactants with different charged head groups: sodium bis(2-ethylhexyl) sulfosuccinate (AOT) (anionic), didodecyldimethylammonium bromide (DDAB) (cationic) and Igepal CO-520 (Igepal) (nonionic) in cyclohexane (Cy) have been studied as a function of hydration (defined by ). Sub-diffusive slow (sub-ns) relaxation dynamics of water has been measured by the time resolved fluorescence spectroscopy (TRFS) technique using two fluorophores, namely 8-anilino-1-naphthalenesulfonic acid (ANS) and coumarin-343 (C-343). The hydrogen bonded connectivity network of water confined in these RMs has been investigated by monitoring the hydrogen bond stretching and libration bands of water using far-infrared FTIR spectroscopy. In addition, the ultrafast collective relaxation dynamics of water inside these RMs has been determined by dielectric relaxation in the THz region (0.2-2.0 THz) using THz time domain spectroscopy (THz-TDS). While TRFS measurements establish the retardation of water dynamics for all the RM systems, FTIR and THz-TDS measurements provide with signature of charge specificity.

Polarizing effect of aligned nanoparticles in terahertz frequency region

We report the polarizing behavior of aligned Ni nanoparticles (NPs) having average diameter of 165±15 nm in ~210 μm thick polyvinyl alcohol (PVA) matrix in the frequency range of 0.2-1.6 THz. The NPs have been prepared via a wet chemical route and then aligned in PVA film by using an external magnetic field. When the polarization of THz electric field is parallel to the NPs alignment direction, a strong THz absorption is observed whereas a minimum THz absorption is noticed for the corresponding perpendicular configuration. Degree of polarization is calculated to be 0.9±0.08. Considering the good polarizing performance, ease of preparation, durability, and low maintenance, this aligned NP system is a perfect candidate to emerge as a potential THz polarizer.

Sequence Dependent Ultrafast Electron Transfer of Nile Blue in Oligonucleotides

In this contribution we report studies on the nature of binding of nile blue (NB), a well known DNA intercalating drug, with three synthetic DNA oligonucleotides, (CGCAAATTTGCG)2, (GCGCGCGCGCGC)2 and (ATATATATATAT)2. The nature of fluorescence quenching of the ligand upon complexation with the DNAs has been studied using steady state and picosecond-resolved optical spectroscopic techniques. The geometrical restriction on the probe in the DNA microenvironment is measured using picosecond-resolved rotational anisotropy measurements. Our experiments identify both non-specific electrostatic and intercalative modes of interaction of the probe with the DNAs at lower and higher DNA concentrations, respectively. This dual nature of binding is also confirmed through gel electrophoresis experiments. The nature of electron transfer (ET) reaction of GC base pairs with intercalated NB has also been explored. Competitive binding study reveals that binding affinity of the probe is higher with SDS micelles than with the DNAs within its structural integrity in presence of the micelles, as evidenced from circular dichroism (CD) measurements. The complex rigidity of NB with various DNAs and its fluorescence quenching with DNAs elucidate a strong recognition mechanism between NB and DNA.

Do the physical properties of water in mixed reverse micelles follow a synergistic effect: a spectroscopic investigation.

In this contribution we have tried to investigate whether the mechanical properties of the reverse micellar (RM) interface dictate the physical properties of entrapped water molecules in the RM waterpool. We choose AOT/Igepal-520/cyclohexane (Cy) mixed RM as a model system which exhibits synergistic water solubilization behavior as a function of interfacial stoichiometry. Such a phenomenon associates systematic modification of the interface curvature. Dynamic light scattering (DLS) studies reveal linear increase in the droplet size and aggregation number of the RMs with increasing XIgepal (mole fraction of Igepal in the surfactant mixture). FTIR study in the 3000-3800 cm(-1) region identifies that the relative population of the surface-bound water molecules is higher in AOT RM compared to that in Igepal RM, and in mixed systems it also follows a linear trend with XIgepal. Water relaxation dynamics as probed by time-resolved fluorescence spectroscopy using Coumarin-500 also reveals an overall linear trend with no characteristic feature around the solubilization inflation point. Our study clearly identifies that the physical properties of water in RM are mostly governed by the interfacial stoichiometry and water content, and merely bares any dependence on the mechanical properties of the interface.