Time-dependent Fluorescence Stokes Shift Research Articles

Spectroscopic studies on the supramolecular interactions of methyl benzoate derivatives with p-sulfocalix[6]arene macrocycles

This paper is a continuation of our previous research and aims to further investigate and elucidate the nature and mechanisms of noncovalent supramolecular interactions between four methyl benzoate derivatives (I-IV), which are capable of exhibiting Twisted Intramolecular Charge Transfer (TICT) and/or Excited State Intramolecular Proton Transfer (ESIPT)‐type behavior, and chemical and biological nanocavities. Photophysical and photochemical properties of molecules I-IV in aqueous solution in the presence of well-recognized macrocyclic host p-sulfocalix[6]arenes (SCA[6]) have been studied using steady-state, time-resolved and 1H NMR spectroscopic techniques. The changes in the ground- and excited-state spectroscopic characteristics (absorption and fluorescence spectra, time-resolved fluorescence spectra, fluorescence decay times and 1H NMR spectra) undergo significant modifications upon encapsulation of the investigated methyl benzoate derivative in the macromolecular cavity. For the two compounds (I and II), the interactions with the macrocycles with a hydrophobic SCA[6] cavity lead to the formation of stable inclusion complexes with 1:1 stoichiometry, both in the ground and excited state, while the stoichiometry of the III-SCA[6] and IV-SCA[6] complexes in the ground and excited states is 1:2. The values of the equilibrium constants have been determined from the spectroscopic data using Benesi-Hildebrand and nonlinear regression procedures. The location of the organic molecule inside the SCA[6] has been investigated by 1H NMR experiments. The changes in macrocyclic compound-induced NMR chemical shifts clearly indicate that the chemical structure of inclusion complexes is very different for methyl benzoate derivative-SCA[6] and methyl benzoate derivative-CB[7] systems. Finally, we have shown, using time-dependent fluorescence Stokes shift, that very fast solvation dynamics of pure water is markedly different from that of the confined water molecule in SCA[6] system.

Dynamic Electric Field Alignment Determines the Water Rotational Motion around Protein.

Water rotational dynamics in biomolecular solution is crucial to evaluating and controlling biomolecule stability. In this molecular dynamics simulation (MD) study on lysozyme solutions, we present how the exerted internal electric field determines water rotational dynamics. We find that the relaxation time of water rotation is equivalent to that of the reorientation of the exerted overall electric field for every single water molecule, regardless of its translation mode. Namely, water molecular rotation synchronizes with the exerted field reorientation. We also map the reorientation process of the electric field at fixed points relative to protein in the solution, which displays the local hydration dynamics commensurate with the reported time-dependent fluorescence Stokes shift (TDFSS) measurements. Comparing the spatial distribution of local field reorientation relaxation time with that of rotational relaxation time, we further suggest that water rotation dynamics are subject to the reorientation of the local overall field within the hydration layer. While outside the hydration layer, the relaxation time of the local electric field reorientation is short enough (subpicosecond) to assume the δ function, showing the electric force with randomly changing orientation is applied to each water molecule.

Ultraslow Biological Water-Like Dynamics in Waterless Liquid Protein.

Fluorescence correlation spectroscopy and time-dependent fluorescence Stokes shift have been employed to elucidate dynamics in different time scales, ranging from picoseconds to nanoseconds, for human serum albumin, in its native and cationized forms as well as in the self-assembled complex of the cationized protein with the polymer surfactant (PS) glycolic acid ethoxylate lauryl ether. The effect of crowding in this complex, especially in the waterless condition, is of prime importance in this context. Excellent correlation of the dynamics with the structures, obtained by circular dichroism and Fourier transform infrared spectroscopy, has been observed. Slow solvation, associated classically with biological water, has been observed in these systems, even in the waterless condition. This apparently intriguing observation has been rationalized by the relaxation of segments of the protein and the PS in the microenvironment of the fluorescent probe.

Time-Resolved Probing of the Nonequilibrium Structural Solvation Dynamics by the Time-Dependent Stokes Shift.

The time-dependent fluorescence Stokes shift monitors the relaxation of the polarization of a polar solvent in the surroundings of a photoexcited solute molecule but also the structural variation of the solute following photoexcitation and the subsequent molecular charge redistribution. Here, we formulate a simple nonequilibrium quantum theory of solvation for an explicitly time-dependent continuous solvent. The time-dependent solvent induces nonequilibrium fluctuations on the solvent dynamics which are directly reflected in different time components in the time-dependent Stokes shift. We illustrate the structural dynamics in the presence of an explicitly time-dependent solvent by the example of a dynamically shrinking solute which leads to a bimodal Stokes shift. Interestingly, both contributions are mutually coupled. Furthermore, we can explain a prominent long-tail decay of the Stokes shift associated with slow structural dynamical variations.

Complete Solvation Dynamics of Coumarin 153 in Tetraalkylammonium Bromide-Based Deep Eutectic Solvents.

Deep eutectic solvents (DESs) are novel environment-friendly media for a variety of applications. In order to obtain insight into the structure and dynamics of some less-explored DESs comprising ethylene glycol and tetraalkylammonium bromide salts with variable alkyl chain length, we have captured complete dynamics occurring in these solvents in a timescale of few femtoseconds to several nanoseconds by monitoring the time-dependent fluorescence Stokes shift of coumarin 153 employing a combination of time-correlated single-photon counting and fluorescence upconversion techniques. The solvent response function constructed from the measured data reveals a sub-picosecond component (∼0.8 ps, 20-35%) in addition to a slow component (180-475 ps) with a distribution of relaxation time. The slow time component is found to be strongly dependent on the viscosity of the medium, indicating that it arises from the diffusive motions of the solvent constituents into and out of the solvation shell, whereas the ultrafast time component, which is nearly independent of the solvent viscosity, arises from fast local motions of the constituents in the immediate vicinity of the solute molecule.

Modulation of Kinetic Pathways of Enzyme-Substrate Interaction in a Microfluidic Channel: Nanoscopic Water Dynamics as a Switch.

Enzyme-mediated catalysis is attributed to enzyme-substrate interactions, with models such as "induced fit" and "conformational selection" emphasizing the role of protein conformational transitions. The dynamic nature of the protein structure, thus, plays a crucial role in molecular recognition and substrate binding. As large-scale protein motions are coupled to water motions, hydration dynamics play a key role in protein dynamics, and hence, in enzyme catalysis. Here, microfluidic techniques and time-dependent fluorescence Stokes shift (TDFSS) measurements are employed to elucidate the role of nanoscopic water dynamics in the interaction of an enzyme, α-Chymotrypsin (CHT), with a substrate, Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC) in the cationic reverse micelles of benzylhexadecyldimethylammonium chloride (BHDC/benzene) and anionic reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT/benzene). The kinetic pathways unraveled from the microfluidic setup are consistent with the "conformational selection" fit for the interaction of CHT with AMC in the cationic reverse micelles, whereas an "induced fit" mechanism is indicated for the anionic reverse micelles. In the cationic reverse micelles of BHDC, faster hydration dynamics (≈550 ps) aid the pathway of "conformational selection", whereas in the anionic reverse micelles of AOT, the significantly slower dynamics of hydration (≈1600 ps) facilitate an "induced fit" mechanism for the formation of the final enzyme-substrate complex. The role of water dynamics in dictating the mechanism of enzyme-substrate interaction becomes further manifest in the neutral reverse micelles of Brij-30 and Triton X-100. In the former, the faster water dynamics aid the "conformational selection" pathway, whereas the significantly slower dynamics of water molecules in the latter are conducive to the "induced fit" mechanism in the enzyme-substrate interaction. Thus, nanoscopic water dynamics act as a switch in modulating the pathway of recognition of an enzyme (CHT) by the substrate (AMC) in reverse micelles.

Role of excited state solvent fluctuations on time-dependent fluorescence Stokes shift.

We explore the connection between the solvation dynamics of a chromophore upon photon excitation and equilibrium fluctuations of the solvent. Using molecular dynamics simulations, fluorescence Stokes shift for the tryptophan in Staphylococcus nuclease was examined using both nonequilibrium calculations and linear response theory. When the perturbed and unperturbed surfaces exhibit different solvent equilibrium fluctuations, the linear response approach on the former surface shows agreement with the nonequilibrium process. This agreement is excellent when the perturbed surface exhibits Gaussian statistics and qualitative in the case of an isomerization induced non-Gaussian statistics. However, the linear response theory on the unperturbed surface breaks down even in the presence of Gaussian fluctuations. Experiments also provide evidence of the connection between the excited state solvent fluctuations and the total fluorescence shift. These observations indicate that the equilibrium statistics on the excited state surface characterize the relaxation dynamics of the fluorescence Stokes shift. Our studies specifically analyze the Gaussian fluctuations of the solvent in the complex protein environment and further confirm the role of solvent fluctuations on the excited state surface. The results are consistent with previous investigations, found in the literature, of solutes dissolved in liquids.

Time-dependent fluorescence Stokes shift and molecular-scale dynamics in alginate solutions and hydrogels

Alginates are water-soluble polysaccharides that bind metal cations like Ca2+, producing hydrogels. Here, we have determined time-dependent fluorescence Stokes shift of a guest fluorophore to elucidate molecular length-scale local dynamics within alginate-based solutions and hydrogels. We find a major bulk water-like fast response emanating from large water pools interspersed between the polysaccharide chains, together with a minor but significant slow response. The possible origin of the latter is discussed in terms of either water molecules constituting the polysaccharide hydration shells or ion distribution and diffusion around the fluorophore dipole, or microscopic structural inhomogeneity inside the alginate-based media.

Anomalous power law decay in solvation dynamics of DNA: a mode coupling theory analysis of ion contribution

Several time dependent fluorescence Stokes shift (TDFSS) experiments have reported a slow power law decay in the hydration dynamics of a DNA molecule. Such a power law has neither been observed in computer simulations nor in some other TDFSS experiments. Here we observe that a slow decay may originate from collective ion contribution because in experiments DNA is immersed in a buffer solution, and also from groove bound water and lastly from DNA dynamics itself. In this work we first express the solvation time correlation function in terms of dynamic structure factors of the solution. We use mode coupling theory to calculate analytically the time dependence of collective ionic contribution. A power law decay in seen to originate from an interplay between long-range probe–ion direct correlation function and ion–ion dynamic structure factor. Although the power law decay is reminiscent of Debye–Falkenhagen effect, yet solvation dynamics is dominated by ion atmosphere relaxation times at longer length scales (small wave number) than in electrolyte friction. We further discuss why this power law may not originate from water motions which have been computed by molecular dynamics simulations. Finally, we propose several experiments to check the prediction of the present theoretical work.

Charge Invariant Protein–Water Relaxation in GB1 via Ultrafast Tryptophan Fluorescence

The protein–water interface is a critical determinant of protein structure and function, yet the precise nature of dynamics in this complex system remains elusive. Tryptophan fluorescence has become the probe of choice for such dynamics on the picosecond time scale (especially via fluorescence “upconversion”). In the absence of ultrafast (“quasi-static”) quenching from nearby groups, the TDFSS (time-dependent fluorescence Stokes shift) for exposed Trp directly reports on dipolar relaxation near the interface (both water and polypeptide). The small protein GB1 contains a single Trp (W43) of this type, and its structure is refractory to pH above 3. Thus, it can be used to examine the dependence of dipolar relaxation upon charge reconfiguration with titration. Somewhat surprisingly, the dipolar dynamics in the 100 fs to 100 ps range were unchanged with pH, although nanosecond yield, rates, and access all changed. These results were rationalized with the help of molecular dynamics (including QM-MM) simulations that reveal a balancing, sometimes even countervailing influence of protein and water dipoles. Interestingly, these simulations also showed the dominant influence of water molecules which are associated with the protein interface for up to 30 ps yet free to rotate at approximately “bulk” water rates.

Ultrafast Time-Resolved Broadband Fluorescence Studies of the Benzene-Tetracyanoethylene Complex: Solvation, Vibrational Relaxation, and Charge Recombination Dynamics

The charge-transfer (CT) state relaxation dynamics of the benzene-tetracyanoethylene (BZ-TCNE) complex was studied with broadband ultrafast time-resolved fluorescence spectroscopy implemented by optical Kerr gating in three solvents of different polarities. The CT state of the BZ-TCNE complex is reached via femtosecond laser excitation, and the subsequent temporal evolutions of the fluorescence spectra were measured. Analyses of various time-dependent spectral properties revealed rapid relaxations along solvent and vibrational coordinates in competition with charge recombination (CR). By comparing the results in solvents of different polarities, we partially separated solvation and vibrational relaxation dynamics and explored the solvent-dependent CR dynamics. Time-dependent dynamic fluorescence Stokes shift (TDFSS) measurements unveiled the solvation and vibrational relaxation contributions to the observed spectral relaxation. The biphasic and slow time scales of the vibrational contributions identified in TDFSS suggested nonstatistical and hindered intramolecular vibrational-energy redistribution that can be attributed to the unique structural properties of EDA complexes. The slowest spectral relaxation of 10-15 ps identified in TDFSS was ascribed to relaxation of the BZ(+)-TCNE(-) intermolecular vibrations, which is equivalent to a structural relaxation from the initial Franck-Condon configuration to the equilibrium CT-state structure. The time scales of vibrational relaxation indicate that a fraction of the CT-state population undergoes CR reactions before complete vibrational/structural equilibrium is achieved. In carbon tetrachloride, a nonexponential temporal profile was observed and attributed to vibrational nonequilibrium CR. In dichloromethane, polar solvation greatly accelerates CR reactions, and a slower reaction-field-induced structural relaxation gives rise to a pronounced biexponential decay. The equilibrium CR time constants of the BZ-TCNE CT state are 29 ps, 150 ps, and 68 ps in dichloromethane, carbon tetrachloride, and cyclohexane, respectively.

Fluorescence Response of Coumarin-153 in N-Alkyl-N-methylmorpholinium Ionic Liquids: Are These Media More Structured than the Imidazolium Ionic Liquids?

The fluorescence behavior of coumarin-153 (C153) has been studied in four N-alkyl-N-methylmorpholinium ionic liquids differing in the alkyl chain length attached to the N-methylmorpholinium cation as a function of the excitation wavelength and temperature to understand some of the physicochemical characteristics of these largely unexplored ionic liquids. While the polarity of the ionic liquid with the smallest alkyl chain length is found comparable to that of the commonly used imidazolium ionic liquids, the probe molecule experiences a less polar environment with increasing chain length of the alkyl group attached to the morpholinium cation. The room temperature steady-state fluorescence spectrum of C153 in these solvents is found to be dependent on the excitation wavelength, and this effect is most pronounced in long chain containing ionic liquids. A bathochromic shift of the fluorescence maximum is observed at higher temperature. The excitation wavelength and temperature dependence of the fluorescence of C153 is explained considering a domain structure of these ionic liquids. The time-resolved fluorescence anisotropy measurements indicate the microviscosity around the probe molecule to be significantly different from the bulk viscosity of the long-chain ionic liquids. The solvent reorganization dynamics, as studied by monitoring the time-dependent fluorescence Stokes shift of C153 in these ionic liquids, is found to be slow and similar to that in imidazolium ionic liquids. The time-resolved measurements under isoviscous conditions seem to provide additional support to the organized domain structure of these ionic liquids.

Solvation and rotational relaxation of coumarin 153 in a new hydrophobic ionic liquid: An excitation wavelength dependence study

The solvent relaxation and rotational dynamics of coumarin 153 have been investigated in a new room temperature ionic liquid, 1-(2-methoxyethyl)-1-methylpyrrolidiniumtris(pentafluorethyl)trifluorophosphate [[MOEMPL][FAP]], at three different excitation wavelengths with a variation in temperature. Wavelength -dependent fluorescence decay behavior of the probe molecule in the present medium has been investigated by studying the time dependent fluorescence Stokes shift in the ps–ns time scale. The dynamic fluorescence Stokes shift measurements suggest that the time-resolvable part of the solvation dynamics is biphasic in nature and the average solvation time depends on the excitation wavelengths. Rotational coupling constants, obtained from the time- resolved anisotropy data, indicate no specific interaction between the probe molecule and the ionic liquids. The excitation wavelength dependent solvation dynamics is attributed to the heterogeneous nature of the present ionic liquid.

Excited state dynamics of 9,9′-bianthryl in room temperature ionic liquids as revealed by picosecond time-resolved fluorescence study

Picosecond time-resolved fluorescence measurements have been carried out on 9,9′-bianthryl in three imidazolium ionic liquids to probe the excited state dynamics. In the early time-scale, the fluorescence spectra of bianthryl have been found to consist of emission from both locally excited (LE) and charge transfer (CT) states. The LE → CT relaxation time, as estimated from the decay of the fluorescence intensity of the LE emission, is found to vary between 230 and 390 ps, while the average solvent relaxation time, as estimated from the analysis of time-dependent fluorescence Stokes shift, is found to vary between 620 ps and 1840 ps, depending on the viscosity of the ionic liquids. The results confirm that while in conventional less viscous solvents the CT formation kinetics of bianthryl occurs simultaneously with the solvation dynamics, in ionic liquids the two processes mostly occur in different time scales.

Hydration Dynamics As Revealed By The Fluorescence Stokes Shift: The Origin Of Slow Hydration Dynamics And Breakdown Of Linear Response

Hydration dynamics in the immediate vicinity of a protein, probed by time-dependent fluorescence Stokes shift experiments, is critical to understand its biological function. Protein Stokes shifts typically exhibit biphasic relaxation following photo-excitation: fast relaxation occurs on a time scale of several picoseconds while slower components indicate additional hydration dynamics on a time scale of tens of picoseconds, or longer. Theoretical studies using both linear response and non-equilibrium molecular dynamics (MD) calculation qualitatively reproduce the observed biphasic behavior of time dependent Stokes shift for Trp-7 (W7) in myoglobin. Comparison with constrained MD simulations with protein frozen at the instant of photo-excitation reveals the molecular mechanism of slow hydration process and establishes the critical role of protein flexibility. Coupled protein-water motion is shown to be necessary for the observation of the slow component of hydration dynamics. Qualitatively similar results are found for a series of additional cases, such as monellin and staph. nuclease. We illustrate why tracking the separate contributions to the Stokes shift without constrained MD studies may not yield an accurate interpretation of protein hydration dynamics. Additionally, we examine the extent to which protein fluctuations obey Gaussian statistics and the linear response approximation to the Stokes shift is valid. Equilibrium fluctuations of the ground-excited energy difference, which control the absorption and fluorescence line shapes, in the ground and excited electronic states are not independent of each other. We illustrate how small differences from Gaussian statistics in one electronic state can be a signature of very significant deviations from linear response theory, such as isomerization, in the other electronic state.

Separation and Analysis of Dynamic Stokes Shift with Multiple Fluorescence Environments: Coumarin 153 in Bovine β-Lactoglobulin A

We use time-dependent fluorescence Stokes shift (TDFSS) information to study the fluctuation rates of the lipocalin, beta-lactoglobulin A in the vicinity of an encapsulated coumarin 153 molecule. The system has three unique dielectric environments in which the fluorophore binds. We develop a method to decompose the static and dynamic contributions to the spectral heterogeneity. This method is applied to temperature-dependent steady-state fluorescence spectra providing us with site-specific information about thermodynamic transitions in beta-lactoglobulin. We confirm previously reported transitions and discuss the presence of an unreported transition of the central calyx at 18 degrees C. Our method also resolves the contributions to the TDFSS from the coumarin 153 centrally located in the calyx of beta-lactoglobulin despite overlapping signals from solvent exposed dyes. Our experiments show dynamics ranging from 3-12,00 ps. The analysis shows a decrease in the encapsulated dye's heterogeneity during the relaxation, which is taken as evidence of the breakdown of linear response.

Solute Rotation and Solvation Dynamics in an Alcohol-Functionalized Room Temperature Ionic Liquid

Steady-state and time-resolved fluorescence behaviors of two dipolar solutes, coumarin 153 and 4-aminophthalimide, have been studied in an alcohol-functionalized room-temperature ionic liquid, 1-(hydroxyethyl)-3-methylimidazolium bis(trifluoromethanesulfonyl)imide. The steady-state fluorescence parameters have been exploited for the estimation of the polarity of this ionic liquid and to obtain information on the hydrogen bonding interaction between the ionic liquid and the probe molecules. The time-resolved measurements have been focused on the dynamics of solvation by studying the dynamic Stokes shift in the ps-ns time scale and solute rotation by measuring the time dependence of the fluorescence anisotropy. The time-resolved anisotropy studies reveal a significant slow down of the rotational motion of one of the probe molecules. The time-dependent fluorescence Stokes shift measurements suggest that the time-resolvable part of the dynamics is biphasic in nature, highly dependent on the probe molecule and the ultrafast component is comparatively less than that in other ionic liquids. The influence of the hydrogen bonding interaction between the probe molecules and the ionic liquids on the solute rotation and the various components of the solvation dynamics is carefully analyzed in an attempt to obtain further insight into the mechanism of solvation in these novel media.

Edge excitation red shift and energy migration in quinine bisulphate dication

Photoinduced excited state dynamical processes in quinine sulphate dication (QSD) have been studied over a wide range of solute concentrations using steady state and nanosecond time-resolved fluorescence spectroscopic techniques. The edge excitation red shift (EERS) of emission maximum, emission wavelength dependence of fluorescence lifetimes and the time dependence of emission maximum are known to occur due to the solvent relaxation process. With increase in solute concentration, the emission spectrum shifts towards the lower frequencies accompanied with decrease in fluorescence intensity, however, absorption spectrum remains unchanged. A decrease in EERS, fluorescence lifetimes, time dependent fluorescence Stokes shift (TDFSS), fluorescence polarization and the solvent relaxation time ( τ r) is observed with the increase in solute concentration. The process of energy migration among the QSD ions along with solvent relaxation has been found responsible for the above experimental findings.

Slow Solvation Dynamics at the Active Site of an Enzyme: Implications for Catalysis

Solvation dynamics at the active site of an enzyme, glutaminyl-tRNA synthetase (GlnRS), was studied using a fluorescence probe, acrylodan, site-specifically attached at cysteine residue C229, near the active site. The picosecond time-dependent fluorescence Stokes shift indicates slow solvation dynamics at the active site of the enzyme, in the absence of any substrate. The solvation dynamics becomes still slower when the substrate (glutamine or tRNA(Gln)) binds to the enzyme. A mutant Y211H-GlnRS was constructed in which the glutamine binding site is disrupted. The mutant Y211H-GlnRS labeled at C229 with acrylodan exhibited significantly different solvent relaxation, thus demonstrating that the slow dynamics is indeed associated with the active site. Implications for catalysis and specificity have been discussed.

Investigation Of Tetherable Distilbazolium Compounds As Fluorescent Probes In Nanostructured Silica Sol-gel Materials

Distibazolium dyes are investigated by steady-state and time-resolved fluorescence techniques in a series of low- and high-viscosity polar solvents and in a silica sol-gel matrix. In all solvents and the sol-gel matrix, an interplay of photoinduced switching between different cis-trans isomers and solvation dynamics is observed. Even in a viscous solution (glycerol) and in silica gel, cis-trans isomerization is solvent-controlled. Whereas in glycerol the solvation results in a time-dependent fluorescence Stokes shift, the solvation-induced spectral heterogeneity in silica gel is mostly static, possibly due to a close proximity of dye molecules to the silica surfaces of the nanopores. Compared to low-viscosity solvents, where the cis-trans isomerization process takes place with a solvent-dependent rate on the timescale of about 120-150 ps, it slows down to about 1100-1400 ps in glycerol and about 1500 ps in a sol-gel matrix. Additionally, fluorescence kinetics of the dyes in the sol-gel reveals the presence of a range of different "frozen-in" conformers exhibiting a broad spectrum of lifetimes from 20 to 300 ps.