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Study is conducted using femtosecond time-resolved fluorescence spectroscopy, to show that the solvation dynamics in the two partially folded states (I(sub s)(super ') and I(sub s)(super '') of a protein, cytochrome C, are very different. Solvation dynamics of I(sub s)(super '') displays a major component of 1.3 ps which indicates that the structure of I(sub s)(super '') is much more open and exposed compared to that of I(sub s)(super ').

The solvation dynamics of 4-(dicyanomethylene)-2methyl-6(p-dimethylaminostyryl) 4H-pyran (DCM) inside a dipalmitoyl-phosphatidyaholine (DPPC) entrapped sodium silicate sol-gel matrix is different from that in DPPC lipid vesicles in bulk water or DMPC entrapped in a tetraethyl orthosilicate (TEOS) derived sol-gel matrix. The anisotropy decay of DCM in DPPC vesicles in bulk water and sol-gel glass is strongly biexponential.

The solvation dynamics in the molten globule state of GlnRS is studied using both a covalent probe and a noncovalent probe, bisanilinosulfonate. It is demonstrated that the solvation dynamics in the molten globule state of GlnRS is quite slow.

The solvation dynamics in the water pool of an aerosol-OT (AOT) microemulsion becomes 2 times faster on addition of 1 mM sodium salicylate (Na-sal) and 4-fold slower on addition of 100 mM sodium cholate (Na-cholate). The spectral width of the time-resolved emission spectra of 4-(dicyanomethylene)-2-methyl-6-(p-dimethyl-aminostyryl)-4H-pyran (DCM) in the water pool is time dependent, while no such time-dependent spectral width is detected in bulk water.

Macromolecular crowding bridges in vivo and in vitro studies by simulating cellular complexities such as high viscosity and limited space while maintaining the experimental feasibility. Over the last two decades, the impact of macromolecular crowding on protein stability and activity has been a significant topic of study and discussion, though still lacking a thorough mechanistic understanding. This article investigates the role of associated water dynamics on protein stability and activity within crowded environments, using bromelain and Ficoll-70 as the model systems. Traditional crowding theory primarily attributes protein stability to entropic effects (excluded volume) and enthalpic interactions. However, our recent findings suggest that water structure modulation plays a crucial role in a crowded environment. In this report, we strengthen the conclusion of our previous study, i.e., rigid-associated water stabilizes proteins via entropy and destabilizes them via enthalpy, while flexible water has the opposite effect. In the process, we addressed previous shortcomings with a systematic concentration-dependent study using a single-domain protein and component analysis of solvation dynamics. More importantly, we analyze bromelain's hydrolytic activity using the Michaelis-Menten model to understand kinetic parameters like maximum velocity ( V max ) achieved by the system and the Michaelis-Menten coefficient ( K M ). Results indicate that microviscosity (not the bulk viscosity) controls the enzyme-substrate ( ES ) complex formation, where an increase in the microviscosity makes the ES complex formation less favorable. On the other hand, flexible associated water dynamics were found to favor the rate of product formation significantly from the ES complex, while rigid associated water hinders it. This study improves our understanding of protein stability and activity in crowded environments, highlighting the critical role of associated water dynamics.

Traditionally, deviation from Stokes-Einstein-Debye (SED) relation in terms of viscosity dependence of medium dynamics, i.e., τ x ∝ ( η T ) p with p ≠ 1, is taken as a signature of dynamic heterogeneity. However, it does not guarantee medium heterogeneity, as the decoupling may also originate from the deviation of the basic assumption of SED. Here, we developed a method to find a stronger relation between viscosity decoupling ( p ≠ 1) and dynamic heterogeneity in terms of rotational motion. Our approach exploited the fact that in heterogeneous media, a solvatochromic probe will be solvated to a different extent at different microdomains (subpopulations), and photoselection of these subpopulations can be achieved by excitation or emission wavelength-dependent measurements. We hypothesized that the dynamics of a homogeneous system might show viscosity decoupling, but the extent of decoupling at different excitations (or at different emissions) should not be different. On the other hand, in a heterogeneous medium, this extent of viscosity decoupling ( p -value) should be different at different excitations (or at different emissions). As proof of concept, we investigated three versatile solvent media: squalane (viscous molecular liquid), 1-ethyle-3-methylimidazolium ethyl sulfate ionic liquid (IL), and [0.78 acetamide + 0.22 LiNO 3 ] deep eutectic solvent (DES). We found that squalane is homogeneous, although it shows fractional viscosity dependence ( p ≠ 1). Interestingly, mild heterogeneity in IL and significant heterogeneity in the DES were observed. Overall, we conclude that the difference in the p -value as a function of excitation (or emission) wavelength-dependent might be a superior way for the detection of dynamic heterogeneity.

Deep eutectic solvents (DESs) are new-generation media that can be fine-tuned to have desired properties circumventing economic and environmental issues. Typically, these are ionic, and only recently, nonionic DESs, having interesting properties, are being explored. In this report, we examined the structure and dynamics of a nonionic lauric acid/menthol (LA/Men) DES through steady-state emission, solvation dynamics, time-resolved fluorescence anisotropy, and translational diffusion dynamics. The zero shift in the emission spectra of coumarin 153 (a solvatochromic dye) as a function of the excitation wavelength suggests that LA/Men DES is spatially homogenous. Decoupling ( p = 0.63) of the average solvation time, ⟨τ s ⟩, from medium viscosity suggests the presence of mild dynamic heterogeneity in the system. Rotational time, ⟨τ r ⟩, which reflects the nature of the first solvation shell, shows little decoupling ( p = 0.81), suggesting it to be fairly dynamically homogeneous at a shorter length scale. An Arrhenius-type analysis also proves that rotation is mainly controlled by medium viscosity. Translational diffusion time, ⟨τ D ⟩, which provides information at a larger length scale, is strongly decoupled from medium viscosity ( p = 0.29). This indicates that at a larger length scale, the DES is quite dynamically heterogeneous. The slow component of solvation time, which is believed to originate at a larger length scale, correlates well with the translational diffusion timescale having similar activation energies. This suggests that their origin is same. Expectedly, for the long component of solvation time, the decoupling is quite strong ( p = 0.30). Overall, our result demonstrates the structure and dynamics of the nonionic LA/Men DES, and the existence of length scale-dependent heterogeneity has been proposed.

One of the main differences in the intercellular environment compared to the laboratory condition is the presence of macromolecular crowders of various compositions, sizes, and shapes. In this article, we have contemplated a systematic shape dependency of macromolecular crowders on the thermodynamics and microsecond conformational fluctuation dynamics of protein unfolding by taking human serum albumin (HSA) as the model protein and similar-sized crowders, namely, dextran-40, ficoll-70, and PEG-35 as macromolecular crowders of different shapes, to mimic the cell environment. We observed that dextran-40 and ficoll-70 counteract the thermal denaturation and PEG-35 assists it. A complete thermodynamic analysis suggests that the stabilization by dextran-40 and ficoll-70 occurs mainly through stabilizing entropic effect, which is somewhat counteracted by the destabilizing enthalpic effect, in line with what is expected from the traditional interpretation of excluded volume and soft interaction. Surprisingly, the destabilizing effect of PEG-35 is not through unfavorable interaction but through a destabilizing entropic effect, which is opposite to the excluded volume prediction. Our speculation is that the modulation of the associated water structure due to crowder-induced distortion plays a crucial role in modulating the entropic component. Moreover, while a two-state model can approximate the overall thermal denaturation of HSA in the absence and presence of various crowders, the thermal denaturation profile of domain III of HSA involves a distinct intermediate state. The active-site dynamics of HSA are altered significantly in the presence of all the three differently shaped crowders used in the study. Rod-shaped dextran-40 and spherical ficoll-70 hinder the microsecond conformational dynamics of domain III of HSA in all states except the intermediate state. Mesh-like PEG-35 in addition to hindering the microsecond conformational dynamics shifts the intermediate state from 40 to 30 °C. Overall, our results provide new insight into deciphering the mechanism of crowder-induced changes in protein. Through our interpretation, we not only explain the unfavorable entropic contribution but also provide a physical basis to explain the entropy-enthalpy compensation.

The influence of mono- and bivalent anions and micelle/submicellar clusters of different ionic and nonionic surfactants on the hydrolysis of malachite green (MG + ) was studied in the presence of low alkali concentrations. Large polarizable I - ion and bivalent SO 4 2- ion show specific interaction toward MG + with the formation of dye-anion ion-pair resulting in a retardation of the reaction rate. Tensiometric studies for anionic surfactants sodium dodecyl sulfate (SDS) and dioctyl sodium sulfosuccinate (AOT) in the presence of dye indicate the formation of ion-pair micelles which subsequently break down to form normal micelles. A change of the absorbance value accompanied by a red shift in the electronic spectra with increase in surfactant concentration also evidenced the formation of ion-pair. The observed rate inhibition by the anionic surfactants and the interaction behavior/nature of binding between the dye and surfactant in the premicellar region have been analyzed in terms of different kinetic models. The Piszkiewicz cooperativity index ( n ) suggests 1:1 association between the anionic surfactant monomer and MG + in the submicellar cluster. A catalyzing effect for the cationic surfactant, cetyltrimethylammonium bromide (CTAB), was noticed in the post-CMC region leading toward a limiting reaction rate, and the Menger-Portonoy pseudophase model was employed to explain the behavior with evaluation of the binding parameter. The nonionic surfactant poly(oxyethylene) (20) sorbitan monolaurate (Tween 20) exerts inhibition effect on the hydrolysis reaction which may be explained qualitatively by the dearth of HO - ions in the vicinity of the micelle-bound dye and quantitatively in light of the pseudophase model. Different equilibrium constants and binding constant parameters suggest appreciable binding between the dye and surfactant monomer/micelles owing to electrostatic/hydrophobic interactions.

Fluorescence up-conversion (∼250 fs instrumental response) coupled with time correlated single photon counting measurements was performed to explore the complete Stokes shift dynamics of a dipolar solute probe, coumarin 153 (C153), in several ionic acetamide deep eutectic solvents (DESs) that contained lithium nitrate/bromide/perchlorate as electrolyte. Combined measurements near room temperature reflected a total dynamic Stokes shift of approximately 800-1100 cm -1 and triexponential solvation response functions. Interestingly, the average rate of solvation became faster upon successive replacement of bromide by nitrate in these deep eutectics, and a subpicosecond time scale emerged in the measured solvation response when bromide was fully replaced by nitrate. Temperature dependent solute diffusion in these deep eutectics at the single molecule level, monitored by tracking the translational motion of rhodamine 6G (R6G) via fluorescence correlation spectroscopic (FCS) technique, revealed pronounced fractional viscosity dependence of the solute's translational motion. Subsequently, this partial decoupling of solute translation was attributed to the microheterogeneous nature of these ionic DESs after examining the diffusion-viscosity relationship via the FCS measurements of R6G in several normal solvents at room temperature and in a liquid amide solvent at different temperatures.

Fluorescence quenching studies through steady-state and time-resolved measurements are inadequate to quantify the bimolecular electron transfer rate in bulk homogeneous solution due to constraints from diffusion. To nullify the effect of diffusion, direct evaluation of the rate of formation of a transient intermediate produced upon the electron transfer is essential. Methyl viologen, a well-known electron acceptor, produces a radical cation after accepting an electron, which has a characteristic strong and broad absorption band centered at 600 nm. Hence it is a good choice to evaluate the rate of photoinduced electron transfer reaction employing femtosecond broadband transient absorption spectroscopy. The time constant of the aforementioned process between pyrene and methyl viologen in methanol has been estimated to be 2.5 ± 0.4 ps using the same technique. The time constant for the backward reaction was found to be 14 ± 1 ps. These values did not change with variation of concentration of quencher, i.e., methyl viologen. Hence, we can infer that diffusion has no contribution in the estimation of rate constants. However, on changing the solvent from methanol to ethanol, the time constant of the electron transfer reaction has been found to increase and has accounted for the change in solvent reorganization energy.

Steady-state absorption, emission, and femtosecond transient absorption spectroscopies were used to ascertain the static and dynamic nature of the solvent response of methanol-chloroform binary solvent mixtures of different stoichiometric ratios using 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM) as the probe molecule. The appearance of synergistic solvation behavior in the steady-state absorption measurements can be explained in terms of solvent-solvent interactions through an extended hydrogen-bonding network. The disappearance of such synergistic behavior in the excited state of the DCM dye was recently proposed by us to be due to the weak nature of the intermolecular interactions present in binary solvent mixtures (J. Phys. Chem. B 2012, 116, 1345). It was anticipated and subsequently confirmed by the dynamics of the solvent response that the disruption of the weak interactive solvent network is the main reason for the absence of the synergism in the excited state. As expected, we observed the slowest dynamics for the mixture with X(MeOH) = 0.45, with an average solvation time of 12.03 ps, which is much higher than the values for the pure bulk counterparts (⟨τ(s)⟩(Methanol) = 4.32 ps and ⟨τ(s)⟩(Chloroform) = 1.32 ps). The unprecedented slowing of solvation for DCM is probably due to the rigid interactive methanol-chloroform solvent system in the first solvation shell, followed by solvent rearrangements around the solute dipole. Overall interactions present within the methanol-chloroform binary solvent mixture furnish clear evidence of solvent association through weak hydrogen bonding.

The present study elucidates the involvement of conformational fluctuation dynamics during chemically and thermally induced unfolding of human serum albumin (HSA) by fluorescence correlation spectroscopic (FCS) study, time-resolved fluorescence measurements, and circular dichroism (CD) spectroscopic methods. Two fluorescent probes, tetramethylrhodamine-5-maleimide (TMR) and N-(7-dimethylamino-4-methylcoumarin-3-yl) iodoacetamide (DACIA) were used to selectively label the domain I of HSA through the reaction with cys-34 for these studies. The guanidine hydrochloride (GnHCl) induced global structural change of HSA is monitored through its hydrodynamic radius (r(H)) and CD response, which is found to be two step in nature. In FCS experiment, along with the diffusion time component we have observed an exponential relaxation time component (τ(R)) that has been ascribed to the concerted chain dynamics of HSA. Unlike in the global structural change, we found that the τ(R) value changes in a different manner in the course of the unfolding. The dependence of τ(R) on the concentration of GnHCl was best fitted with a four state model, indicating the involvement of two intermediate states during the unfolding process, which were not observed through the CD response and r(H) data. The fluorescence lifetime measurement also supports our observation of intermediate states during the unfolding of HSA. However, no such intermediate states were observed during thermally induced unfolding of HSA.

Femtosecond fluorescence up-conversion measurements of malachite green (MG) have been carried out to confirm the relaxation pathway and subsequently to probe the microviscosity of water trapped in a nanoconfined environment using an AOT (sodium dioctylsulfosuccinate, aerosol-OT) reverse micelle as a model system. The experimental results reveal a strong dependence of S(1) state relaxation dynamics of MG on solvent viscosity while a very weak dependence has been observed for the S(2) state relaxation. The time-dependent density functional theory (TD-DFT) calculations have been used to construct potential energy surfaces of MG by pursuing an intramolecular rotation along the torsional coordinate of the phenyl rings. On synchronization with the experimental observations, the computational results comprehend the existence of a conical intersection along the S(1) and S(0) potential energy surfaces, which leads to mixed vibrational levels of S(1) and S(0) characteristics. The results suggest that the conical intersection is along the torsional coordinate of N,N-dimethyl substituted phenyl ring. Correlating the observed dynamics of MG in a confined system with the relaxation time of MG in different glycerol-water mixtures, we assert the determination of the microviscosity of water inside the AOT reverse micelle. The data confer that the microviscosity of water in an AOT water pool of w(0) = 2 (9 cP) is almost 9 times higher than the bulk water. As we increase the w(0) from 2 to 40, the microviscosity decreases monotonically to 5.68 cP, and the decrease is observed to be exponential in nature.