Nikhil Guchhait

Exploring Hydrophobic Subdomain IIA of the Protein Bovine Serum Albumin in the Native, Intermediate, Unfolded, and Refolded States by a Small Fluorescence Molecular Reporter

A simple intramolecular charge transfer (ICT) compound, 5-(4-dimethylamino-phenyl)-penta-2,4-dienoic acid methyl ester (DPDAME), has been documented to be a potential molecular reporter for probing microheterogeneous environments of a model transport protein bovine serum albumin (BSA) using spectroscopic techniques. Meteoric modifications to the emission profile of DPDAME upon addition of BSA come out to be a result of its binding to hydrophobic subdomain IIA. The highly polarity-sensitive ICT emission of DPDAME is found to be a proficient extrinsic molecular reporter for efficient mapping of native, intermediate, unfolded, and refolded states of the protein. Experimental data coupled with a reinforcing support from theoretical simulation using CHARMM22 software confirm the binding site of the probe to be the subdomain IIA of BSA, while FRET study reveals a remarkably close approach of our extrinsic molecular reporter to Trp-212 (in domain IIA): the distance between DPDAME and Trp-212 is 1.437 nm. The caliber of DPDAME as an external fluorescence marker also extends to the depiction of protein-surfactant (BSA-SDS) interaction to commendable fruition. Additionally, the protective action of small amounts of SDS on urea-denatured protein is documented by polarity-sensitive ICT emission of the probe. The present study also reflects the enhancement of the stability of BSA with respect to chemically induced denaturation by urea as a result of binding to the probe DPDAME.

Exploring the Strength, Mode, Dynamics, and Kinetics of Binding Interaction of a Cationic Biological Photosensitizer with DNA: Implication on Dissociation of the Drug–DNA Complex via Detergent Sequestration

The present study aims at exploring a detailed characterization of the binding interaction of a promising cancer cell photosensitizer, harmane (HM), with DNA extracted from herring sperm. The polarity-sensitive prototropic transformation of HM, a naturally occurring, fluorescent, drug-binding alkaloid, β-carboline, is remarkably modified upon interaction with DNA and is manifested through significant modulations on the absorption and emission profiles of HM. From the series of studies undertaken in the present program, for example, absorption; steady-state emission; the effect of chaotrope (urea); iodide ion-induced steady-state fluorescence quenching; circular dichroism (CD); and helix melting from absorption spectroscopy; the mode of binding of HM into the DNA helix has been substantiated to be principally intercalative. Concomitantly, a discernible dependence of the photophysics of the DNA-bound drug on the medium ionic strength indicates that electrostatic attraction should not be ignored in the interaction. Efforts have also been delivered to delineate the dynamical aspects of the interaction, such as modulation in time-resolved fluorescence decay and rotational relaxation dynamics of the drug within the DNA environment. In view of the prospective biological applications of HM, the issue of facile dissociation of intercalated HM from the DNA helix also comprises a crucial prerequisite for the functioning as an effective therapeutic agent. In this context, our results imply that the concept of detergent-sequestered dissociation of the drug from the drug-DNA complex can be a prospective strategy through an appropriate choice of the detergent molecule. The utility of the present work resides in exploring the potential applicability of the fluorescence property of HM for studying its interactions with a relevant biological target, for example, DNA. In addition, the methods and techniques used in the present work can also be exploited to study the interaction of HM with other biological, biomimicking assemblies and drug delivery vehicles, and so forth.

Modulation of Excited-State Intramolecular Proton Transfer Reaction of 1-Hydroxy-2-naphthaldehyde in Different Supramolecular Assemblies

The excited-state intramolecular proton transfer (ESIPT) reaction of 1-hydroxy-2-naphthaldehyde (HN12) has been studied within the interior of the supramolecular assemblies of alpha-, beta-, and gamma-cyclodextrins (CD) and biomimicking environments of ionic (SDS) and non-ionic (TW-20) micelles. Fluorescence measurements are used to investigate the effect of various supramolecular assemblies on the ESIPT reaction by monitoring the large Stokes-shifted tautomer emission of HN12. Enhanced tautomer emission in the microencapsulated state predicts favorable ESIPT reaction in the supramoleuclar assemblies. Benesi-Hildebrand plots have been employed to ascertain that the stoichiometric ratios of the complexes formed between HN12 and CDs are 1:2, 1:1, and 1:1 for alpha-, beta-, and gamma-CD, respectively. The binding constants (K(1)) and free-energy change (DeltaG) for inclusion complexation are also determined from the linearized Benesi-Hildebrand plots. Steady-state fluorescence anisotropy, REES, excitation anisotropy, and fluorescence lifetime measurements are in line with other experimental findings. Differential action of urea on SDS and TW-20-bound probe has also been investigated.

A DFT-Based Theoretical Study on the Photophysics of 4-Hydroxyacridine: Single-Water-Mediated Excited State Proton Transfer

Study of intra- and intermolecular hydrogen-bonding interaction and excited state proton transfer reaction has been carried out in 4-hydroxyacridine (4-HA) and its hydrated clusters theoretically. Density functional theory [B3LYP/6-311++G(d,p)] has been exploited to calculate structural parameters and relative energies of different conformers of 4-HA and its hydrates. The substantial impact of solvent reaction field on hydrogen-bond energies, conformational equilibrium, and tautomerization reaction in aqueous medium have been realized by employing Onsager and PCM reaction field methods, and the stability of the conformers of 4-HA is found to be profusely modulated by the electrostatic influence of the solvent. A deeper insight into the nature of H-bonding in 4-HA and its hydrated clusters has been achieved under the provision of natural bond orbital and atoms in molecule analysis. Elucidation of potential energy curves for proton transfer reaction reveals that an intrinsic and two-water-molecule-assisted proton transfer (PT) reaction in 4-HA is hindered by high energy barrier in the S(1) surface, whereas single-water-assisted PT reaction is practically rendered barrierless. At the same time, the appreciably high barrier height of the ground state potential energy curve in all the cases unambiguously rules out the possibility of ground state proton transfer reaction.

Modulation of prototropic activity and rotational relaxation dynamics of a cationic biological photosensitizer within the motionally constrained bio-environment of a protein.

The present work describes the interaction of a promising cancer cell photosensitizer, harmane (HM), with a model transport protein, Bovine Serum Albumin (BSA). The studied molecule of interest (HM) belongs to the family of naturally occurring fluorescent drug-binding alkaloids, the β-carbolines. A combined use of steady-state and time-resolved fluorescence techniques is applied to follow and characterize the binding interaction. The polarity-dependent prototropic activity of HM is found to be responsible for the commendable sensitivity of the probe to the protein environments and is distinctly reflected on the emission profile. Steady-state fluorescence anisotropy study reveals the impartation of a considerable degree of motional restriction on the drug molecule as a result of binding to the protein. Contrary to the single-exponential nature of fluorescence anisotropy decay of HM in aqueous buffer, they are found to be biexponential in the protein environment. The rotational relaxation dynamics of HM within the protein has been interpreted on the lexicon of the Two-Step and Wobbling-in-Cone model. The probable binding location for the cationic drug is found to be the hydrophilic binding zone of BSA, i.e., domain I (characterized by a net negative charge). The AutoDock-based blind docking simulation has been explored for evaluating an unbiased result of the probable interaction site of HM in the protein. To unfold the effect of binding of the drug on the secondary structural content of the protein, circular dichroism (CD) spectroscopy has been exploited to see that binding of the drug accompanies some decrease in α-helical content of BSA, and the effect gradually saturates toward a higher drug/protein molar ratio.

Modulated Photophysics of an ESIPT Probe 1-Hydroxy-2-naphthaldehyde within Motionally Restricted Environments of Liposome Membranes Having Varying Surface Charges

The present work demonstrates the modulation of excited state intramolecular proton transfer (ESIPT) emission of 1-hydroxy-2-naphthaldehyde (HN12) upon its interaction with the liposomal vesicles/bilayer of dimyristoyl-l-α-phosphatidylcholine (DMPC) and dimyristoyl-l-α-phosphatidylglycerol (DMPG) and its subsequent implementation as an efficient molecular reporter for probing of microheterogeneous environments of lipid-bilayer system. Modifications on the emission profile of HN12 in terms of remarkable emission intensity enhancement coupled with a hypsochromic shift induced by the presence of DMPC and DMPG membranes have been interpreted to be vivid manifestations of the interactions between the two partners. Steady-state anisotropy, red-edge excitation shift (REES), and time-resolved fluorescence measurements have been fruitfully exploited to complement other experimental findings. Probable binding site of HN12 in the lipid-bilayers has been assessed on the basis of intertwining the results of fluorescence quenching with other experimental results and is further substantiated from docking studies.

Spectroscopic probe analysis for exploring probe–protein interaction: A mapping of native, unfolding and refolding of protein bovine serum albumin by extrinsic fluorescence probe

Steady state and dynamic fluorescence measurements have been used to investigate interaction between Bovine Serum Albumin (BSA) and fluorescence probe para-N,N-dimethylamino orthohydroxy benzaldehyde (PDOHBA), a structurally important molecule exhibiting excited state coupled proton transfer (PT) and charge transfer (CT) reaction. Fluorescence anisotropy, acrylamide quenching, and time resolved fluorescence measurements corroborate the binding nature of the probe with protein. The binding constant between BSA and PDOHBA has been determined by using Benesi-Hildebrand and Stern-Volmer equations. The negative value of ΔG indicates the spontaneity of this probe-protein complexation process. Observations from synchronous, three dimensional fluorescence spectra and circular dichroism spectra point toward the fact that the hydrophobicity as well as α-helix content of BSA are altered in presence of probe PDOHBA. The PT band of PDOHBA is found to be an excellent reporter for the mapping of destructive and protective behavior of SDS with variation of chaotrope concentration.

Rhodamine-based molecular clips for highly selective recognition of Al3+ ions: synthesis, crystal structure and spectroscopic properties

A novel fluorescent chemosensor based on a rhodamine derivative (L) was designed, synthesized, and used as a selective Al+3 ion sensor. Upon addition of Al3+ to an aqueous-acetonitrile solution of L, the development of a strong fluorescence signal by a chelation-enhanced fluorescence (CHEF) process was observed with an attractive glowing orange emission. This sensor shows high selectivity towards Al3+ ions in the presence of other competing metal ions. The fluorescence quantum yield of L–Al3+ (Φf = 0.30) was found to be very high compared to the bare ligand. The limit of detection (LOD) of Al3+ ions was calculated to be 2 × 10−8 M by fluorescence titration. The 1 : 1 binding stoichiometry of the metal complex was established by combined UV-vis, fluorescence and TOF-MS spectroscopy.

Domain Specific Association of Small Fluorescent Probe trans-3-(4-Monomethylaminophenyl)-Acrylonitrile (MMAPA) with Bovine Serum Albumin (BSA) and Its Dissociation from Protein Binding Sites by Ag Nanoparticles: Spectroscopic and Molecular Docking Study

Photoinduced intramolecular charge transfer produced a polar excited state in trans-3-(4-monomethylaminophenyl)acrylonitrile (MMAPA), rendering the resulting emission sensitive to the medium polarity. Strong binding interaction of silver nanoparticles with the probe was observed, causing fluorescence quenching through the static quenching process. The probe MMAPA was found to bind to the less polar hydrophobic, restricted proteinous environment of bovine serum albumin (BSA) resulting in the blue shift of the emission maximum with an increase in emission intensity and fluorescence anisotropy. Studies using site markers of flufenamic acid and phenylbutazone coupled with molecular docking results predicted that the binding site of the probe is in between subdomains IIIA and IB of BSA and is different from the conventional Sudlow sites. The denaturation of the probe-bound BSA by urea or heat released the probe from this proteinous environment to water marked by exactly reverse spectral changes. On the interaction of silver nanoparticles with the probe bound protein, the probe was observed to move from its binding site in the protein to the Ag(0) nanoparticle surface involving conformational changes of the protein near the probe binding site.

Inequivalence of substitution pairs in hydroxynaphthaldehyde: A theoretical measurement by intramolecular hydrogen bond strength, aromaticity, and excited-state intramolecular proton transfer reaction

The inequivalence of substitution pair positions of naphthalene ring has been investigated by a theoretical measurement of hydrogen bond strength, aromaticity, and excited state intramolecular proton transfer (ESIPT) reaction as the tools in three substituted naphthalene compounds viz 1‐hydroxy‐2‐naphthaldehyde (HN12), 2‐hydroxy‐1‐naphthaldehyde (HN21), and 2‐hydroxy‐3‐naphthaldehyde (HN23). The difference in intramolecular hydrogen bond (IMHB) strength clearly reflects the inequivalence of substitution pairs where the calculated IMHB strength is found to be greater for HN12 and HN21 than HN23. The H‐bonding interactions have been explored by calculation of electron density ρ(r) and Laplacian ∇2ρ(r) at the bond critical point using atoms in molecule method and by calculation of interaction between σ* of OH with lone pair of carbonyl oxygen atom using NBO analysis. The ground and excited state potential energy surfaces (PESs) for the proton transfer reaction at HF (6‐31G**) and DFT (B3LYP/6‐31G**) levels are similar for HN12, HN21 and different for HN23. The computed aromaticity of the two rings of naphthalene moiety at B3LYP/6‐31G** method also predicts similarity between HN12 and HN21, but different for HN23.