Tanumoy Mondol

Picosecond-resolved solvent reorganization and energy transfer in biological and model cavities

Water molecules in hydrophobic biological cleft/cavities are of contemporary interest for the biomolecular structure and molecular recognition of hydrophobic ligands/drugs. Here, we have explored picosecond-resolved solvation dynamics of water molecules and associated polar amino acids in the hydrophobic cleft around Cys-34 position of Endogenous Serum Albumin (ESA). While site selective acrylodan labeling to Cys-34 allows us to probe solvation in the cleft, Förster resonance energy transfer (FRET) from intrinsic fluorescent amino acid Trp 214 to the extrinsic acrylodan probes structural integrity of the protein in our experimental condition. Temperature dependent solvation in the cleft clearly shows that the dynamics follows Arrhenius type behavior up to 60 °C, after which a major structural perturbation of the protein is evident. We have also monitored polarization gated dynamics of the acrylodan probe and FRET from Trp 214 to acrylodan at various temperatures. The dynamical behavior of the immediate environments around the probe acrylodan in the cleft has been compared with a model biomimetic cavity of a reverse micelle (w0 = 5). Using same fluorescent probe of acrylodan, we have checked the structural integrity of the model cavity at various temperatures using picosecond-resolved FRET from Trp to acrylodan in the cavity. We have also estimated possible distribution of donor-acceptor distances in the protein and reverse micelles. Our studies reveal that the energetics of the water molecules in the biological cleft is comparable to that in the model cavity indicating a transition from bound state to quasibound state, closely consistent with a recent MD simulation study.

Ultrafast dynamics of solvation and charge transfer in a DNA-based biomaterial.

Charge migration along DNA molecules is a key factor for DNA-based devices in optoelectronics and biotechnology. The association of a significant amount of water molecules in DNA-based materials for the intactness of the DNA structure and their dynamic role in the charge-transfer (CT) dynamics is less documented in contemporary literature. In the present study, we have used a genomic DNA-cetyltrimethyl ammonium chloride (CTMA) complex, a technological important biomaterial, and Hoechest 33258 (H258), a well-known DNA minor groove binder, as fluorogenic probe for the dynamic solvation studies. The CT dynamics of CdSe/ZnS quantum dots (QDs; 5.2 nm) embedded in the as-prepared and swollen biomaterial have also been studied and correlated with that of the timescale of solvation. We have extended our studies on the temperature-dependent CT dynamics of QDs in a nanoenvironment of an anionic, sodium bis(2-ethylhexyl)sulfosuccinate reverse micelle (AOT RMs), whereby the number of water molecules and their dynamics can be tuned in a controlled manner. A direct correlation of the dynamics of solvation and that of the CT in the nanoenvironments clearly suggests that the hydration barrier within the Arrhenius framework essentially dictates the charge-transfer dynamics.

Simultaneous binding of anti-tuberculosis and anti-thrombosis drugs to a human transporter protein: A FRET study

Although rifampicin (Rf) is one of the most effective antibiotics against infection caused by Mycobacterium tuberculosis, interaction of the drug with universal carrier protein in human blood plasma is not fully understood. Reduction of medicinal efficacy of other drugs, including anti-thrombosis drug warfarin (Wf), to the patients on Rf therapy also needs molecular understanding. In the present work we have studied interaction of Rf with one of the model carrier protein (human serum albumin). By using circular dichroism (CD) spectroscopy we have characterized the change in the secondary structure of the protein. The consequence of the simultaneous binding of the two drugs, Rf and Wf, on the structure of the protein has also been explored. Picosecond resolved Förster resonance energy transfer (FRET) from Wf to Rf explores possible binding sites of the anti-tuberculosis drug on the protein. In this report, we have discussed the potential problem of using the single tryptophan of the protein (Trp 214) as energy donor in FRET experiment for the characterization of the binding site of the drug Rf on the protein.

Recognition of different DNA sequences by a DNA-binding protein alters protein dynamics differentially

λ‐Repressor–operator sites interaction, particularly OR1 and OR2, is a key component of the λ‐genetic switch. FRET from the dansyl bound to the C‐terminal domain of the protein, to the intercalated EtBr in the operator DNA indicates that the structure of the protein is more compact in the OR2 complex than in the OR1 complex. Fluorescence anisotropy reveals enhanced flexibility of the C‐terminal domain of the repressor at fast timescales after complex formation with OR1. In contrast, OR2 bound repressor shows no significant enhancement of protein dynamics at these timescales. These differences are shown to be important for correct protein–protein interactions. Altered protein dynamics upon specific DNA sequence recognition may play important roles in assembly of regulatory proteins at the correct positions.

Ultrafast electron transfer in the recognition of different DNA sequences by a DNA-binding protein with different dynamical conformations

Ultrafast electron transfer (ET) phenomenon in protein and protein–DNA complex is very much crucial and often leads to the regulation of various kinds of redox reactions in biological system. Although, the conformation of the protein in protein–DNA complex is concluded to play the key role in the ET process, till date very little evidences exist in the literature. λ-repressor–operator DNA interaction, particularly OR1 and OR2, is a key component of the λ-genetic switch and is a model system for understanding the chemical principles of the conformation-dependent ET reaction, governed by differential protein dynamics upon binding with different DNA target sequences. Here, we have explored the photoinduced electron transfer from the tryptophan moieties of the protein λ-repressor to two operators DNA of different sequences (OR1 and OR2) using picosecond-resolved fluorescence spectroscopy. The enhanced flexibility and different conformation of the C-terminal domain of the repressor upon complexation with OR1 DNA compared to OR2 DNA are found to have pronounced effect on the rate of ET. We have also observed the ET phenomenon from a dansyl chromophore, bound to the lysine residue, distal from the DNA-binding domain of the protein to the operator DNA with a specific excitation at 299 nm wavelength. The altered ET dynamics as a consequence of differential protein conformation upon specific DNA sequence recognition may have tremendous biological implications.

Interaction of an Antituberculosis Drug with a Nanoscopic Macromolecular Assembly: Temperature-Dependent Förster Resonance Energy Transfer Studies on Rifampicin in an Anionic Sodium Dodecyl Sulfate Micelle

In this contribution, we report studies on the nature of binding of a potent antituberculosis drug, Rifampicin (RF) with a model drug delivery system, sodium dodecyl sulfate (SDS) micelle. Temperature dependent dynamic light scattering (DLS), conductometry, and circular dichroism (CD) spectroscopy have been employed to study the binding interaction of the drug with the micelle. The absorption spectrum of the drug RF in the visible region has been employed to study Förster resonance energy transfer (FRET) from another fluorescent drug Hoechst 33258 (H33258), bound to the micelle. Picosecond-resolved FRET studies at room temperature confirm the simultaneous binding of the two drugs to the micelle and the distance between the donor-acceptor pair is found to be 34 Å. The temperature dependent FRET study also confirms that the location and efficiency of drug binding to the micelle changes significantly at the elevated temperature. The energy transfer efficiency of the donor H33258, as measured from time-resolved studies, decreases significantly from 76% at 20 °C to 60% at 55 °C. This reveals detachment of some amount of the drug molecules from the micelles and increased donor-acceptor distance at elevated temperatures. The estimated donor-acceptor distance increases from a value of 33 Å at 20 °C to 37 Å at 55 °C. The picosecond resolved FRET studies on a synthesized DNA bound H33258 in RF solution have been performed to explore the interaction between the two. Our studies are expected to find relevance in the exploration of a potential vehicle for the vital drug rifampicin.

Ultrafast interfacial solvation dynamics in specific protein DNA recognition

An overwhelming number of structural and functional studies on specific protein-DNA complexes reveal the existence of water molecules at the interaction interface. What role does the interfacial water molecules play in determining the specificity of association is thus a critical question. Herein, we have explored the dynamical role of minor groove water molecules and DNA side chain flexibility in lambda repressor-operator DNA interaction using well-characterized DNA minor groove binder dye, Hoechst 33258. The most striking finding of our studies reveals that the solvation time scale corresponding to the minor groove water molecules (∼50 ps) and DNA side chain flexibility (∼10 ns) remain unaltered even in protein-DNA complex in comparison to unbound operator DNA. The temperature dependent study further reveals the slower exchange of minor grove water molecules with bulk water in DNA-protein complex in comparison to the unbound DNA. Detailed structural studies including circular dichroism (CD) and Förster resonance energy transfer (FRET) have also been performed to elucidate the interaction between protein and DNA.

Interaction of an Antituberculosis Drug with Nano-sized Cationic Micelle: Forster Resonance Energy Transfer from Dansyl to Rifampicin in the Microenvironment

In this contribution, we report studies on the interaction of an antituberculosis drug rifampicin (RF) in a macromolecular assembly of CTAB with an extrinsic fluorescent probe, dansyl chloride (DC). The absorption spectrum of the drug RF has been employed to study Förster resonance energy transfer (FRET) from DC, bound to the CTAB micelle using picosecond resolved fluorescence spectroscopy. We have applied a kinetic model developed by Tachiya to understand the kinetics of energy transfer and the distribution of acceptor (RF) molecules around the donor (DC) molecules in the micellar surface with increasing quencher concentration. The mean number of RF molecules associated with the micelle increases from 0.24 at 20 μm RF concentration to 1.5 at 190 μm RF concentration and consequently the quenching rate constant (kq) due to the acceptor (RF) molecules increases from 0.23 to 0.75 ns−1 at 20 and 190 μm RF concentration, respectively. However, the mean number of the quencher molecule and the quenching rate constant does not change significantly beyond a certain RF concentration (150 μm), which is consistent with the results obtained from time resolved FRET analysis. Moreover, we have explored the diffusion controlled FRET between DC and RF, using microfluidics setup, which reveals that the reaction pathway follows one‐step process.

Dynamical perspective of protein-DNA interaction.

Abstract The interactions between protein-DNA are essential for various biological activities. In this review, we provide an overview of protein-DNA interactions that emphasizes the importance of dynamical aspects. We divide protein-DNA interactions into two categories: nonspecific and specific and both the categories would be discussed highlighting some of our relevant work. In the case of nonspecific protein-DNA interaction, solvation studies (picosecond and femtosecond-resolved) explore the role environmental dynamics and change in the micropolarity around DNA molecules upon complexation with histone protein (H1). While exploring the specific protein-DNA interaction at λ-repressor-operator sites interaction, particularly OR1 and OR2, it was observed that the interfacial water dynamics is minimally perturbed upon interaction with DNA, suggesting the labile interface in the protein-DNA complex. Förster resonance energy transfer (FRET) study revealed that the structure of the protein is more compact in repressor-OR2 complex than in the repressor-OR1 complex. Fluorescence anisotropy studies indicated enhanced flexibility of the C-terminal domain of the repressor at fast timescales after complex formation with OR1. The enhanced flexibility and different conformation of the C-terminal domain of the repressor upon complexation with OR1 DNA compared to OR2 DNA were found to have pronounced effect on the rate of photoinduced electron transfer.

Förster resonance energy transfer in a nanoscopic system on a dielectric interface

We investigate picosecond-resolved energy transfer between a quantum dot (donor) and an organic molecule (acceptor) in the proximity of a reflecting metallic/non-metallic surface. We demonstrate experimentally that the Förster resonance energy transfer (FRET) is significantly influenced by the proximity of the mirror. Locating a cadmium selenide quantum dot (donor: D) attached to an organic dye merocyanine (acceptor: A) at well-defined positions from the reflecting silver/silicon surface allows the transfer rate to be determined as a function of distance from the surface. An attempt to fit the experimental data to a model relying upon the change of the apparent energy transfer rate due to interference of direct and reflected light waves reveals reasonably good results. The results show that the observed FRET rate in a D-A pair on the mirror surface is oscillating in nature, providing information for the measured energy transfer, which could be potentially different from that of the actual transfer due to optical interference.