• Volume 119, Issue 2

March 2007,   pages  59-215

• Foreword

• Identification of intermediate species in protein-folding by quantitative analysis of amplitudes in time-domain fluorescence spectroscopy

In protein-folding studies it is often required to differentiate a system with only two-states, namely the native (N) and unfolded (U) forms of the protein present at any condition of the solvent, from a situation wherein intermediate state(s) could also be present. This differentiation of a two-state from a multi-state structural transition is non-trivial when studied by the several steady-state spectroscopic methods that are popular in protein-folding studies. In contrast to the steady-state methods, time-resolved fluorescence has the capability to reveal the presence of heterogeneity of structural forms due to the fingerprint’ nature of fluorescence lifetimes of various forms. In this work, we establish this method by quantitative analysis of amplitudes associated with fluorescence lifetimes in multiexponential decays. First, we show that we can estimate, accurately, the relative population of species from two-component mixtures of non-interacting molecules such as fluorescent dyes, peptides and proteins. Subsequently, we demonstrate, by analysing the amplitudes of fluorescence lifetimes which are controlled by fluorescence resonance energy transfer (FRET), that the equilibrium folding-unfolding transition of the small singledomain protein barstar is not a two-step process.

• Excited-state proton transfer from pyranine to acetate in methanol

Excited-state proton transfer (ESPT) of pyranine (8-hydroxypyrene-1,3,6-trisulphonate, HPTS) to acetate in methanol has been studied by steady-state and time-resolved fluorescence spectroscopy. The rate constant of direct proton transfer from pyranine to acetate ($k_1$) is calculated to be $\sim 1 \times 10^9$ M-1 s-1. This is slower by about two orders of magnitude than that in bulk water ($8 \times 10^{10}$ M-1 s-1) at 4 M acetate.

• Fluorescence resonance energy transfer from tryptophan in human serum albumin to a bioactive indoloquinolizine system

The interaction between a bioactive molecule, 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ), with human serum albumin (HSA) has been studied using steady-state absorption and fluorescence techniques. A 1 : 1 complex formation has been established and the binding constant (𝐾) and free energy change for the process have been reported. The AODIQ-HSA complex results in fluorescence resonance energy transfer (FRET) from the tryptophan moiety of HSA to the probe. The critical energy-transfer distance ($R_0$) for FRET and the Stern-Volmer constant ($K_{sv}$) for the fluorescence quenching of the donor in the presence of the acceptor have been determined. Importantly, $K_{SV}$ has been shown to be equal to the binding constant itself, implying that the fluorescence quenching arises only from the FRET process. The study suggests that the donor and the acceptor are bound to the same protein at different locations but within the quenching distance.

• Effects of polar protic solvents on dual emissions of 3-hydroxychromones

3-Hydroxychromones (3HC), exhibit dual emissions highly sensitive to solvent properties due to excited state intramolecular proton transfer (ESIPT). Therefore, 3HCs find wide applications as fluorescence probes in biological systems. Here, it is particularly important to understand the fluorescence behaviour of 3HCs in polar environments. Herein, we studied 3-hydroxyflavone, 2-(2-furyl)-3-hydroxychromone and 2-(2-benzofuryl)-3-hydroxychromone in high polarity solvents characterized by different H-bond donor abilities, donor concentrations and acceptor abilities. Our results show that the dual emissions of the dyes are insensitive to solvent basicity but strongly depend on the two other parameters. Moreover, furyl- and benzofuryl-substituted dyes were significantly more sensitive than the 3-hydroxyflavone to H-bond donor ability, while all three dyes showed roughly equivalent high sensitivity to H-bond donor concentration. These results can be explained by different mechanisms. Thus, the sensitivity of all three dyes to increasing concentrations of H-bond donors probably results from increase in the population of solvated dye with disrupted intramolecular H-bonds. Meantime, the sensitivity to Hbond donor ability of the solvent, observed mainly with furyl and benzofuryl dyes, is probably related to the strength of the H-bonds between the solvent and the 4-carbonyl group of the dye with intact intramolecular H-bonds. The present results provide new insights for further applications of 3HC derivatives as environment-sensitive probes and labels of biological molecules.

• Ratiometric fluorescence signalling of fluoride ions by an amidophthalimide derivative

Fluorescence behaviour of 4-benzoylamido-N-methylphthalimide (1), designed and developed for selective detection of fluoride ions, is reported. 1 displays $F^-$-induced colour change that allows its detection with the naked eye. The $F^-$ specificity of the sensor system is evident from the fact that unlike $F^-$, other halides do not affect the absorption characteristics of 1. Apart from the colorimetric response, the fluorescence output of 1 is also modulated by $F^-$ in a manner that permits ratiometric fluorescence signalling of $F^-$ as well. It is found that the system can detect $F^-$ in the concentration range of $10^-$ 60 𝜇M. The results of the experiments and theoretical calculations unambiguously suggest that the changes of the electronic absorption and fluorescence behaviour of 1, which have been exploited for signalling purpose, are due to $F^-$-induced deprotonation of the 4-amido moiety of the sensor system.

• Evidence for covalent binding of epicocconone with proteins from synchronous fluorescence spectra and fluorescence lifetimes

Synchronous fluorescence and time-resolved fluorescence spectroscopic studies that reveal the interaction of epicocconone with human serum albumin is significantly different from its interaction with surfactant assemblies. This observation, along with steady-state fluorescence data, indicates groundstate interaction between the fluorophore epicocconone and the protein. Similarity in fluorescence properties with the adduct of the fluorophore with 𝑛-butylamine indicates that bonding occurs at the Nterminus of the protein.

• Solvent relaxation of a room-temperature ionic liquid [bmim][PF6] confined in a ternary microemulsion

In this paper we have reported the solvent and rotational relaxation of 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF6]) confined in tween 20/([bmim][PF6]/water microemulsion using coumarin 153 (C-153) as probe. The most interesting feature of our experiment was that we observed an increase in solvent relaxation time with increase in 𝑅 (𝑅 = tween 20-to-[bmim][PF6] molar ratio). This is due to the fact that with increase in [bmim][PF6] content of the microemulsions, the microviscosity of the pool of the microemulsions increases, and motion of ions of [bmim][PF6] is hindered in the pool of microemulsions. Since motion of ions is responsible for solvation in room-temperature ionic liquids (RTILs), solvent-relaxation time increases with increase in 𝑅.

• On the origin of the anomalous ultraslow solvation dynamics in heterogeneous environments

Many recent experimental studies have reported a surprising ultraslow component (even &gt;10 ns) in the solvation dynamics of a polar probe in an organized assembly, the origin of which is not understood at present. Here we propose two molecular mechanisms in explanation. The first one involves the motion of the buried water’ molecules (both translation and rotation), accompanied by cooperative relaxation (‘local melting’) of several surfactant chains. An estimate of the time is obtained by using an effective Rouse chain model of chain dynamics, coupled with a mean first passage time calculation. The second explanation invokes self-diffusion of the (di)polar probe itself from a less polar to a more polar region. This may also involve cooperative motion of the surfactant chains in the hydrophobic core, if the probe has a sizeable distribution inside the core prior to excitation, or escape of the probe to the bulk from the surface of the self-assembly. The second mechanism should result in the narrowing of the full width of the emission spectrum with time, which has indeed been observed in recent experiments. It is argued that both the mechanisms may give rise to an ultraslow time constant and may be applicable to different experimental situations. The effectiveness of solvation as a dynamical probe in such complex systems has been discussed.

• Time-resolved fluorescence analysis of the mobile flavin cofactor in 𝑝-hydroxybenzoate hydroxylase

Conformational heterogeneity of the FAD cofactor in 𝑝-hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations; the in’ conformation with the isoalloxazine ring located in the active site, and the out’ conformation with the isoalloxazine ring disposed towards the protein surface. Fluorescence-lifetime analysis of these complexes revealed similar lifetime distributions for the in’ and out’ conformations. The reason for this is twofold. First, the active site of PHBH contains various potential fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222 is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these quenching sites and the isoalloxazine ring differs only little on average, these aromatic residues are likely to contribute to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluorescence quenching. The flavin conformation is therefore only reflected in the small relative contributions of the longer lifetimes.

• Wavelength-selective fluorescence in ion channels formed by gramicidin A in membranes

Gramicidins are linear peptides that form ion channels that are specific for monovalent cations in membranes. The tryptophan residues in the gramicidin channel play a crucial role in the organization and function of the channel. The natural mixture of gramicidins, denoted as gramicidin A', consists of mostly gramicidin A, but also contains gramicidins B, C and D as minor components. We have previously shown that the tryptophan residues in ion channels formed by the naturally occurring peptide, gramicidin A', display wavelength-dependent fluorescence characteristics due to the motionally restricted environment in which they are localized. In order to check the influence of ground-state heterogeneity in the observed wavelength-selective fluorescence of gramicidin A' in membranes, we performed similar experiments with pure gramicidin A in model membranes. Our results show that the observed wavelength-selective fluorescence characteristics of naturally occurring gramicidin A' are not due to groundstate heterogeneity.

• Employing the fluorescence anisotropy and quenching kinetics of tryptophan to hunt for residual structures in denatured proteins

Residual structures in denatured proteins have acquired importance in recent years owing to their role as protein-folding initiation sites. Locating these structures in proteins has proved quite formidable, requiring techniques like NMR. Here in this report, we take advantage of the ubiquitous presence of tryptophan residues in residual structures to hunt for their presence using steady-state fluorescence spectroscopy. The surface accessibility and rotational dynamics of tryptophan in putative residual structures among ten different proteins, namely glucagon, melittin, subtilisin carlsberg, myelin basic protein, ribonuclease T1, human serum albumin, barstar mutant, bovine serum albumin, lysozyme and Trp-Met-Asp-Phe-NH2 peptide, was studied using steady state fluorescence quenching and anisotropy, respectively. Five proteins, namely ribonuclease T1, bovine serum albumin, melittin, barstar and hen egg white lysozyme appear likely to possess tryptophan(s) in hydrophobic clusters based on their reduced bimolecular quenching rates and higher steady-state anisotropy in proportion to their chain length. We also show that the fluorescence emission maximum of tryptophan is insensitive to the presence of residual structures.

• A concise review of dynamical processes in polymorphic environments of a block copolymer: Rotational diffusion and photoisomerization

This article describes our ongoing efforts to understand dynamical processes such as rotational diffusion and photoisomerization in polymorphic environments of a block copolymer. The objective is to explore how the typical properties of a block copolymer solution such as critical micelle temperature (CMT) and temperature-induced sol-gel transition influence the rotational diffusion of hydrophobic solute molecules. Rotational diffusion of solute molecules differs significantly below and above the CMT of a block copolymer solution, while there is no influence of sol-gel transition on solute rotation. This is rationalized on the basis of the site of solubilization of the solute molecules which is the palisade layer of the micelles in both phases and unaffected by gelation. A similar result has been obtained in case of photoisomerization studies carried out with a carbocyanine derivative in the sol and gel phases of the block copolymer. The isomerization studies have been extended to the reverse phases (sol and gel phases) of the block copolymer to explore the nature of the water present in the cores of the reverse micelles. Our results provide evidence for the existence of water droplets with properties resembling bulk water. In essence, we show that despite having vastly differing bulk properties, both the solution and gel phases (normal as well as reverse) offer identical microscopic environment.

• Fluorescence anisotropy of acridinedione dyes in glycerol: Prolate model of ellipsoid

Time-dependent reorientations of resorcinol-based acridinidione (ADR) dyes in glycerol were studied using steady-state and time-resolved fluorescence studies. The difference between fluorescence anisotropy decays recorded at 460 nm when exciting at 250 nm and those obtained when exciting at 394 nm are reported. When exciting at 394 nm, the fluorescence anisotropy decay is bi-exponential, while on exciting at 250 nm a mono-exponential fluorescence anisotropy decay is observed. We interpret this in terms of different directions of the absorption dipole at 394 and 250 nm with the emission dipole respectively, which is experimentally validated and further analysed as a prolate model of ellipsoid.

• Effect of sodium deoxycholate and sodium cholate on DPPC vesicles: A fluorescence anisotropy study with diphenylhexatriene

Effects of two bile salts, namely sodium deoxycholate (NaDC) and sodium cholate (NaC), on DPPC small unilamellar vesicles have been investigated using the steady-state fluorescence anisotropy ($r_{ss}$) of diphenylhexatriene (DPH) as a tool. It was found that the variation of $r_{ss}$ is sensitive enough to monitor different stages of interaction of bile salts with DPPC vesicles. NaDC induced significant changes in the membrane well below its CMC (6 mM). Even at 4 mM, which is still lower than the CMC, the phospholipids were completely solubilised by the NaDC micelles. The effect of NaC on DPPC vesicles, however, was much less significant, especially in the sub-micellar concentration regime. Being more hydrophilic NaC does not interact with the membrane efficiently. Complete solubilisation of phospholipids took place only when the concentration of NaC was above its CMC (16 mM). The experiments also showed that the bile salt-induced changes of vesicle structure were strongly dependent on the concentration of the bile salt and not on the molar ratio of lipid and bile salt.

• Comparative photophysical behaviour of naphthalene-linked crown ethers and aza crown ethers of varying cavity dimensions

A comparative time-resolved emission studies of several naphtho-crown ethers I-V, where metal ions can be complexed in a predetermined orientation with respect to the naphthalene (Naph) 𝜋- plane and naphthalene-linked aza crown ethers (L1 and L2) have been presented. In both the systems, crown ethers and aza crown ethers, naphthalene fluorescence gets quenched. In the systems I to V, the quenching is mainly due to efficient spin-orbit coupling (SOC) leading to greater population of the lowest triplet state of naphthalene. This SOC depends on the orientation of the crown ring with respect to the Naph-𝜋-plane. However, in the systems L1 and L2, the quenching is due to photoinduced electron transfer (PET) from nitrogen lone pair of the aza crown ring to naphthalene moiety and consequent exciplex formation. The results have been interpreted using the time-resolved emission studies of all the compounds in various solvents, their alkali metal ion complexes, and protonated ligands.

• Effect of surfactants on the fluorescence spectra of water-soluble MEHPPV derivatives having grafted polyelectrolyte chains

Poly(2-methoxy-5-[2'-ethylhexyoxy]-1,4-phenylenevinylene) (MEHPPV) derivatives with polyacrylic acid (PAA) chains grafted onto their backbone were found to be water soluble, and they exhibited a dramatic increase in their fluorescence intensity in the presence of a variety of surfactants, even at concentrations far below their critical micelle concentrations (CMC). This increase was accompanied by a blue-shift in the emission maximum. These observations are rationalized based on the postulate that the backbone conformation of the conjugated polymer is modulated upon interaction of the surfactant molecules with the polyelectrolytic tethers, which in turn results in a significant depletion of intra-chain interchromophore interactions that are known to cause red-shifted emission bands with significantly lower emission yields.

• Photoinduced intramolecular charge-transfer reactions in 4-amino-3-methyl benzoic acid methyl ester: A fluorescence study in condensedphase and jet-cooled molecular beams

Photoinduced intramolecular charge-transfer reactions in 4-amino-3-methyl benzoic acid methyl ester (AMBME) have been investigated spectroscopically. AMBME, with its weak charge donor primary amino group, shows dual emission in polar solvents. Absorption and emission measurements in the condensed phase support the premise that the short wavelength emission band corresponds to local emission and the long wavelength emission band to the charge transfer emission. Laser-induced fluorescence excitation spectra show the presence of two low-energy conformers in jet-cooled molecular beams. Theoretical calculations using density functional theory help to determine structure, vibrational modes, potential energy surface, transition energy and oscillator strength for correlating experimental findings with theoretical results.

• Effect of strong coupling on interfacial electron transfer dynamics in dye-sensitized TiO2 semiconductor nanoparticles

Dynamics of interfacial electron transfer (ET) in ruthenium polypyridyl complex [{bis-(2,2'-bpy)-(4-[2-(4'-methyl-[2,2']bipyridinyl-4-yl)-vinyl]-benzene-1,2-diol)}ruthenium(II) hexafluorophosphate] (Ru-cat) and 5,10,15-tris phenyl-20-(3,4-dihydroxy benzene) porphyrin (TPP-cat)-sensitized TiO2 nanoparticles have been investigated using femtosecond transient absorption spectroscopic detection in the visible and near-infrared region. We have observed that both Ru-cat and TPP-cat are coupled strongly with the TiO2 nanoparticles through their pendant catechol moieties. We have observed a single exponential and pulse-width limited (&lt; 100 fs) electron injection from nonthermalized-excited states of Ru-complex. Here electron injection competes with the singlet-triplet manifold relaxation due to strong coupling of catecholate binding, which is a unique observation.

Optical absorption measurements indicate that the catechol moiety interacts with TiO2 nanoparticles showing the characteristic pure catechol-TiO2 charge-transfer (CT) band in the visible region. Transient absorption studies on TPP-cat/TiO2 system exciting both the Soret band at 400 nm and the Q-band at 800 nm have been carried out to determine excitation wavelength-dependence on ET dynamics. The reaction channel for the electron-injection process has been found to be different for both the excitation wavelengths. Excitation at 800 nm, is found directly populate directly the excited CT state from where diffusion of electrons into the conduction band takes place. On the other hand, excitation at 400 nm light excites both the CT band of cat-TiO2 and also Soret band of TPP-cat.

• # Journal of Chemical Sciences

Volume 132, 2020
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• # Editorial Note on Continuous Article Publication

Posted on July 25, 2019