Kankan Bhattacharyya

Solvation dynamics of DCM in micelles

The solvation dynamics of 4-(dicyanomethylene)-2-methyl-6(p-dimethylaminostyryl) 4H-pyran (DCM) have been studied in neutral (Triton X-100, TX), cationic (cetyl trimethyl ammonium bromide, CTAB) and anionic (sodium dodecyl sulfate, SDS) micelles using picosecond time-resolved Stokes shift. Above the critical micellar concentration for all three micelles, DCM exhibits wavelength-dependent fluorescence decays. At short wavelengths, a fast decay is observed while, at long wavelengths, a distinct growth precedes the decay. The time-dependent Stokes shift indicates that the water molecules in the Stern layer of the micelles relax on a timescale which is markedly slower than the sub-picosecond relaxation dynamics in pure water.

Selective Killing of Breast Cancer Cells by Doxorubicin‐Loaded Fluorescent Gold Nanoclusters: Confocal Microscopy and FRET

Fluorescent gold nanoclusters (AuNCs) capped with lysozymes are used to deliver the anticancer drug doxorubicin to cancer and noncancer cells. Doxorubicin-loaded AuNCs cause the highly selective and efficient killing (90u2009%) of breast cancer cells (MCF7) (IC50 =155u2005nm). In contrast, the killing of the noncancer breast cells (MCF10A) by doxorubicin-loaded AuNCs is only 40u2009% (IC50 =4500u2005nm). By using a confocal microscope, the fluorescence spectrum and decay of the AuNCs were recorded inside the cell. The fluorescence maxima (at ≈490-515u2005nm) and lifetime (≈2u2005ns), of the AuNCs inside the cells correspond to Au10-13 . The intracellular release of doxorubicin from AuNCs is monitored by Förster resonance energy transfer (FRET) imaging.

Excited state proton transfer in the lysosome of live lung cells: normal and cancer cells.

Dynamics of excited state proton transfer (ESPT) in the lysosome region of live lung cells (normal and cancer) is studied by picosecond time-resolved confocal microscopy. For this, we used a fluorescent probe, pyranine (8-hydroxy-pyrene-1,3,6-trisulfonate, HPTS). From the colocalization of HPTS with a lysotracker dye (lysotracker yellow), we confirmed that HPTS resides in the lysosome for both of the cells. The diffusion coefficient (Dt) in the lysosome region was obtained from fluorescence correlation spectroscopy (FCS). From Dt, the viscosity of lysosome is estimated to be ∼40 and ∼30 cP in the cancer and normal cells, respectively. The rate constants of the elementary steps of ESPT in a normal lung cell (WI38) are compared with those in a lung cancer cell (A549). It is observed that the time constant of the initial proton transfer process in a normal cell (τ(PT) = 40 ps) is similar to that in a cancer cell. The recombination of the geminate ion pair is slightly faster (τ(rec) = 25 ps) in the normal cell than that (τ(rec) = 30 ps) in a cancer cell. The time constant of the dissociation (τ(diss)) of the geminate ion pair for the cancer cell (τ(diss) = 80 ps) is 1.5 times faster compared to that (τ(diss) = 120 ps) in a normal cell.

Ionic liquid induced dehydration and domain closure in lysozyme: FCS and MD simulation

Effect of a room temperature ionic liquid (RTIL, [pmim][Br]) on the structure and dynamics of the protein, lysozyme, is investigated by fluorescence correlation spectroscopy (FCS) and molecular dynamic (MD) simulation. The FCS data indicate that addition of the RTIL ([pmim][Br]) leads to reduction in size and faster conformational dynamics of the protein. The hydrodynamic radius (rH) of lysozyme decreases from 18 Å in 0 M [pmim][Br] to 11 Å in 1.5 M [pmim][Br] while the conformational relaxation time decreases from 65 μs to 5 μs. Molecular origin of the collapse (size reduction) of lysozyme in aqueous RTIL is analyzed by MD simulation. The radial distribution function of water, RTIL cation, and RTIL anion from protein clearly indicates that addition of RTIL causes replacement of interfacial water by RTIL cation ([pmim](+)) from the first solvation layer of the protein providing a comparatively dehydrated environment. This preferential solvation of the protein by the RTIL cation extends up to ∼30 Å from the protein surface giving rise to a nanoscopic cage of overall radius 42 Å. In the nanoscopic cage of the RTIL (42 Å), volume fraction of the protein (radius 12 Å) is only about 2%. RTIL anion does not show any preferential solvation near protein surface. Comparison of effective radius obtained from simulation and from FCS data suggests that the dry protein (radius 12 Å) alone diffuses in a nanoscopic cage of RTIL (radius 42 Å). MD simulation further reveals a decrease in distance (domain closure) between the two domains (alpha and beta) of the protein leading to a more compact structure compared to that in the native state.

Role of Red-Ox Cycle in Structural Oscillations and Solvation Dynamics in the Mitochondria of a Live Cell

Structural oscillations and solvation dynamics in the mitochondria of a live cell are studied by time-resolved microscopy using a covalent fluorescence probe. We compared the dynamics in a human breast cancer cell (MCF-7) with that in a normal breast cell MCF-10A. The probe, CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin), binds with the free thiol groups. In MCF-10A cell, CPM binds with the discrete mitochondria. In MCF-7, CPM labels the clustered mitochondria in the peri-nuclear region. Location of the CPM in the mitochondria is confirmed by colocalization with a mitochondria-tracker dye. The red-ox cycle in the mitochondria causes periodic fluctuation in the microenvironment in the discrete mitochondria. This is manifested in fluctuations in fluorescence intensity of CPM bound to mitochondria. The magnitude of oscillation is much less for CPM bound to the clustered mitochondria (in which the red-ox cycle is inefficient) in the cancer cell (MCF-7). In both of the cells (MCF-10A and MCF-7) CPM bound to thiol-containing proteins in mitochondria exhibits ultraslow response with average solvation time (⟨τs⟩) of 850 and 1400 ps in MCF-10A and MCF-7, respectively.

Intermittent Fluorescence Oscillations in Lipid Droplets in a Live Normal and Lung Cancer Cell: Time-Resolved Confocal Microscopy.

Intermittent structural oscillation in the lipid droplets of live lung cells is monitored using time-resolved confocal microscopy. Significant differences are observed between the lung cancer cell (A549) and normal (nonmalignant) lung cell (WI38). For this study, the lipid droplets are covalently labeled with a fluorescent dye, coumarin maleimide (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin, CPM). The number of lipid droplets in the cancer cell is found to be ∼20-fold higher than that in the normal (nonmalignant) cell. The fluctuation in the fluorescence intensity of the dye (CPM) is attributed to the red-ox processes and periodic formation/rupture of the S-CPM bond. The amount of reactive oxygen species (ROS) is much higher in a cancer cell. This is manifested in faster oscillations (0.9 ± 0.3 s) in cancer cells compared to that in the normal cells (2.8 ± 0.7 s). Solvation dynamics in the lipid droplets of cancer cells is slower compared to that in the normal cell.

Solvation Dynamics of DCM in Dipalmitoyl Phosphatidylcholine Lipid

Solvation dynamics of 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl) 4H-pyran (DCM) in dipalmitoylphosphatidylcholine (DPPC) vesicles in water is studied using picosecond emission spectroscopy. The solvation dynamics of DCM in DPPC vesicles is found to be bi-exponential with two components of 120±20 ps (20%) and 5.5±0.5 ns (80%). This indicates slow relaxation of the water molecules inside the water pool of the lipid vesicles.

Cytochrome c-Capped Fluorescent Gold Nanoclusters: Imaging of Live Cells and Delivery of Cytochrome c.

Cytochromeu2005c-capped fluorescent gold nanoclusters (Au-NCs) are used for imaging of live lung and breast cells. Delivery of cytochromeu2005c inside the cells is confirmed by covalently attaching a fluorophore (Alexa Fluoru2005594) to cytochromeu2005c-capped Au-NCs and observing fluorescence from Alexau2005594 inside the cell. Mass spectrometry studies suggest that in bulk water, addition of glutathione (GSH) to cytochromeu2005c-capped Au-NCs results in the formation of glutathione-capped Au-NCs and free apo-cytochromeu2005c. Thus glutathione displaces cytochromeu2005c as a capping agent. Using confocal microscopy, the emission spectra and decay of Au-NCs are measured in live cells. From the position of the emission maximum it is shown that the Au-NCs exist as Au8 in bulk water and as Au13 inside the cells. Fluorescence resonance energy transfer from cytochromeu2005c-Au-NC (donor) to Mitotracker Orange (acceptor) indicates that the Au-NCs localise in the mitochondria of live cells.

Effect of alcohol on the structure of cytochrome C: FCS and molecular dynamics simulations

Effect of ethanol on the size and structure of a protein cytochrome C (Cyt C) is investigated using fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations. For FCS studies, Cyt C is covalently labeled with a fluorescent probe, alexa 488. FCS studies indicate that on addition of ethanol, the size of the protein varies non-monotonically. The size of Cyt C increases (i.e., the protein unfolds) on addition of alcohol (ethanol) up to a mole fraction of 0.2 (44.75% v/v) and decreases at higher alcohol concentration. In order to provide a molecular origin of this structural transition, we explore the conformational free energy landscape of Cyt C as a function of radius of gyration (Rg) at different compositions of water-ethanol binary mixture using MD simulations. Cyt C exhibits a minimum at Rg ∼ 13 Å in bulk water (0% alcohol). Upon increasing ethanol concentration, a second minimum appears in the free energy surface with gradually larger Rg up to χEtOH ∼ 0.2 (44.75% v/v). This suggests gradual unfolding of the protein. At a higher concentration of alcohol (χEtOH > 0.2), the minimum at large Rg vanishes, indicating compaction. Analysis of the contact map and the solvent organization around protein indicates a preferential solvation of the hydrophobic residues by ethanol up to χEtOH = 0.2 (44.75% v/v) and this causes the gradual unfolding of the protein. At high concentration (χEtOH = 0.3 (58% v/v)), due to structural organization in bulk water-ethanol binary mixture, the extent of preferential solvation by ethanol decreases. This causes a structural transition of Cyt C towards a more compact state.

Single-molecule Spectroscopy: Exploring Heterogeneity in Chemical and Biological Systems.

Many chemical and biological systems are heterogeneous in the molecular length scale (∼ 1 nm). Heterogeneity in many chemical systems and organized assemblies may be monitored using single-molecule spectroscopy (SMS). In SMS, the size of the focal spot (i.e., the smallest region to be probed) is nearly half of the excitation wavelength (λ/2, i.e., 200-375 nm) for visible light (400-750 nm). We discuss how one can get spatial resolutions better than 200 nm using molecules as nanometric probes. We show that polymer hydrogels, lipid vesicles, room temperature ionic liquids (RTILs), and binary liquid mixtures exhibit such heterogeneity. Another important observation is solute-dependent friction in RTILs. In an RTIL, diffusion of an ionic solute is slower than that of a neutral solute.