An inducible amphipathic helix within the intrinsically disordered C terminus can participate in membrane curvature generation by peripherin-2/rds.

Peripherin-2/rds is required for biogenesis of vertebrate photoreceptor outer segment organelles. Its localization at the high-curvature rim domains of outer segment disk membranes suggests that it may act to shape these structures; however, the molecular function of this protein is not yet resolved. Here, we apply biochemical, biophysical, and imaging techniques to elucidate the role(s) played by the protein's intrinsically disordered C-terminal domain and an incipient amphipathic α-helix contained within it. We investigated a deletion mutant lacking only this α-helix in stable cell lines and Xenopus laevis photoreceptors. We also studied a soluble form of the full-length ∼7-kDa cytoplasmic C terminus in cultured cells and purified from Escherichia coli The α-helical motif was not required for protein biosynthesis, tetrameric subunit assembly, tetramer polymerization, localization at disk rims, interaction with GARP2, or the generation of membrane curvature. Interestingly, however, loss of the helical motif up-regulated membrane curvature generation in cellulo, introducing the possibility that it may regulate this activity in photoreceptors. Furthermore, the incipient α-helix (within the purified soluble C terminus) partitioned into membranes only when its acidic residues were neutralized by protonation. This suggests that within the context of full-length peripherin-2/rds, partitioning would most likely occur at a bilayer interfacial region, potentially adjacent to the protein's transmembrane domains. In sum, this study significantly strengthens the evidence that peripherin-2/rds functions directly to shape the high-curvature rim domains of the outer segment disk and suggests that the protein's C terminus may modulate membrane curvature-generating activity present in other protein domains.

GsMTx4: Mechanism of Inhibiting Mechanosensitive Ion Channels.

GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (MSCs). It has six lysine residues that have been proposed to affect membrane binding. We synthesized six analogs with single lysine-to-glutamate substitutions and tested them against Piezo1 channels in outside-out patches and independently measured lipid binding. Four analogs had ∼20% lower efficacy than the wild-type (WT) peptide. The equilibrium constants calculated from the rates of inhibition and washout did not correlate with the changes in inhibition. The lipid association strength of the WT GsMTx4 and the analogs was determined by tryptophan autofluorescence quenching and isothermal calorimetry with membrane vesicles and showed no significant differences in binding energy. Tryptophan fluorescence-quenching assays showed that both WT and analog peptides bound superficially near the lipid-water interface, although analogs penetrated deeper. Peptide-lipid association, as a function of lipid surface pressure, was investigated in Langmuir monolayers. The peptides occupied a large fraction of the expanded monolayer area, but that fraction was reduced by peptide expulsion as the pressure approached the monolayer-bilayer equivalence pressure. Analogs with compromised efficacy had pressure-area isotherms with steeper slopes in this region, suggesting tighter peptide association. The pressure-dependent redistribution of peptide between "deep" and "shallow" binding modes was supported by molecular dynamics (MD) simulations of the peptide-monolayer system under different area constraints. These data suggest a model placing GsMTx4 at the membrane surface, where it is stabilized by the lysines, and occupying a small fraction of the surface area in unstressed membranes. When applied tension reduces lateral pressure in the lipids, the peptides penetrate deeper acting as "area reservoirs" leading to partial relaxation of the outer monolayer, thereby reducing the effective magnitude of stimulus acting on the MSC gate.

Joint refinement of FRET measurements using spectroscopic and computational tools.

The variability of the orientation factor is a long-standing challenge in converting FRET efficiency measurements into donor-acceptor distances. We propose the use of molecular dynamics (MD) simulations to characterize orientation distributions and thus improve the accuracy of distance measurements. Here, we test this approach by comparing experimental and simulated FRET efficiencies for a model donor-acceptor pair of enhanced cyan and enhanced yellow FPs connected by a flexible linker. Several spectroscopic techniques were used to characterize FRET in solution. In addition, a series of atomistic MD simulations of a total length of 1.5 µs were carried out to calculate the distances and the orientation factor in the FRET-pair. The resulting MD-based and experimentally measured FRET efficiency histograms coincided with each other, allowing for direct comparison of distance distributions. Despite the fact that the calculated average orientation factor was close to 2/3, the application of the average κ2 to the entire histogram of FRET efficiencies resulted in a substantial artificial broadening of the calculated distribution of apparent donor-acceptor distances. By combining single pair-FRET measurements with computational tools, we demonstrate that accounting for the donor and acceptor orientation heterogeneity is critical for accurate representation of the donor-acceptor distance distribution from FRET measurements.

Experimental Estimation of Membrane Tension Induced by Osmotic Pressure.

Osmotic pressure (Π) induces the stretching of plasma membranes of cells or lipid membranes of vesicles, which plays various roles in physiological functions. However, there have been no experimental estimations of the membrane tension of vesicles upon exposure to Π. In this report, we estimated experimentally the lateral tension of the membranes of giant unilamellar vesicles (GUVs) when they were transferred into a hypotonic solution. First, we investigated the effect of Π on the rate constant, kp, of constant-tension (σex)-induced rupture of dioleoylphosphatidylcholine (DOPC)-GUVs using the method developed by us recently. We obtained the σex dependence of kp in GUVs under Π and by comparing this result with that in the absence of Π, we estimated the tension of the membrane due to Π at the swelling equilibrium, σosmeq. Next, we measured the volume change of DOPC-GUVs under small Π. The experimentally obtained values of σosmeq and the volume change agreed with their theoretical values within the limits of the experimental errors. Finally, we investigated the characteristics of the Π-induced pore formation in GUVs. The σosmeq corresponding to the threshold Π at which pore formation is induced is similar to the threshold tension of the σex-induced rupture. The time course of the radius change of GUVs in the Π-induced pore formation depends on the total membrane tension, σt; for small σt, the radius increased with time to an equilibrium one, which remained constant for a long time until pore formation, but for large σt, the radius increased with time and pore formation occurred before the swelling equilibrium was reached. Based on these results, we discussed the σosmeq and the Π-induced pore formation in lipid membranes.

Spontaneous transition of micelle-vesicle-micelle in a mixture of cationic surfactant and anionic surfactant-like ionic liquid: a pure nonlipid small unilamellar vesicular template used for solvent and rotational relaxation study.

The micelle-vesicle-micelle transition in aqueous mixtures of the cationic surfactant cetyl trimethyl ammonium bromide (CTAB) and the anionic surfactant-like ionic liquid 1-butyl-3-methylimidazolium octyl sulfate, [C4mim][C8SO4] has been investigated by using dynamic light scattering (DLS), transmission electron microscopy (TEM), surface tension, conductivity, and fluorescence anisotropy at different volume fractions of surfactant. The surface tension value decreases sharply with increasing CTAB concentration up to ∼0.38 volume fraction and again increases up to ∼0.75 volume fraction of CTAB. Depending upon their relative amount, these surfactants either mixed together to form vesicles and/or micelles, or both of these structures were in equilibrium. Fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), incorporated in this system at different composition of surfactant indicates the formation of micelle and vesicle structures. The apparent hydrodynamic diameter of these large multilamellar vesicles is about ∼200 nm-300 nm obtained by DLS measurement and finally confirmed by TEM micrographs. The large multilamellar vesicles are transformed into small unilamellar ones by sonication using a Lab-line instruments probe sonicator with a diameter of ∼90-125 nm. To investigate the heterogeneity, solvent, and rotational relaxation of coumarin-153 (C-153) have been investigated in these unilamellar vesicles by using picosecond time-resolved fluorescence spectroscopic technique. The solvation dynamics of C-153 in these vesicles is found to be biexponential with average time constant ∼580 ps. This indicates the slow relaxation of water molecules in the surfactant bilayer. In accordance with solvation dynamics, fluorescence anisotropy analysis of C-153 in unilamellar vesicles also indicates hindered rotation compared to bulk water.

The chameleon-like nature of zwitterionic micelles: the effect of ionic liquid addition on the properties of aqueous sulfobetaine micelles.

We used fluorescence probing, ζ potentials, and dynamic light scattering measurements to study the interactions between the zwitterionic surfactant N-hexadecyl-N,N-dimethylammonio-1-propanesulfonate (SB-16) and three ionic liquids (ILs), 1-ethyl-3-methylimidazolium ethylsulfate ([C(2)mim][C(2)SO(4)]), 1-ethyl-3-methylimidazolium n-butylsulfate ([C(2)mim][C(4)SO(4)]), and 1-ethyl-3-methylimidazolium n-hexylsulfate ([C(2)mim][C(6)SO(4)]). The three ILs have the same cationic part and their anionic parts differ only in the length of the alkyl chain. The aim of our work is to offer a comparative study and establish the role of the alkyl chain length of the anion of ILs on 1) the incorporation of these anions in the zwitterionic micelles of SB-16 (selectivity of anions) and 2) the physicochemical properties of aqueous solutions of SB-16. Results show that, at lower concentrations (i.e. ≤20 mM), the different ILs modify the properties of the aqueous SB-16 solution in similar manner. All of them bring about a decrease in the critical micelle concentration (CMC) and also in size, and increase the aggregation number of the SB-16 micelles; these effects are more dramatic when [C(2)mim][C(6)SO(4)] is used as the additive rather than [C(2)mim][C(4)SO(4)] and [C(2)mim][C(2)SO(4)]. It is proposed that, in case of [C(2)mim][C(6)SO(4)], the presence of a hexyl chain on the hexylsulfate ion allows the ion to align itself with the tail part of SB-16, whereas, in the case of [C(2)mim][C(2)SO(4)], the presence of ethyl chain in the ethylsulfate ion is not sufficient to bring about a similar alignment of the ethylsulfate anion with the tail part of SB-16. This difference in the location of the anions of the ILs is responsible for the different behavior of the ILs.

Photoinduced electron transfer between various coumarin analogues and N,N-dimethylaniline inside niosome, a nonionic innocuous polyethylene glycol-based surfactant assembly.

Photoinduced electron transfer (ET) reactions between coumarin dyes and N,N-dimethylaniline have been investigated inside niosome, a nonionic innocuous polyethylene glycol (PEG)-based surfactant assembly using steady state and time-resolved fluorescence measurements. The location of coumarin dyes inside the bilayer headgroup region of niosome has been reported and it was verified by determination of the high distribution coefficient of all the dyes inside niosome compared to bulk water. Fluorescence anisotropy parameters of the dyes inside niosome are also in good correlation with the above inference about their location. Bimolecular diffusion guided rates inside niosome were determined by comparing the microviscosities inside niosome and in acetonitrile and butanol solutions and it was found that diffusion of the donor and the acceptor is much slower than the ET rates, implying insignificant role of reactant diffusion in ET reaction inside niosome. We have observed a Marcus inversion region in our restricted media, which shows maxima at lower exergonicity. Such behavior has been demonstrated by the presence of nonequilibrium solvent excited state using two dimensional ET (2DET) theory. Unusually high quenching rates of two coumarins C-152 and C-152A inside niosome were explained by the presence of a stable non-fluorescent twisted intramolecular charge transfer (TICT) state along with an emissive intramolecular charge transfer (ICT) state. Moreover, intermolecular hydrogen bonding between carbonyl oxygens of these two dyes and water in their non-emissive and emissive charge transfer states also plays a key role in their dynamical exchange with each other [G.-J. Zhao and K.-L. Han, Acc. Chem. Res., 2011].

The effect of membrane fluidity on FRET parameters: an energy transfer study inside small unilamellar vesicle.

The fluorescence resonance energy transfer (FRET) in a lipid bilayer system containing two different donors and one common acceptor at below and above transition temperature has been studied and all the FRET parameters are analyzed using steady state and time-resolved fluorescence spectroscopy. Using dynamic light scattering measurement, we have followed the process of preparation of small unilamellar vesicles, and by following the FRET parameters of C-153-Rh6G and C-151-Rh6G pairs inside SUVs at 16 °C and 33 °C (T(m) = 23.9 °C) we have noticed that there is greater effect of temperature on the FRET parameters in case of the C-153-Rh6G pair than that of the C-151-Rh6G pair. Finally we have concluded that this difference is due to their different location inside the lipid bilayer in which fluidity of the long alkyl chain markedly affects the FRET parameters for C-153-Rh6G pair embedded inside a small unilamellar vesicle of size 20-50 nm.

Characterization of 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Emim][Tf2N])∕TX-100∕cyclohexane ternary microemulsion: investigation of photoinduced electron transfer in this RTIL containing microemulsion.

In this study we have characterized a ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethyl- sulfonyl)imide containing ternary nonaqueous microemulsion ([Emim][Tf(2)N]∕∕TX-100∕cyclo- hexane). The phase behavior and dynamic light scattering study show that the [Emim][Tf(2)N]∕TX-100∕cyclohexane three component system can form microemulsion with [Emim][Tf(2)N] as polar core at suitable condition. We have investigated photoinduced electron transfer (PET) using dimethyl aniline as electron donor and several Coumarin dyes as electron acceptor molecules at two different R values (R = [ionic liquid]∕[surfactant]) to observe how the dynamics of the PET rate is affected in this type of confined microenvironment compared to that of the PET dynamics in neat ionic liquid and other pure solvent media. The plot of observed k(q) values with the free energy change (ΔG(0)) for electron transfer reaction shows an apparent inversion in the observed rate as predicted by the Marcus theory.

Effect of polymer, poly(ethylene glycol)(PEG-400), on solvent and rotational relaxation of coumarin-480 in an ionic liquid containing microemulsions.

The effect of a polymer, polyethylene glycol with a molecular weight of 400 (PEG-400), on the dynamics of solvent and rotational relaxation have been investigated in [bmim][BF4]/TX100/cyclohexane microemulsions using steady state and time resolved fluorescence spectroscopy as a tool and coumarin 480 (C-480) as a fluorescence probe. The size of the microemulsions increases with addition of PEG-400, which was revealed by dynamic light scattering (DLS) measurement. This leads to faster collective motion of the cations and anions of [bmim][BF4], which contributes to faster solvent relaxation in microemulsions.

Photophysical studies of a hemicyanine dye (LDS-698) in dioxane-water mixture, in different alcohols, and in a room temperature ionic liquid.

In this paper, we have investigated the photophysics of 1-ethyl-2-(4-(p-dimethylaminophenyl)-1,3-butadienyl)pyridinium perchlorate (LDS-698) molecule in dioxane-water mixture and in a room temperature ionic liquid (RTIL) 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF4]). Both photoisomerization and twisted intramolecular charge transfer (TICT) are possible in this molecule. We have shown that TICT is favorable in this molecule in the dioxane-water mixture and in neat [bmim][BF4]. The change in absorption energy of LDS-698 molecule with mole fraction of water is nonlinear in these systems. The Stern-Volmer plot also deviates from linearity in these systems. This nonlinearity is due to the specific solvation of water molecules in the mixture.

To probe the interaction of methanol and acetonitrile with the ionic liquid N,N,N-Trimethyl-N-propyl ammonium bis(trifluoromethanesulfonyl) imide at different temperatures by solvation dynamics study.

The dynamics of solvent and rotational relaxation of coumarin 153 (C-153) has been studied in neat N,N,N-trimethyl-N-propyl ammonium bis(trifluoromethanesulfonyl) imide ([N(3111)][Tf(2)N]) and its mixtures with polar solvents, namely, methanol and acetonitrile at three different temperatures from 294 to 303 K. Both the solvent and rotational relaxation dynamics of C-153 in neat [N(3111)][Tf(2)N] are linearly correlated with the bulk viscosity at different temperatures. The solvent relaxation time and rotational relaxation time of C-153 decrease with gradual addition of cosolvents in [N(3111)][Tf(2)N]. The gradual addition of cosolvent decreases the viscosity of the medium, and consequently, the solvation and rotational relaxation time also decrease. The decrease of solvation time is more pronounced on addition of acetonitrile compared to methanol.

Computational refinement of spectroscopic FRET measurements.

This article supplies raw data related to a research article entitled "Joint refinement of FRET measurements using spectroscopic and computational tools" (Kyrychenko et al., 2017) [1], in which we demonstrate the use of molecular dynamics simulations to estimate FRET orientational factors in a benchmark donor-linker-acceptor system of enhanced cyan (ECFP) and enhanced yellow (EYFP) fluorescent proteins. This can improve the recalculation of donor-acceptor distance information from single-molecule FRET measurements.

Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain.

Cellular entry of diphtheria toxin is a multistage process involving receptor targeting, endocytosis, and translocation of the catalytic domain across the endosomal membrane into the cytosol. The latter is ensured by the translocation (T) domain of the toxin, capable of undergoing conformational refolding and membrane insertion in response to the acidification of the endosomal environment. While numerous now classical studies have demonstrated the formation of an ion-conducting conformation-the Open-Channel State (OCS)-as the final step of the refolding pathway, it remains unclear whether this channel constitutes an in vivo translocation pathway or is a byproduct of the translocation. To address this question, we measure functional activity of known OCS-blocking mutants with H-to-Q replacements of C-terminal histidines of the T-domain. We also test the ability of these mutants to translocate their own N-terminus across lipid bilayers of model vesicles. The results of both experiments indicate that translocation activity does not correlate with previously published OCS activity. Finally, we determined the topology of TH5 helix in membrane-inserted T-domain using W281 fluorescence and its depth-dependent quenching by brominated lipids. Our results indicate that while TH5 becomes a transbilayer helix in a wild-type protein, it fails to insert in the case of the OCS-blocking mutant H322Q. We conclude that the formation of the OCS is not necessary for the functional translocation by the T-domain, at least in the histidine-replacement mutants, suggesting that the OCS is unlikely to constitute a translocation pathway for the cellular entry of diphtheria toxin in vivo.

Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain.

The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8-TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8-TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

Curcumin in reverse micelle: an example to control excited-state intramolecular proton transfer (ESIPT) in confined media.

In this Article, we focused on the modulation of the photophysical properties of curcumin, an anti-cancer drug, in aqueous and nonaqueous reverse micelles of AOT in n-heptane using steady-state and time-resolved fluorescence spectroscopy. The instability of curcumin is a common problem which restricts its numerous applications like Alzheimer disease, HIV infections, cystic fibrosis, etc. Our study reveals that curcumin shows comparatively higher stability after encapsulation into the interfacial region of the reverse micelle. To get a vivid description of the microenvironment, we added hydrogen-bond-donor (HBD) as well as non-hydrogen-bond-donor (NBD) core solvents. For experimental purposes, we used water, ethylene glycol (EG), glycerol (GY) as HBD solvents and N,N-dimethyl formamide (DMF) as a NBD solvent. With increasing amount of core solvents, irrespective of HBD or NBD, the fluorescence intensity and lifetime of curcumin increase with remarkable red-shift inside the reverse micelle. This is attributed to the modulation of the nonradiative rates associated with the excited-state intermolecular hydrogen bonding between the pigment and the polar solvents. We obtained a high partition constant at W0 = 0 (W0 = [core solvent]/[AOT]) which is certainly due to the hydrogen bonding between the negatively charged sulfonate group of AOT and hydroxyl groups of curcumin. Steady-state anisotropy and time-resolved results give an idea about the microenvironment sensed by the curcumin molecules. The red-shift of emission spectra, increase in the value of ET(30), as well as the increase in the fluorescence lifetime were interpreted as being caused by the partition of the probe between the micellar interface and the polar core solvent. Indeed, we show here that it is possible to control the excited state intramolecular proton transfer (ESIPT) process of curcumin by simply changing the properties of the AOT reverse micelle interfaces by choosing the appropriate polar solvents to make the reverse micelle media.

Designing a new strategy for the formation of IL-in-oil microemulsions.

Due to the increasing applicability of ionic liquids (ILs) as different components of microemulsions (as the polar liquid, the oil phase, and the surfactant), it would be advantageous to devise a strategy by which we can formulate a microemulsion of our own interest. In this paper, we have shown how we can replace water from water-in-oil microemulsions by ILs to produce IL-in-oil microemulsions. We have synthesized AOT-derived surface-active ionic liquids (SAILs) which can be used to produce a large number of IL-in-oil microemulsions. In particular, we have characterized the phase diagram of the [C(4)mim][BF(4)]/[C(4)mim][AOT]/benzene ternary system at 298 K. We have shown the formation of IL-in-oil microemulsions using the dynamic light scattering (DLS) technique and using methyl orange (MO), betaine 30, and coumarin-480 (C-480) as probe molecules.

Ionic-liquid-induced changes in the properties of aqueous zwitterionic surfactant solution: solvent and rotational relaxation studies.

In the recent past, the chameleon-like nature of zwitterionic micelles has been utilized for performing electrophilic, nucleophilic, base, and acid catalyzed reactions. But the use of simple salts to induce the zwitterionic character limits the variation to inorganic cations and anions only. To overcome this problem, we have used room temperature ionic liquids (RTILs), which can be tailored according to need. More precisely, we have shown the effect of added RTILs on the nature of water molecules in the palisade layer of a zwitterionic (N-hexadecyl-N,N-dimethylammonio-1-propanesulfonate (SB-16)) micelle using solvation and rotational relaxation studies of C-153 dye. We have carried out a comparative study of changes in the solvent and rotational relaxation parameters of C-153 in an aqueous solution of SB-16 upon addition of three different ionic liquids (ILs): 1-ethyl-3-methylimidazolium ethyl sulfate [C(2)mim][C(2)SO(4)], 1-ethyl-3-methylimidazolium n-butyl sulfate [C(2)mim][C(4)SO(4)], and 1-ethyl-3-methylimidazolium n-hexyl sulfate [C(2)mim][C(6)SO(4)]. It has been observed that in the presence of added RTILs the solvation dynamics become faster and the change in solvation dynamics is more pronounced in the case of [C(2)mim][C(6)SO(4)] compared to that for [C(2)mim][C(4)SO(4)] and [C(2)mim][C(2)SO(4)]. This can be accounted for by considering the increased water penetration (increased microfluidity) with the addition of ILs. In accordance with solvation dynamics results, fluorescence anisotropy studies also indicate an increase in microfluidity of the palisade layer of the SB-16 micelle with the added RTILs. The average rotational relaxation time in 28 mM SB-16 was found to be 1.12 ns. With the addition of 800 mM [C(2)mim][C(2)SO(4)], the average rotational relaxation time remains the same (1.12 ns), whereas with the addition of 800 mM [C(2)mim][C(6)SO(4)] it decreases to 0.40 ns. This observation is in agreement with our earlier report on the microfluidity of SB-16 solution with the addition of [C(2)mim][C(2)SO(4)] and [C(2)mim][C(6)SO(4)] (Rao, V. G.; Ghatak, C.; Ghosh, S.; Mandal, S.; Sarkar, N. Chem. Phys. Chem. DOI: 10.1002/cphc.201100866).

Dynamics of solvation and rotational relaxation of coumarin 480 in pure aqueous-AOT reverse micelle and reverse micelle containing different-sized silver nanoparticles inside its core: a comparative study.

In this work, we have synthesized different-sized silver nanoparticles in an aqueous-AOT reverse micellar system under the same condition by choosing different reduction processes. We chose two different reducing agents, glucose (mild) and sodium borohydride (strong). In the glucose reduction process, we obtained smaller size nanoparticles in comparison to the nanoparticles obtained in the borohydride reduction process under the same condition. Solvation dynamics study showed that reverse micellar aggregated structures were present after the nanoparticles' formation in a perturbed state. Nanoparticles inside the reverse micellar core were responsible for this perturbation. Larger size nanoparticles were triggering larger perturbation than the smaller size nanoparticles. These changes in perturbation were also reflected clearly in solvation dynamics and rotational relaxation measurements.