Deciphering the evolution of supramolecular nanofibers in solution and solid-state: a combined microscopic and spectroscopic approach.

Supramolecular self-assembly of small organic molecules has emerged as a powerful tool to construct well-defined micro- and nanoarchitecture through fine-tuning a range of intermolecular interactions. The size, shape, and optical properties of these nanostructures largely depend on the specific assembly of the molecular building units, temperature and polarity of the medium, and external stimuli. The engineering of supramolecular self-assembled nanostructures with morphology-dependent tunable emission is in high demand due to the promising scope in nanodevices and molecular machines. However, probing the evolution of molecular aggregates from the solution and directing the self-assembly process in a pre-defined fashion are challenging. In the present study, we have deciphered the sequential evolution of supramolecular nanofibers from solution to spherical and oblong-shaped nanoparticles through the variation of solvent polarity, tuning the hydrophobic-hydrophilic interactions. An intriguing case of molecular self-assembly has been elucidated employing a newly designed π-conjugated thiophene derivative (TPAn) through a combination of steady-state absorption, emission measurements, fluorescence correlation spectroscopy (FCS), and electron microscopy. The FCS analysis and microscopy results revealed that the small-sized nanofibers in the dispersion further agglomerated upon solvent evaporation, resulting in a network of nanofibers. Stimuli-responsive reversible interconversion between a network of nanofibers and spherical nanoaggregates was probed both in dispersion and solvent-evaporated state. The evolution of organic nanofibers and a subtle control over the self-assembly process demonstrated in the current investigation provide a general paradigm to correlate the size, shape, and emission properties of fluorescent molecular aggregates in complex heterogeneous media, including a human cell.

Time-dependent enhancement of fluorescence from Rhodobacter capsulatus SB1003 and its critical dependence on concentration temperature and static magnetic field.

Continuous exposure of 395 nm light increases the fluorescence emission intensity of photosynthetic purple non-sulphur bacteria, Rhodobacter capsulatus (SB1003). We show that such an increase in fluorescence emission of extracellular pigment complexes (PC) from these photosynthetic bacteria depends on the concentration of the pigment and temperature and can also be modulated by the static magnetic field. The time-dependent enhanced emission disappears either at or below 300 K or below a threshold sample concentration (0.1 mg/ml). The enhanced emission reappears at this condition (T < 278 K) if a static magnetic field (395 mT) is introduced during fluorescence measurement. The time dependence of emission is expressed in terms of a first-order rate constant, k = dF/(Fdt). The sign of k shifts from positive to negative as PC concentration is lowered than a threshold value, implying onset of fluorescence decay (k < 0) rather than amplification (k > 0). At PC concentration higher than a threshold, k becomes negative if the temperature is lowered. But, surprisingly, at low temperature, a static magnetic field reverts the k value to positive. We explain the logical nature of k-switching and photo-dynamics of the aforesaid microbial fluorescence emission by aggregation of protoporphyrin rings present in the PC. While the simultaneous presence of decay in fluorescence and susceptibility to static magnetic field suggests the dominance of triplet states at low temperatures, the process is reversed by SMF-induced removal of spin degeneracy. At higher temperatures, the optical excitability and lack of magnetic response suggest the dominance of singlet states. We propose that the restructuring of the singlet-triplet distribution by intersystem crossing may be the basis of this logical behaviour. In context with microbial function, time-dependent enhancement of fluorescence also implies relay of red photons to the neighbouring microbes not directly exposed to the incident radiation, thus serving as an indirect photosynthetic regulator.

Probing Viscosity of Co-Polymer Hydrogel and HeLa Cell Using Fluorescent Gold Nanoclusters: Fluorescence Correlation Spectroscopy and Anisotropy Decay.

Fluorescence dynamics of gold nanoclusters (Au9 and Au25 ) are studied in the complex and crowded environment of a triblock co-polymer (F127) hydrogel and inside cervical cancer cell, HeLa. In the hydrogel, spherical micelles of F127 remain immobilized with a hydrophobic core (PPO) and a hydrophilic corona (PEO) region. The fluorescence anisotropy decay suggests that the timescale of rotational relaxation in the hydrogel is similar to that in bulk water (viscosity ∼1 cP). From fluorescence correlation spectroscopy (FCS) it is inferred that the local viscosity in the hydrogel is 12 cP for Au9 and 18 cP for Au23 . These results indicate that gold nanoclusters (AuNCs) localize in the corona region of the hydrogel. Evidently, frictions against rotation and translation are different inside the gel. It is suggested that rotation of the AuNCs senses the immediate water-like "void" region while translation motion involves in-and-out movement of the AuNCs at the periphery of the gel. Finally, the gold nanoclusters are used for cell imaging and estimation of intracellular viscosity of HeLa cells.

Time Evolution of Local pH Around a Photo-Acid in Water and a Polymer Hydrogel: Time Resolved Fluorescence Spectroscopy of Pyranine.

In this work, we propose a new analysis of the time resolved emission spectra of a photo-acid, HA, pyranine (8-hydroxypyrene-1,3,6-trisulphonic acid, HPTS) based on time resolved area normalized emission spectra (TRANES). Presence of an isoemissive point in TRANES confirms the presence of two emissive species (HA and A- ) inside the system in bulk water and inside a co-polymer hydrogel [F127, (PEO)100 -(PPO)70 -(PEO)100 ]. We show that following electronic excitation, the local pH around HPTS, is much lower than the bulk pH presumably because of ejection of proton from the photo-acid in the excited state. With increase in time, the local pH increases and reaches the bulk value. We further, demonstrate that the excited state pKa of HPTS may be estimated from the emission intensities of HA and A- at long time. The time constant for time evolution of pH is ∼630 ps in water, ∼1300 ps in F127 gel and ∼4700 ps in CTAB micelle. The location and local viscosity sensed by the probe is ascertained using fluorescence correlation spectroscopy (FCS) and fluorescence anisotropy decay. The different values of the local viscosity reported by these two methods are reconciled.

Probing Deviation of Adhered Membrane Dynamics between Reconstituted Liposome and Cellular System.

The dynamics of cell-cell adhesion are complicated due to complexities in cellular interactions and intra-membrane interactions. In the present work, we have reconstituted a liposome-based model system to mimic the cell-cell adhesion process. Our model liposome system consists of one fluorescein-tagged and one TRITC (tetramethyl-rhodamine isothiocyanate)-tagged liposome, adhered through biotin-neutravidin interaction. We monitored the adhesion process in liposomes using Förster Resonance Energy Transfer (FRET) between fluorescein (donor) and TRITC (acceptor). Occurrence of FRET is confirmed by the decrease in donor lifetime as well as distinct rise time of the acceptor fluorescence. Interestingly, the acceptor's emission exhibits fluctuations in the range of ≈3±1 s. This may be attributed to structural oscillations associated in two adhered liposomes arising from the flexible nature of biotin-neutravidin interaction. We have compared the dynamics in a cell-mimicking liposome system with that in an in vitro live cell system. In the adhered live cell system, we used CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin, donor) and nile red (acceptor), which are known to stain the membrane of CHO (Chinese Hamster Ovary) cells. The dynamics of the adhered membranes of two live CHO cells were observed through FRET between CPM and nile red. The acceptor fluorescence intensity exhibits an oscillation in the time-scale of ≈1±0.75 s, which is faster compared to the reconstituted liposome system, indicating the contributions and involvement of multiple dynamic protein complexes around the cell membrane. This study offers simple reconstituted model systems to understand the complex membrane dynamics using a FRET-based physical chemistry approach.

Structure, Activity, and Dynamics of Human Serum Albumin in a Crowded Pluronic F127 Hydrogel.

Structure, activity, and dynamics of a plasma protein, human serum albumin (HSA), inside a crowded environment of F127 gel are studied by circular dichroism (CD), fluorescence correlation spectroscopy (FCS), and picosecond time-resolved fluorescence spectroscopy. For this purpose, the protein is covalently labeled by a maleimide dye, 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methyl-coumarin (CPM). The circular dichroism (CD) spectra suggest that the protein is more structured in the gel reflecting about the biological activities of the protein. FCS results demonstrate that compared to that in bulk water (buffer solution), translational diffusion is about 59 times slower inside the F127 gel. This indicates higher translational friction (viscosity) sensed by the probe (CPM). On the contrary, rotational relaxation (and hence, rotational friction) is more or less similar in F127 gel and in bulk water. FCS results further indicate that the time scales of conformational relaxation of the protein are substantially slow inside the crowded environment of F127 gel. The fast component of conformational relaxation is retarded by ∼55 times, and the slow component by ∼20 times. Fluorescence maximum of CPM bound to HSA show a ∼5 nm red shift, implying that the microenvironment of the probe, CPM, is more polar inside the gel. Solvation dynamics of CPM-labeled HSA inside the gel (⟨τs⟩ ∼ 300 ps) is faster compared to that for the protein in bulk water (⟨τs⟩ ∼ 600 ps).

Local environment of organic dyes in an ionic liquid-water mixture: FCS and MD simulation.

The composition dependent local environment of three organic dyes in binary mixtures of a room temperature ionic liquid (1-methyl-3-pentylimidazolium bromide, [pmim][Br]) and water is studied by fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations. We used three dyes-neutral coumarin 480 (C480), anionic coumarin 343 (C343), and highly hydrophobic 4-(dicyanomethylene)-2-methyl-6-(p-dimethyl-aminostyryl)-4H-pyran (DCM)-to probe different environments in the binary mixtures. The heterogeneity of the [pmim][Br]-water mixture leads to multiple values (i.e., distribution) of diffusion coefficients (Dt). In these binary mixtures, the effective viscosity (ηeff, obtained from FCS) and the local concentration of the [pmim][Br] around the three dyes (revealed by MD simulations) are found to be quite different than that in bulk. The viscosity experienced by the C480 and C343 dyes is almost twice as large as that experienced by DCM dye. Through rigorous MD simulation, we show that in the vicinity of the less hydrophobic coumarin dyes (C480 and C343) compared to DCM dye, the local concentration of the [pmim][Br] is ∼3-7 times larger than that in bulk. In the case of the most hydrophobic dye, DCM, the local concentration of [pmim][Br] is almost similar to bulk-like. Further analysis reveals the formation of hydrogen bond between the imidazolium ring of [pmim][Br] and the carbonyl oxygen atom of the coumarin dyes (C-H[pmim][Br]⋯O=CDye). Finally, computer simulation indicates a slow component of solvation dynamics in the [pmim][Br]-water mixture in the time scale of ∼100-200 ps, which is similar to the experimental observation.

Live Cell Microscopy: A Physical Chemistry Approach.

Probing dynamics of intracellular components using physical chemistry techniques is a remarkable bottom-up approach for understanding the structures and functions of a biological cell. In this "Feature Article", we give an overview on local polarity, solvation, viscosity, acid-base property, red-ox processes (thiol-disulfide exchange), and gene silencing at selected intracellular components inside a live cell. Significant differences have been observed between cancer cells and their noncancer counterparts. We demonstrate that thiol-disulfide exchange, calcium oscillation, and gene silencing are manifested in time dependence of fluorescence intensity. We show that fluorescent gold nanoclusters may be used in drug delivery (e.g., doxorubicin) and selective killing of cancer cells. Further, we discuss dynamics and structural changes of DNA quadruplexes and i-motifs, induced by different external conditions (e.g., pH) and additives (e.g., K+ and other target specific small molecules). We demonstrate that peptidomimetic analogues have high specificity over double-stranded DNA for binding with i-motifs and G-quadruplexes. These results may have significant biological implications.

Interaction of proteins with ionic liquid, alcohol and DMSO and in situ generation of gold nano-clusters in a cell.

In this review, we give a brief overview on how the interaction of proteins with ionic liquids, alcohols and dimethyl sulfoxide (DMSO) influences the stability, conformational dynamics and function of proteins/enzymes. We present experimental results obtained from fluorescence correlation spectroscopy on the effect of ionic liquid or alcohol or DMSO on the size (more precisely, the diffusion constant) and conformational dynamics of lysozyme, cytochrome c and human serum albumin in aqueous solution. The interaction of ionic liquid with biomolecules (e.g. protein, DNA etc.) has emerged as a current frontier. We demonstrate that ionic liquids are excellent stabilizers of protein and DNA and, in some cases, cause refolding of a protein already denatured by chemical denaturing agents. We show that in ethanol-water binary mixture, proteins undergo non-monotonic changes in size and dynamics with increasing ethanol content. We also discuss the effect of water-DMSO mixture on the stability of proteins. We demonstrate how large-scale molecular dynamics simulations have revealed the molecular origin of this observed phenomenon and provide a microscopic picture of the immediate environment of the biomolecules. Finally, we describe how favorable interactions of ionic liquids may be utilized for in situ generation of fluorescent gold nano-clusters for imaging a live cell.

Probing the conformational dynamics of photosystem I in unconfined and confined spaces.

The fluorescence dynamics of Photosystem I (PSI) in bulk water and inside a confined environment like a liposome have been investigated using time resolved confocal microscopy. In bulk water, PSI exhibits a major emission peak at ∼680 nm, while in the liposome it exhibits a markedly blue shifted emission maximum at ∼485 nm. This is indicative of conformational changes due to entrapment and emergence of a stressed conformation of PSI inside the liposome. The observed time constants for the fluorescence lifetime of PSI inside the liposome are significantly high as opposed to PSI in bulk water. More interestingly, the fluorescence intensity of PSI in bulk water exhibits strong fluctuations with many high intensity jumps and these are anti-correlated with the fluorescence lifetime of PSI. In contrast, inside the liposome, no such anti-correlated behaviour is observed. We further demonstrated that PSI exhibits at least two conformational states in bulk water, whereas a single conformation is observed inside the liposome, indicating the conformational rigidity and locking of the PSI complex inside a liposome.

Preferential targeting of i-motifs and G-quadruplexes by small molecules.

i-Motifs and G-quadruplexes are dynamic nucleic acid secondary structures, which are believed to play key roles in gene expression. We herein report two peptidomimetic ligands (PBP1 and PBP2) that selectively target i-motifs and G-quadruplexes over double-stranded DNA. These peptidomimetics, regioisomeric with respect to the position of triazole/prolinamide motifs, have been synthesized using a modular method involving Cu(i)-catalyzed azide and alkyne cycloaddition. The para-isomer, PBP1 exhibits high selectivity for i-motifs while the meta-isomer PBP2 binds selectively to G-quadruplex structures. Interestingly, these ligands have the ability to induce G-quadruplex or i-motif structures from the unstructured single-stranded DNA conformations, as observed using single molecule Förster resonance energy transfer (smFRET) studies. The quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and dual-luciferase assays indicate that PBP1 upregulates and PBP2 downregulates BCL-2 gene expression in cancer cells.

Physical chemistry in a single live cell: confocal microscopy.

A live cell is a complex, yet extremely important container. Understanding the dynamics in a selected intracellular component is a challenging task. We have recently made significant progress in this direction using a confocal microscope as a tool. The smallest size of the focused spot in a confocal microscope is ∼0.2 µm (200 nm). This is nearly one hundred times smaller than the size of a live cell. Thus, one can selectively study different intracellular components/organelles in a live cell. In this paper, we discuss how one can image different intracellular components/organelles, record fluorescence spectra and decay at different locations, ascertain local polarity and viscosity, and monitor the dynamics of solvation, proton transfer, red-ox and other phenomena at specified locations/organelles inside a cell. We will highlight how this knowledge enriched us in differentiating between cancer and non-cancer cells, 3D tumor spheroids and towards drug delivery.

Differential role of nonmuscle myosin II isoforms during blebbing of MCF-7 cells.

Bleb formation has been correlated with nonmuscle myosin II (NM-II) activity. Whether three isoforms of NM-II (NM-IIA, -IIB and -IIC) have the same or differential roles in bleb formation is not well understood. Here we report that ectopically expressed, GFP-tagged NM-II isoforms exhibit different types of membrane protrusions, such as multiple blebs, lamellipodia, combinations of both, or absence of any such protrusions in MCF-7 cells. Quantification suggests that 50% of NM-IIA-GFP-, 29% of NM-IIB-GFP-, and 19% of NM-IIC1-GFP-expressing MCF-7 cells show multiple bleb formation, compared with 36% of cells expressing GFP alone. Of interest, NM-IIB has an almost 50% lower rate of dissociation from actin filament than NM-IIA and -IIC1 as determined by FRET analysis both at cell and bleb cortices. We induced bleb formation by disruption of the cortex and found that all three NM-II-GFP isoforms can reappear and form filaments but to different degrees in the growing bleb. NM-IIB-GFP can form filaments in blebs in 41% of NM-IIB-GFP-expressing cells, whereas filaments form in only 12 and 3% of cells expressing NM-IIA-GFP and NM-IIC1-GFP, respectively. These studies suggest that NM-II isoforms have differential roles in the bleb life cycle.

Enzyme activity of α-chymotrypsin: Deactivation by gold nano-cluster and reactivation by glutathione.

Effect of gold nanoclusters (Au-NCs) on the circular dichroism (CD) spectra and enzymatic activity of α-chymotrypsin (ChT) (towards hydrolysis of a substrate, N-succinyl-l-phenylalanine p-nitroanilide) are studied. The CD spectra indicate that on binding to Au-NC, ChT is completely unfolded, resulting in nearly zero ellipticity. α-chymotrypsin (ChT) coated gold nano-clusters exhibit almost no enzymatic activity. Addition of glutathione (GSH) or oxidized glutathione (GSSG) restore the enzyme activity of α-chymotrypsin by 30-45%. ChT coated Au-NC exhibits two emission maxima-one at 480nm (corresponding to Au10) and one at 640nm (Au25). On addition of glutathione (GSH) or oxidized glutathione (GSSG) the emission peak at 640nm vanishes and only one peak at 480nm (Au10) remains. MALDI mass spectrometry studies suggest addition of glutathione (GSH) to α-chymotrypsin capped Au-NCs results in the formation of glutathione-capped Au-NCs and α-chymotrypsin is released from Au-NCs. CD spectroscopy indicates that the conformation of the released α-chymotrypsin is different from that of the native α-chymotrypsin.

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.

Amyloid beta peptides inside a reconstituted cell-like liposomal system: aggregation, FRET, fluorescence oscillations and solvation dynamics.

Aggregations of amyloid-beta (Aß) peptides were studied inside a reconstituted cell like liposomal system using time-resolved confocal microscopy. Fluorescence correlation spectroscopy (FCS) and confocal images indicate that Aß forms a very large aggregate in bulk and more efficiently, in the bilayer region of the liposome, respectively. The aggregates formed inside the liposome gradually migrate out to bulk water. FRET, from HiLyte Fluor 488 (covalently attached to an Aß peptide) to TRITC (tetramethylrhodamine isothiocyanate) covalently attached to a DHPE lipid present in the bilayer, reveals intermittent oscillations in the time scale of ∼0.5 s. This is attributed to the structural fluctuations of the membrane of the liposome. The solvation dynamics of Aß in monomer and in oligomeric state is studied by monitoring the emission of HiLyte Fluor 488. The solvation dynamics of the Aß monomer is similar to that of oligomeric aggregates in the liposome.