Shyamtanu Chattoraj

Role of ionic liquid on the conformational dynamics in the native, molten globule, and unfolded states of cytochrome c: a fluorescence correlation spectroscopy study.

The role of a room temperature ionic liquid (RTIL, [pmim][Br]) on the size and conformational dynamics of a protein, horse heart cytochrome c (Cyt C) in its native, molten globule (MG-I and II), and unfolded states is studied using fluorescence correlation spectroscopy (FCS). For this purpose, the protein was covalently labeled by a fluorescent dye, Alexa Fluor 488. It is observed that the addition of the RTIL leads to an increase in the hydrodynamic radius (r(H)) of the protein, Cyt C in the native or MG-I state. In contrast, the addition of RTIL causes a decrease in the size (hydrodynamic radius, r(H)) of Cyt C unfolded by GdnHCl or MG-II state. The decrease in size indicates the formation of a relatively compact structure. We detected two types of conformational relaxation of the protein. The shorter relaxation time component (~3-5.5 μs) corresponds to the protein folding or intrachain contact formation, while the relatively longer time component (~63-122 μs) may be assigned to the motion of the protein side chains or concerted chain dynamics. The burst integrated fluorescence lifetime histograms indicate that the increase in size of the protein is accompanied by an increase in the contribution of the shorter component (~0.3-0.4 ns) with a concomitant decrease of the contribution of the longer component (~2.8-3.6 ns). An opposite trend is observed during the decrease in size of the protein.

Dynamics in Cytoplasm, Nucleus, and Lipid Droplet of a Live CHO Cell: Time-Resolved Confocal Microscopy

Different regions of a single live Chinese hamster ovary (CHO) cell are probed by time-resolved confocal microscopy. We used coumarin 153 (C153) as a probe. The dye localizes in the cytoplasm, nucleus, and lipid droplets, as is clearly revealed by the image. The fluorescence correlation spectroscopy (FCS) data shows that the microviscosity of lipid droplets is ~34 ± 3 cP. The microviscosities of nucleus and cytoplasm are found to be 13 ± 1 and 14.5 ± 1 cP, respectively. The average solvation time () in the lipid droplets (3600 ± 50 ps) is slower than that in the nucleus ( = 750 ± 50 ps) and cytoplasm ( = 1100 ± 50 ps). From the position of emission maxima of C153, the polarity of the nucleus is estimated to be similar to that of a mixture containing 26% DMSO in triacetin (η ~ 11.2 cP, ε ~ 26.2). The cytoplasm resembles a mixture of 18% DMSO in triacetin (η ∼ 12.6 cP, ε ∼ 21.9). The polarity of lipid droplets is less than that of pure triacetin (η ~ 21.7 cP, ε ~ 7.11).

Heterogeneity in binary mixtures of dimethyl sulfoxide and glycerol: Fluorescence correlation spectroscopy

Diffusion of four coumarin dyes in a binary mixture of dimethyl sulfoxide (DMSO) and glycerol is studied using fluorescence correlation spectroscopy (FCS). The coumarin dyes are C151, C152, C480, and C481. In pure DMSO, all the four dyes exhibit a very narrow (almost uni-modal) distribution of diffusion coefficient (Dt). In contrast, in the binary mixtures all of them display a bimodal distribution of Dt with broadly two components. One of the components of D(t) corresponds to the bulk viscosity. The other one is similar to that in pure DMSO. This clearly indicates the presence of two distinctly different nano-domains inside the binary mixture. In the first, the micro-environment of the solute consists of both DMSO and glycerol approximately at the bulk composition. The other corresponds to a situation where the first layer of the solute consists of DMSO only. The burst integrated fluorescence lifetime (BIFL) analysis also indicates presence of two micro-environments one of which resembles DMSO. The relative contribution of the DMSO-like environment obtained from the BIFL analysis is much larger than that obtained from FCS measurements. It is proposed that BIFL corresponds to an instantaneous environment in a small region (a few nm) around the probe. FCS, on the contrary, describes the long time trajectory of the probes in a region of dimension ~200 nm. The results are explained in terms of the theory of binary mixtures and recent simulations of binary mixtures containing DMSO.

Effect of ionic liquid on the native and denatured state of a protein covalently attached to a probe: solvation dynamics study.

Effect of a room temperature ionic liquid (RTIL, [pmim][Br]) on the solvation dynamics of a probe covalently attached to a protein (human serum albumin (HSA)) has been studied using femtosecond up-conversion. For this study, a solvation probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) has been covalently attached to the lone cysteine group (cys-34) of the protein HSA. Addition of 1.5 M RTIL or 6 M GdnHCl causes a red shift of the emission maxima of CPM bound to HSA by 3 nm and 12 nm, respectively. The average solvation time 〈τ(s)〉 decreases from 650 ps (in native HSA) to 260 ps (~2.5 times) in the presence of 1.5 M RTIL and to 60 ps (~11 times) in the presence of 6 M GdnHCl. This is ascribed to unfolding of the protein by RTIL or GdnHCl and therefore making the probe CPM more exposed. When 1.5 M RTIL is added to the protein denatured by 6 M GdnHCl in advance, a further ~5 nm red shift along with further ~2 fold faster solvent relaxation ( ~30 ps) is observed. Our previous fluorescence correlation spectroscopy study [D. K. Sasmal, T. Mondal, S. Sen Mojumdar, A. Choudhury, R. Banerjee, and K. Bhattacharyya, J. Phys. Chem. B 115, 13075 (2011)] suggests that addition of RTIL to the protein denatured by 6 M GdnHCl causes a reduction in hydrodynamic radius (r(h)). It is demonstrated that in the presence of RTIL and GdnHCl, though the protein is structurally more compact, the local environment of CPM is very different from that in the native state.

Effect of ethanol-water mixture on the structure and dynamics of lysozyme: A fluorescence correlation spectroscopy study

Effect of ethanol-water mixture on the hydrodynamic radius (r(H)) and conformational dynamics of lysozyme has been studied by circular dichroism, emission spectra, and fluorescence correlation spectroscopy. For this purpose, the protein lysozyme is covalently labeled near the active site with a fluorescent probe, alexa 488. The ethanol molecules are sequestered near the hydrophobic tryptophan residues as indicated by the blue shift of the emission maximum of tryptophan. It is observed that both size (r(H)) and time constant of conformational relaxation (τ(R)) of lysozyme oscillate with increase in ethanol concentration. The r(H) of the protein fluctuates from 19 Å in the native state, to a minimum of 13 Å, and a maximum of 29 Å. It is proposed that the oscillating behavior arises from competition between mutual interaction among protein, ethanol, and water. The fluorescence intensity fluctuates because of quenching of the fluorescence of the probe (alexa) by the free amino group of certain residues (e.g., tryptophan). Rate of inter-conversion (folding dynamics) between the open (fluorescent) and closed (non-fluorescent) form has been determined and is found to exhibit similar oscillation with variation in ethanol content.

Cancer Cell Imaging Using in Situ Generated Gold Nanoclusters

In situ generated fluorescent gold nanoclusters (Au-NCs) are used for bio-imaging of three human cancer cells, namely, lung (A549), breast (MCF7), and colon (HCT116), by confocal microscopy. The amount of Au-NCs in non-cancer cells (WI38 and MCF10A) is 20-40 times less than those in the corresponding cancer cells. The presence of a larger amount of glutathione (GSH) capped Au-NCs in the cancer cell is ascribed to a higher glutathione level in cancer cells. The Au-NCs exhibit fluorescence maxima at 490-530 nm inside the cancer cells. The fluorescence maxima and matrix-assisted laser desorption ionization (MALDI) mass spectrometry suggest that the fluorescent Au-NCs consist of GSH capped clusters with a core structure (Au8-13). Time-resolved confocal microscopy indicates a nanosecond (1-3 ns) lifetime of the Au-NCs inside the cells. This rules out the formation of aggregated Au-thiolate complexes, which typically exhibit microsecond (≈1000 ns) lifetimes. Fluorescence correlation spectroscopy (FCS) in live cells indicates that the size of the Au-NCs is ≈1-2 nm. For in situ generation, we used a conjugate consisting of a room-temperature ionic liquid (RTIL, [pmim][Br]) and HAuCl4. Cytotoxicity studies indicate that the conjugate, [pmim][AuCl4], is non-toxic for both cancer and non-cancer cells.

Solvation Dynamics and Intermittent Oscillation of Cell Membrane: Live Chinese Hamster Ovary Cell

Dynamics of the exofacial thiols (i.e., cell surface thiol containing membrane proteins) of a live Chinese hamster ovary (CHO) cell is probed by time-resolved confocal microscopy. For this purpose, a fluorescent probe, 7-(diethylamino)-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) is covalently attached to the exofacial thiols. The emission maximum of CPM bound exofacial thiols indicates a highly exposed and polar environment. Using CPM, we studied solvation dynamics, for the first time, at the membrane of a live cell. The thiol containing membrane proteins shows ultraslow response with average solvation time, ⟨τs⟩ = 475 ps. CPM labeled exofacial thiols also show spontaneous, intermittent oscillation in fluorescence intensity with a period of 0.5-1.0 s. This is ascribed to reversible, intermittent changes in the structure and conformation of the membrane proteins.

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 (90 %) of breast cancer cells (MCF7) (IC50 =155 nm). In contrast, the killing of the noncancer breast cells (MCF10A) by doxorubicin-loaded AuNCs is only 40 % (IC50 =4500 nm). By using a confocal microscope, the fluorescence spectrum and decay of the AuNCs were recorded inside the cell. The fluorescence maxima (at ≈490-515 nm) and lifetime (≈2 ns), 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.

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.

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

Cytochrome c-capped fluorescent gold nanoclusters (Au-NCs) are used for imaging of live lung and breast cells. Delivery of cytochrome c inside the cells is confirmed by covalently attaching a fluorophore (Alexa Fluor 594) to cytochrome c-capped Au-NCs and observing fluorescence from Alexa 594 inside the cell. Mass spectrometry studies suggest that in bulk water, addition of glutathione (GSH) to cytochrome c-capped Au-NCs results in the formation of glutathione-capped Au-NCs and free apo-cytochrome c. Thus glutathione displaces cytochrome c 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 cytochrome c-Au-NC (donor) to Mitotracker Orange (acceptor) indicates that the Au-NCs localise in the mitochondria of live cells.