Dynamics of Ionic Liquids Assisted Refolding of Denatured

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Dynamics of Ionic Liquids Assisted Refolding of Denatured Cytochrome c: A study of preferential interactions towards Renaturation Upendra Kumar Singh, and Rajan Patel Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00212 • Publication Date (Web): 16 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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Molecular Pharmaceutics

Dynamics of Ionic Liquids Assisted Refolding of Denatured Cytochrome c: A Study of Preferential Interactions towards Renaturation Upendra Kumar Singha and Rajan Patela* a

Biophysical Chemistry Laboratory, Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (A Central University), New Delhi.

*Corresponding author. Tel.: +91 8860634100; fax: +91 11 26983409. Email address: [email protected], [email protected] (Dr. R. Patel)

1 ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT In vitro refolding of denatured protein and influence of the alkyl chain, on refolding of protein were tested using long chain imidazolium chloride salts, 1-methyl-3-octylimidazolium chloride [C8mim][Cl], 1-decyl-3-methylimidazolium chloride [C10mim][Cl]. The horse heart cytochrome c (h-cyt c) was denatured by urea and guanidinium hydrochloride (GdnHCl), as well as base induced denaturation at pH 13 to provide a broad overview of the overall refolding behavior. The variation in alkyl chain of ionic liquids (ILs) showed a profound effect on the refolding of denatured h-cyt c. The ligand-induced refolding was roughly correlated to understand the mechanism of conformational stability in proteins in aqueous solutions of ILs. The results showed that the long chain ILs having [C8mim]+ & [C10mim]+ cations promote the refolding of alkali-denatured h-cyt c. The IL having [C10mim]+ cation efficiently refold the alkali-denatured h-cyt c with the formation of MG state whereas the IL having [C8mim]+ cation, which is known as compatible for protein stability, shows slight refolding and forms different transition state. The life time results shows successful refolding of alkaline denatured h-cyt c by both the ILs, however, more refolding was observed in case of [C10mim][Cl] and this was correlated with the fast and medium lifetimes (τ1 & τ2) obtained show increase accompanied with increase in structure. The hydrophobic interactions plays an important role in the refolding of chemically and alkali-denatured h-cyt c by long chain imidazolium ILs. The formation of MG state by [C10mim][Cl] was also confirmed as some regular structure exists far below the CMC of IL. The overall results suggested that [C10mim]+ cation bound to the unfolded h-cyt c trigger its refolding by electrostatic and hydrophobic interactions that stabilize the MG state. KEYWORDS: Cytochrome c, Ionic liquids, Refolding, MG state, Time resolved fluorescence spectroscopy.


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INTRODUCTION The protein refolding depends on condition to obtain significant amount of active protein. Some known additives having low molecular weights were observed to enhance the refolding process 1. Inclusion body proteins in certain cases have shown refolding 1-3 with the proper additives which triggers the correct folding pathways and thus avoid formation of misfolded species and suppressing unspecific aggregation

4-5

. Often terms like preferentially excluded and bound are

used to understand the process. The additive utilized in refolding should be able to interact strongly with the protein surface than with water molecules that enrich the solvation sphere of protein and are termed preferentially bound. The other one just interacts more with water molecules than with the protein and named preferentially excluded. Preferentially bound one reduces energetic cost of exposing surface area and therefore turns up protein solubility in cosolvents thus favors denatured state over the native state. In case of the excluded co-solvents protein solubility is decreased and the stability of native state is increased. The dichotomy of binding versus exclusion process of additive salts has been used to classify the nature of electrolyte ions into chaotropic and kosmotropic, respectively 6. Stabilization of proteins by different ionic liquids (ILs) has been reported with occasional refolding of denatured proteins and formation of molten globule-like state 7-13. Some works have been extended to the membrane active peptides and proteins demonstrating ILs in membrane specific functions 13-15. ILs were first utilized by Summers and Flowers to explore additives in protein refolding. The neat IL (tetra-alkyl ammonium nitrates) found to denature the protein while 0.5M concentration of the same led to the effective refolding yield of the protein without aggregation 16. Experiments conducted with difference of alkyl chain length, hydrophobicity and substituted cations sprung up with significant factors of ILs required for protein refolding. Previous literature have



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suggested that ILs with larger alkyl side chains that imparts more hydrophobicity acts as effective suppressors while they also exert destabilizing effect on native sate of refolded proteins. 1, 17-19

.

Researchers have characterized the ability of ILs as preservant of enzyme activity and stability when stored at strict challenging conditions or for extreme periods

7, 20-23

. Recently, we have

shown that the surface active ionic liquids (SAILs) performs better in maintaining stability for long-term periods of horse heart cytochrome c (h-cyt c)

23

. So, ILs as stabilizing solvent for

native proteins does not warrant as a refolding enhancer. Experiments by Lange et al. 1 establish ILs as effective refolding additives possessing intermediate capacity to solubilize proteins as well as denaturant strength. The tryptophan solubility could possibly be utilized to characterize ILs in accordance to their refolding capacity. The solubility of the proteinogenic amino acid tryptophan in IL co-solvent systems display effective renaturation of rPA and promote the in vitro refolding 19

. Imidazolium ILs with alkyl chains longer than four are considered as amphiphilic compounds

as they display the interface ordering phenomena depending on their chain length

24-25

. Studies

have reported the molten globule (MG) state exists as major intermediate of protein folding, induced by the refolding additives having long alkyl chain length such as sodium octyl sulfate (SOS), sodium dodecyl sulfate (SDS), alkyl trimethylammonium bromides etc. 26-30. Such studies have demonstrated that MG states formed by additives with hydrophobic chains contained identical amount of electron transferring as N state. Some studies reported earlier to characterized the MG state as function of the increasing alkyl chain length, show enhanced exothermic values of calorimetric enthalpy that induces compact state of the unfolded h-cyt c for the formation of MG state 31. Mostly, the hydrophobic forces were found to play dominant role in stabilizing and preventing the aggregation of the MG state 29-30.


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The urea and guanidinium hydrochloride (GdnHCl) denatured proteins are observed to show refolding by ILs, although the concentration utilized is high (>1 M) with chances of aggregation, ILs used as an additive to induce into act as solvent in the system. Denaturation of h-cyt c by urea and GdnHCl renders primarily random coil structure of h-cyt c 32. In base-induced unfolded state h-cyt c exposes 70-80% of the total surface area to solvent, while the rest remains inaccessible to the solvent.33-34 In high pH induced unfolded states there is no specific intramolecular interaction maintaining stability as in native states due to the strong electrostatic repulsions between charged residues.35 The alkali-pH transitions of h-cyt c in the oxidized state can possibly be stabilized and transformed by cations to alkali molten globule-like (MG) state. A refolded structure of h-cyt c with characteristic features of an MG state after addition of salts that reduces repulsion between charged residues and proteins attains a more compact structure 36

26-28,

. Various researches claim formation of MG state by ionic surfactants through hydrophobic

interactions that stabilizes the MG state.

37-38

ILs have not been checked for the refolding of

alkali-denatured proteins earlier. In this study, the long chain imidazolium ILs have been employed as possible refolding additive for h-cyt c within the reported concentrations. The family of heme proteins contain heme group as active sites and have served prominent role in biophysical research over many decades. Heme groups are iron-protoporphyrin derivatives. The monomeric heme proteins are simpler and serve as prototype in folding/unfolding studies having dominated helical segments in their secondary structure. The h-cyt c is single-chain heme protein of 104 residues have one tryptophan (Trp59). The heme prosthetic group of h-cyt c covalently bound with two thioether bridges to cysteine residues, Cys14 and Cys17. The heme under physiological conditions is axial ligated to His18 and Met80. Cations possessing long alkyl chains facilitate hydrophobic solute surface, but mostly are claimed to be kosmotropic having



high values of viscosities partially due to hydrophobic hydration.

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6

Prolonging the alkyl side

chain makes cation hydrophobic which limits interaction with water molecules but increase the protein hydrophobic surfaces 39. The long alkyl chains of ILs points to "charge center" that offers apolar regions with increased interactions of significant importance

39-40

. The hydrophobic

solutes like apolar amino acids may find favorable interaction partner. The alkyl chains are hydrophobic and have tendency towards the non-polar protein surface

41-43

. Water molecules

arrange themselves in a quasi-crystalline structure (often termed ‘‘iceberg’’). The hydrophobic solvation decreases the water entropy at protein surface compared to bulk water 44. We have used the ILs having imidazolium cations of different alkyl chain length and consecutively showed the effect of ILs chain length on the refolding of h-cyt c. Different biophysical techniques like fluorescence, UV-vis, CD, FT-IR and time-resolved fluorescence spectroscopy were employed to monitor the refolding process of denatured h-cyt c. The work studies the interaction mechanism and effect of length of alkyl chain of IL on the renaturation of urea, GdnHCl, and alkali-denatured h-cyt c. Further, efforts were applied to analyze the role of hydrophobic forces to enhance the refolding ability of ILs. MATERIALS AND METHOD Materials Equine heart cytochrome c (h-cyt c, type IV), sodium hydroxide, Urea, Guanidinium hydrochloride (GdnHCl), sodium monobasic dihydrate and dibasic dihydrate, 1-methyl-3octylimidazolium chloride [C8mim][Cl], 1-decyl-3-methylimidazolium chloride [C10mim][Cl], and potassium chloride were obtained from Sigma Aldrich. Chemicals and reagents utilized in the experiments used were of analytical grade. For maintaining purity during experiments millipore water was used throughout.


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Sample preparation Preparation of stock solution of h-cyt c. The h-cyt c was oxidized completely to ferric form and was dialyzed as mentioned in our previous literature

23, 36

The dialyzed solution of h-cyt c

was checked for concentration using the molar absorbance coefficient (ε) values of 1.06 x 105 M1

cm-1 at 410 nm 45, and for urea and GdnHCl it was determined by refractive index measurement.

46

All the measurements were carried in the degassed phosphate buffer at pH 7.0 and for pH 13,

the KCl-NaOH buffer was used. Addition of [C8mim][Cl] and [C10mim][Cl]. The aqueous stock solution of [C8mim][Cl] and [C10mim][Cl] was prepared in milli-Q water. The h-cyt c was denatured by utilizing the 8 M urea, 6 M GdnHCl and pH 13 KCl-NaOH buffer. To obtain working concentrations, the different amount of stock solution of [C8mim][Cl] and [C10mim][Cl] were added into the solution containing h-cyt c. The samples were incubated in the dark for a period of four hours before each experiment. Methods Circular dichroism measurements. The far-UV CD spectra were measured through Jasco spectropolarimeter (J-1500) of h-cyt c equipped with peltier type temperature controller (PTC100). The spectra were recorded in the absence and presence of [C8mim][Cl] and [C10mim][Cl] at 25.0 ± 0.1 °C and pH 7.0. The near and Soret spectral measurement were measured using cuvette having 1 cm path length, while 0.1 cm pathlength was used for far-UV CD measurements. The raw CD data were converted into [θ]λ, the mean residue ellipticity (deg cm2 dmol-1) at a given wavelength λ using the relation 47. [θ ]λ = θλ Mo /10lc

(1)



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where c is the protein concentration (mg/cm3), Mo is the mean residue weight of the protein, l is the path length (centimetres) and θλ is the observed ellipticity (millidegrees) at wavelength λ. The α-helical content of h-cyt c was calculated from the [θ] value at 222 nm using following equation48.

%α =

[ θ ] 222 − 2340

× 100

30300

(2)

Steady-state fluorescence measurements. The fluorescence measurements were performed on Cary Eclipse spectrofluorimeter (Varian, USA) with a 150W xenon lamp using 1 cm path length quartz cuvettes at 25.0 ± 0.1°C. The excitation and emission slits with a band pass of 5 nm were set for all experiments. Temperature was controlled by constant-temperature water circulator (Varian, USA) using constant-temperature cell holder. Fluorescence of the h-cyt c was measured at an excitation wavelength of 280 nm and the spectra were recorded in the wavelength range of 300-400 nm. For 8-Anilino-1-napthalenesulfonic acid (ANS) measurements excitation was set at 360 nm and the spectra were recorded in range of 450–650 nm, the ratio of 1:20 was used for protein to ANS. In fluorescence measurements h-cyt c was taken as 5 х 10-6 M. The alkalidenatured h-cyt c data were analyzed by fitting of the Stern–Volmer equation 49. F0 =1+ Ksv[Q] =1+ Kq τ0 [Q] F

(3)

where F0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively. Kq is the quenching rate constant of the fluorophore, Ksv is the Stern–Volmer quenching constant, [Q] is the molar concentrations of [C8mim][Cl] and [C10mim][Cl] and τo is the lifetime of the fluorophore without quencher. Estimation of binding parameters of the fluorescence quenching data were calculated from this formulae 50. 8 ACS Paragon Plus Environment

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log

F0 − F

F

= logK b + nlog[Q]

(4)

The binding constant (Kb), binding affinity (n) for alkali-denatured h-cyt c interaction with [C8mim][Cl] and [C10mim][Cl], respectively. Absorbance measurements. The absorption spectra in the range of 350-450 nm was obtained with the help of Specord 210 plus spectrophotometer (Analytik Jena, Jena, Germany) of denatured h-cyt c (urea, GdnHCl and alkaline pH) in the absence and presence of [C8mim][Cl] and [C10mim][Cl]. The constant temperature at 25.0 ± 0.05 °C was maintained throughout experiments with the peltier controlled temperature controller JUMO dTRON 308. FT-IR Spectroscopy. FT-IR spectra were recorded with Specac Golden Gate diamond ATR sampler fitted to a Bruker Tensor 27 with an MCT detector and MIRacle, a single reflection horizontal ATR diamond crystal plate at 25 °C. A total of 128 background-subtracted scans at 2 cm-1 resolution were averaged of alkali-denatured h-cyt c and with [C8mim][Cl] and [C10mim][Cl]. The estimation of the secondary structural compositions of denatured h-cyt c with [C8mim][Cl] and [C10mim][Cl] were performed as reported in previous literature by curve fitting method of the amide I band 23, 41 51. Time resolved fluorescence measurements. Fluorescence lifetimes were calculated from timeresolved fluorescence intensity decays by the single-photon counting spectrometer equipped with pulsed nanosecond LED excitation heads at 280 nm (Horiba, Jobin Yvon, IBH Ltd, Glasgow, UK) at 25.0 °C. The lifetime data were measured to 10,000 counts in the peak with a band pass of 8 nm. The instrumental response function was recorded sequentially using a ludox solution and a time calibration of 114 ps/channel. The decay curves were analyzed by IBH DAS6 software as mentioned in previous literature 23, 52. The mean fluorescence lifetimes for multi-



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exponential iterative fittings and pre-exponential factors were calculated using the following relation 53:

τ

∑a τ = ∑a τ

2

i i

(5)

i i

where αi and τi are the relative contribution and life time of different components to the total decay. For acceptable fit, the autocorrelation function showed random deviation about zero with a minimum goodness of fit (χ2) values. Analysis of folding/refolding transition curves. The data obtained from the absorbance experiment at 25.0 °C was plotted against [C8mim][Cl] and [C10mim][Cl] at 400 nm. The sigmoidal shape of refolding h-cyt c with increasing concentration of [Cnmim][Cl] at temperatures suggest a two-state process based on Pace theory. The linear extrapolation method have been utilized (LEM)

54

to fit the data of sigmoidal curve obtained in the presence of

different concentrations of [Cnmim][Cl]. The Gibbs free energy change associated with reversible denaturation and renaturation, ∆GD , was calculated according to LEM, by using this following relation: ∆ GD = ∆G D ( H 2 O ) - m[[C nmim][Cl]]

(6)

where ∆GD (H2O) represents the extrapolated value of ∆GD in the absence of any denaturant/ renaturant, and m is the slope of the stability curve, i.e., ∂∆GD / ∂[Cnmim][Cl]T,P . It measures the dependency of ∆GD on the [Cnmim][Cl]. At equilibrium, ∆GD is zero and [Cnmim][Cl]is represents by [Cnmim][Cl]1/2

[Cnmim][Cl ]1 / 2 = ∆G D ( H 2O) m

(7)


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[Cnmim][Cl]1/2 represents the concentrations of ILs [C8mim][Cl] and [C10mim][Cl] at which folded and unfolded states of protein are in equilibrium with each other. For a two-state process ∆GD is represented by the following equation:

∆GD = −RT ln KD = −RT ln ( Aobs − AU ) ( AMG − Aobs )

(8)

where R is the gas constant and K D is equilibrium constant at the absolute temperature, T. Aobs is the observed optical property which is used to follow the refolding transition curves, AU and AMG are the properties of Aobs for unfolded state (U) and intermediate state, respectively. RESULTS Absorbance measurements. The absorbance spectra of denatured h-cyt c with [C8mim][Cl] and [C10mim][Cl] at different concentration are shown in Figure 1. The h-cyt c in its denatured state shows Soret bands around 395 nm (reflecting high spin state iron, a form imposed by the heme’s axial ligands, His18 and Met80 of the heme group). The absorbance spectra for the native h-cyt c having Soret band at 409 nm is also shown in Figure 1 (π–π* transition a position denoting low spin state of iron in heme) and denatured h-cyt c at 395 nm. The h-cyt c was denatured by different denaturants urea and guanidine hydrochloride (GdnHCl) at pH 7.0. The h-cyt c was also alkali-denatured at pH 13 and checked for refolding by ILs. The alkali-denatured h-cyt c have the Soret band around 399 nm, the 60’s helix extending from residue 60 to 70

55

are loss structure at high pH due to misligation and it serves as a marker for

the loss of tertiary structure of h-cyt c.

56-57

On the addition of [C10mim][Cl] (below cmc), the

absorbance decreases sharply along with significant peak shift, indicating that within this concentration range no fundamental change in the heme occurs (before the inflection point), although there is possibility of multiple heme microenvironments that broads the Soret band. Figure 1 displays the change in the Soret absorption spectra, as [C10mim][Cl] decreases the 11 ACS Paragon Plus Environment


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unfolded state of h-cyt c shows red shift with decrease in absorbance. However, unfolded state does not completely fold into the native state of h-cyt c. In UV–vis spectra the Soret band for MG is positioned at 407 nm. The heme in MG state is buried inside the hydrophobic core and show mixed spin, with His18 coordinated to iron in both spin states, while the Met80 is coordinated in only low-spin species

37-38, 58-59

. This shift signifies the structural change at the

heme occurs, although with constant secondary structural content as observed from far-UV CD measurements. Chamani et al. have observed similar type of results with cationic surfactants DTAB, TTAB and HTAB upon addition to alkali and acid-denatured h-cyt c. 29, 60-64 The increasing concentration of both ILs with h-cyt c, shows absorbance from the heme group of the h-cyt c. For urea and GdnHCl denatured h-cyt c the absorbance shows decrease of the Soret peak at 395 nm with no shift, suggests no changes in the state of the h-cyt c with addition of ILs, i.e. the h-cyt c remains to be in the denatured form. The GdnHCl denatured h-cyt c shows increase in absorbance after addition with both of the ILs. The increase in absorbance was due to the further denaturation or exposure of heme to little non-polar region of protein that reduces remaining structure of h-cyt c around the heme cleft. In case of urea denatured h-cyt c, the addition of ILs causes decrease in absorbance which was purely due to exposure of heme towards more polar region. Only high pH denatured state show considerable refolding of h-cyt c by the ILs. Steady-state fluorescence measurements. Fluorescence emission provides information about the structural changes in the denatured h-cyt c upon interaction with ILs. Heme group through foster energy transfer quenches the emission intensity of aromatic Trp59. Hence, native h-cyt c produces very weak fluorescence. The fluorescence emission in native h-cyt c by tryptophan shows very low intensity due to quenching by heme group. While, upon unfolding the tryptophan


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moves far from the heme moiety, and this increase in distance between them reduces the quenching and increased tryptophan fluorescence is observed. The peak at nearly 353 nm is observed after denaturation of h-cyt c (Figure 2). The inner filter effect in concentrated samples arises due to macromolecules which scatter and produces incorrect data. Inner filter effect accounts for decrease in the apparent fluorescence intensity that distorts the quenching data 65-66. The inner filter effects were corrected for the observed fluorescence from the samples

49

that

attenuate background signals and produce aberrant readings. The alkali-denatured h-cyt c shows peak around 353 nm, with the addition of both ILs. The decrease of fluorescence intensity in both the case was recorded due to Trp59 that remains at least partially buried. Fluorescence quenching of Trp59 indicates partially unfolded state but significant shift was observed only in case of the [C10mim][Cl] as shown in Figure 2. The blue shift in the maximum emission wavelength from 353 nm to 343 nm is observed, that originates due to the shift of the Trp environment towards non-polar environment. Hence, [C10mim][Cl] addition increases the hydrophobicity around the Trp that remains solvated (polar) in denatured form. The blue shift of emission maximum with decrease in fluorescence intensity (~80%) of h-cyt c-[C10mim][Cl] complex. In case of [C8mim][Cl] the fluorescence intensity decreases ~65% with shift in the peak position from 353 nm to 349 nm. The shift is more for the [C10mim][Cl] compared to the [C8mim][Cl] and also greater reduction in fluorescence intensity were observed. The GdnHCl denatured h-cyt c shows decrease in fluorescence after addition with both of the ILs. The decrease followed by the red shift with [C8mim][Cl] (at 50mM) but almost no shift in case of [C10mim][Cl] at different concentration was observed. The case reversed in the urea denatured h-cyt c where the [C10mim][Cl] (at 50mM) shows red shift but it was not observed with [C8mim][Cl]. The decrease in intensity may be due to the probable quenching of Trp



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intensity by both ILs ~40% (greater in case of urea) and ~20% (less in case of GdnHCl denatured). The MG like state of h-cyt c are familiar having nearly as compact structure as the native state 2930

. The presence of single tryptophan in h-cyt c makes it easy to check for the compaction by

monitoring the fluorescence emission of such compact states that produces lower intensity. At pH 13, the four tyrosine residues of h-cyt c also contribute towards fluorescence. The quenching of intensity, however, produces firm evidence about compaction of alkali-denatured h-cyt c that relates towards the increasing proximity of the fluorophore and the heme after refolding. The Stern-Volmer plot and the double logarithmic plot of alkali-denatured h-cyt c (for refolding of hcyt c) were calculated as shown in Figure 3 (a & b), respectively with [C8mim][Cl] and [C10mim][Cl]. The plots were utilized to calculate the Stern-Volmer constant (Ksv), bimolecular quenching constant (kq), the binding constant (Kb), the stoichiometry of binding (n) for the renaturation of alkali-denatured h-cyt c by ILs. The data (Table 1 (a)) obtained suggests greater values of kq and Ksv for [C10mim][Cl] when compared to the [C8mim][Cl]. Table 1 (b) lists the binding constant (Kb) and the stoichiometry of binding (n), calculated from the double logarithmic plot. The binding of [C10mim][Cl] to alkali-denatured h-cyt c is 10 times greater compared to [C8mim][Cl]. This suggest extent of binding and interaction in case of the [C10mim][Cl] is much greater than [C8mim][Cl] with denatured h-cyt c and therefore more refolding of alkali-denatured h-cyt c was observed in case of [C10mim][Cl]. The 8-Anilino-1-naphthalene sulfonic acid (ANS), a hydrophobic fluorescent probe that indicates presence of hydrophobic patches on protein and is used in characterization of partially folded intermediates

67-69

. It is minimally fluorescent in polar environments, but exposure to

nonpolar environments dramatically influences their emission70. ANS conforms the presence of


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hydrophobic patches by binding to it and also produces a blue shift in fluorescence emission67. The denatured samples have their emission maximum same as that of native h-cyt c i.e. no hydrophobic patch of proteins are exposed, thus have very low affinity for binding to ANS. After addition of [C8mim][Cl] and [C10mim][Cl] to the denatured h-cyt c, the shift in emission maximum with increase in intensity were observed in urea and alkali-denatured h-cyt c, except for GdnHCl denatured h-cyt c. In urea-denatured h-cyt c with [C8mim][Cl] no shift occurs with slight increase in the intensity, but in case of [C10mim][Cl], with increase in intensity shift is observed. The alkali-denatured h-cyt c with [C8mim][Cl] and [C10mim][Cl] shows increase in intensity with peak shift, more in case of [C10mim][Cl]. This suggest formation of refolded states of alkali-denatured h-cyt c by ILs more pertinent with the [C10mim][Cl] with blue shift in the emission peak. Circular dichroism measurements. The secondary structure of proteins can be investigated through monitoring the far-UV spectrum in range 200-250 nm. Alteration in backbone orientation will affect optical transition and hence indicates change in secondary structure content of protein. Here, structural content where estimated by far-UV CD of denatured h-cyt c with both ILs. The far-UV CD spectra of alkali-denatured h-cyt c with [C8mim][Cl] and [C10mim][Cl] were recorded as shown in Figure 5. The helical content calculated from CD spectra of the far-UV region of alkali-denatured h-cyt c matches well with reported literature 33. The addition of [C10mim][Cl] to the denatured h-cyt c refolds h-cyt c and it attains a compact structure. In figure 5, the secondary structure of denatured protein form, evident from the ellipticity gain near 222 nm which is the characteristic of the alpha helix

71-72

. The high

transmission voltage recorded at higher concentration of ILs restrains us to measure data beyond reported concentration in Table 2. The urea and GdnHCl denatured h-cyt c did not showed any



Page 16 of 42

increase in the helical content of h-cyt c (data not shown). The refolding of h-cyt c shows the effect of hydrophobic alkyl chain of ILs. The table 2 shows helical content of alkali-denatured and refolded h-cyt c with [C8mim][Cl] and [C10mim][Cl]. The [C8mim][Cl] at 6 mM show only slight increase in helical content (~12%), while the [C10mim][Cl] at same concentration showed greater increase in helical content (~20%). The refolding of alkali-denatured h-cyt c occurs by increase in helical content after, but the spectra were unable to record beyond 6mM of ILs due to high transmission voltage. So it was unable to find the refolded helical content by far-UV CD spectra, although the results observed by FT-IR, UV-vis and fluorescence spectroscopy suggest that refolding of denatured protein occurs beyond concentration of [C8mim][Cl] & [C10mim][Cl], which comprehends the gain in helical content approximate to native content after refolding and form molten globule-like state. This was not observed even at high concentration of [C8mim][Cl] having short alkyl chain than [C10mim][Cl]. So, these results directly correlate to the length of hydrophobic chain crucial for refolding ability of ILs. Similar increase in helical content at approximate to cationic surfactants shows the formation of molten globule like state. 37-38 The near-UV CD measurement in the range of 250-350 nm was performed to study the monitor the tertiary structure of protein. The tertiary structure is maintained by interactions that are nonspecific between the side chains of amino acid in polypeptide backbone. Peaks in this region are assigned to amino acid residues in peptide backbone according to their relative position in the spectra. Near-UV spectrum produces two sharp minima near 282 and 289 nm, this resembles the tight tertiary structural packing of of Trp59 & Tyr.73-74 The figure 6 shows the tertiary structure upon ILs addition to denatured h-cyt c. The near-UV CD signature of urea, GdnHCl and alkalidenatured h-cyt c are almost same as unlike native h-cyt c no absorption is observed at 282 nm


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and 289 nm, although the alkali-denatured h-cyt c is reported to have nearly half of the native helical content but it is dimensionally expanded. The ILs after addition to urea and GdnHCl denatured h-cyt c as suggested by other results do not show any peak at 282 nm and 289 nm for native protein. These results are augmenting previous observations. Although negative readings increases in GdnHCl and urea-denatured proteins due to the presence of [C8mim][Cl] and [C10mim][Cl] as shown in figure 6. Noted increase in peak occurs in case of the alkali-denatured h-cyt c where addition of [C10mim][Cl] produces small peak around 282 nm and 289 nm, but not in the case of [C8mim][Cl]. The results obtained were in coherent to the results mentioned in different techniques. FT-IR Spectra. FT-IR spectroscopy was employed to characterize the structural insight of protein from gross aspects to subtle rearrangement upon interaction with ligands. The 1600–1700 cm-1 region is assigned to the amide band I peak associated with the mainly C=O stretching, this regionis related to the secondary structure changes of protein. FT-IR results have been preformed to confirm the structural changes obtained by far-UV CD spectra. In the figure S1(a) the second derivative spectrum of alkali-denatured h-cyt c in absence and presence of both ILs showing the amide I band around 1650 cm-1 that ascertain the relative amounts of the secondary structure 75. The second-derivative spectrum distinguishes different peaks associated to secondary structures of the protein. The amide I band appears at 1648 cm-1 of alkali-denatured h-cyt c. Addition of [C8mim][Cl] and [C10mim][Cl] shifts the amide band due to refolding of h-cyt c. The [C8mim][Cl] and [C10mim][Cl] with alkali-denatured h-cyt c , at 15 mM and 20 mM have shown refolding with increase in intensity of band due to increase in the content of helix. The [C10mim][Cl] shift in peak of band and increase in intensity were higher as obtained for



Page 18 of 42

[C8mim][Cl]. This result augments the CD data earlier shown for denatured h-cyt c with the addition of ILs (gain of helical content). In the figure S1 (b, c & d), the curve fitted spectra shown depicts the change in peak position of bands with respective ILs. The curve fitting method and the deconvolution analysis have been performed according to previous literatures 23, 76. The deconvoluted area obtained in the spectral region were found consistent with our CD data and was earlier reported

8, 78

. The square of the

correlation coefficient (R2) were quite appreciable for each fitting and the data are mentioned in the figure S1 and Table S1, respectively. The secondary structural contents of native h-cyt c 23, alkali-denatured and their refolded content by [C8mim][Cl] & [C10mim][Cl] listed in Table S1. The secondary structure associated peaks have been shown in the figure S1 (b, c & d) of alkalidenatured h-cyt c upon interaction with both ILs. These results show, [C10mim][Cl] interacts more strongly and serves as better refolding additive than the [C8mim][Cl]. Time-resolved fluorescence measurements. Lifetime measurement is a sensitive method to monitor the molecular interactions and motions for determining the structure and dynamics of biomacromolecules in local environment. The tryptophan fluorescence of proteins is sensitive to protein conformations, lifetime is highly dependent on local environment around the fluorophore and thus varies with change in segmental motions and the macromolecular conformation. Here, decay of h-cyt c has been measured in presence of both ILs. The single tryptophan containing protein provides the simple interpretation of the lifetime data, although such proteins show multiexponential decay kinetics. The different interactions of tryptophan side chain with local environment show different roatmeric conformation due to which heterogeneous decay is obtained. The overall contribution of the individual components shows greater contribution of shorter lifetimes by pre-exponential factor (αi). The αi relates to the individual rotamer


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population and corresponds to the secondary structure of the tryptophan residue by the main chain on rotamer populations. The denatured h-cyt c showed decreased average lifetime compared to native h-cyt c listed in Table S2. The lifetime experiments of denatured h-cyt c with [C8mim][Cl] and [C10mim][Cl] show multimodal form of distribution shown in Table S2. The results of techniques discussed above shows successful refolding of alkali-denatured h-cyt c by [Cnmim][Cl] (C8 & C10) within decay curve spectra in figure 7, respectively. The results can be correlated with the fast and medium lifetime (τ1 & τ2) obtained from refolded h-cyt c show increase accompanied with increase in structure. The increase in the medium lifetime (τ2) may originate from the refolding of structure of h-cyt c. So, increase in ordered structures of protein after refolding originates and shows a pattern that also is followed by the helical content of refolding protein as shown in figure 8. Another important point we noted is that all denatured protein data fitted into the second exponential while the refolded one fitted triple exponential. The additional component originates probably due to bound ILs to protein molecule as [C10mim][Cl] decreases the size of denatured protein. However, the urea and GdnHCl denatured h-cyt c do not show any refolding at reported concentrations of both the ILs. Studies reported previously of protein with ILs79-80 have also shown similar trend in which the protein capsules of higher helical content showed change in lifetime compared to the protein solution this relates to change in polarity. The fast and medium lifetime components originate from the vicinity of heme region. Thus, alteration of lifetime components suggest change in polarity around heme and the change in conformation can easily be moniotred.81. The results obtained at 50 mM of [C10mim][Cl] showed regular increase of average lifetime with increasing concentration of IL. The slow increase in α3 till 30mM for refolding of alkali-denatured h-cyt c



Page 20 of 42

by [C10mim][Cl] was noted, this signify increase in structural content upto this concentration of h-cyt c, beyond which no further arrangement occurs and steep increase in α3 is observed. Thermodynamic analysis of MG formation. From UV-vis results figure 1 was redrawn for the analysis of sigmoidal curves from the unfolded to MG state of h-cyt c upon addition of [Cnmim][Cl] at alkaline conditions (pH 13). The curves were analyzed using linear extrapolation method (LEM).37 The increase in alkyl chain from 8-10 in [Cnmim][Cl] ILs show different effect on refolding of alkaline denatured h-cyt c. Table 3 lists the thermodynamic parameter calculated through LEM using equations 6-8. The observation suggests that the difference in chain length, the ∆GD (H2O) value changes as the shift in wavelength in figure 9 of the refolded h-cyt c were not same i.e. the refolding ability was affected by the chain length. The figure 9 shows [C8mim][Cl] refolds until 403 nm while the [C10mim][Cl] attains maximum refolding to 406 nm of the Soret band. Also, the decrease in absorbance in not the same for both the ILs ([C10mim][Cl] shows better-refolding ability). Parameters from Stern-Volmer plot in Table 1 further suggests the strength of the binding is greater for the [C10mim][Cl] than [C8mim][Cl]. The plot of ∆GD against [Cnmim][Cl] and the values of the slope (i.e. m-value) in figure 10 are provided in Table 3. The value of m reflects the cooperativity of refolding of alkaline unfolded state of h-cyt c. Here, different refolding ability with the variation of alkyl chain of [Cnmim][Cl] can be related to the m-value that correlates with free energy contributions of protein groups that becomes buried upon folding of protein. Formation of different MG-like state and refolding of hcyt c upon addition of [Cnmim][Cl] (C8 & C10) and the refolding occurs much below of their cmc, respectively. DISCUSSION


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The denatured h-cyt c through urea, GdnHCl and high pH were checked for ligand-induced refolding to understand the mechanism of conformational stability. This paper studies the refolding of denatured h-cyt c to folded structure by [C8mim][Cl] and [C10mim][Cl]. The study provides evidence to substantiate the contribution of the hydrophobic interactions involved in refolding of denatured protein and hence the stability of the MG state. The results mentioned in above section from different sector of techniques illustrate the ability of different alkyl chain length of [Cnmim][Cl] as these ILs are itself hydrophobic and contain a positively charged imidazolium ring. Alkaline denatured protein exposes almost 70-80% of its surface

33

. Upon unfolding, buried

protein surface exposes itself to the solvent is main structure determinant for m-values

38

. The

conformational transitions upon refolding from the denatured state towards native state involve transition states that guide protein folding pathway. The [Cnmim][Cl] ILs were selected for the study because these showed the dual nature of interactions ie. electrostatic as well as hydrophobic interactions. These are surface active ionic liquids (SAILs) having a polar head and non-polar tails, their amphiphatic nature allows refolding of protein towards the formation of MG-like state

26-28

. The calculated values form absorbance graph shown in Table 3, lists m-

values, Cm and the ∆GD (H2O) values. The difference of chain length of an alkyl chain having same counter ion Cl-, unravels the role of electrostatic and hydrophobic interactions responsible for refolding of h-cyt c. The alkali-denatured h-cyt c shows refolding with both [C8mim][Cl] and [C10mim][Cl], but the extent of refolding and formation of transition states are quite different. The increase in the secondary structure after refolding shown by far-UV CD spectra in figure 5 confirms the increase in the helical content of unfolded protein with both the ILs is different. Table 2 shows the gain in



Page 22 of 42

the secondary structure at a certain concentration of [Cnmim][Cl] is differ by ~ 10% of helical content. Similar results were also obtained from FT-IR curve-fitted and the second derivative graph in figure S1, that show different peaks originated after the addition of ILs to unfolded hcyt c. Table S1 lists the helical content after refolding of h-cyt c in presence of [C8mim][Cl] and [C10mim][Cl]. The addition of [C8mim][Cl] to alkali-denatured h-cyt c shows slight refolding and less increase in the helical content of alkali-denatured h-cyt c comparative to the [C10mim][Cl]. In case of [C10mim][Cl], the MG state, shows a native-like amount of α-helix suggesting the significant refolding of denatured h-cyt c. The longer alkyl chain increases the ability of the MG state to be formed shown in Figure 1, which is possibly due to the differential binding ability of IL provided by the difference in length of the alkyl chain might hold the reason for it. Our results show that the renaturing property of the [C10mim][Cl] may be due to the hydrophobic effect dependent on the length of alkyl chain as suggested in literatures37-38. Therefore, hydrophobic forces play a dominant role in inducing the MG state also shown through earlier studies 26-28, 38. The conformation of alkali-denatured proteins is dependent on the balance of charge repulsion between the negative groups. Addition of [Cnmim][Cl], shields the intermolecular electrostatic repulsion forces in the denatured state of protein by the positive polar head of [Cnmim]+ binding as a result of which overriding of intrinsic forces favors the formation of the MG like state. Studies have been reported that confirm the influence of alkyl chain of surfactants on the refolding of proteins due to hydrophobic forces that stabilizes the MG state 2728, 65

. Similar studies by Chamani et al. show formation of MG states of acid unfolded state of h-

cyt c due to increase in alkyl chain length (increase in hydrophobic forces) of alkyl sulfates induces more compact MG states 27-28. The data obtained from other techniques were also found complimentary to the fluorescence lifetime results where the increase in the medium lifetime (τ2)


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corroborates with increased helical content of the unfolded protein, and protein becomes more compact. The h-cyt c consists of single Trp, it shows multimodal form of distribution with refolding by both ILs from its base induced unfolded form. The fluorescence lifetime of h-cyt c is dependent distance between tryptophan and the heme and on their relative orientation 49. At pH 7.0 in the native state, it is reported that 80% fluorescence is due to the heme group while remaining is due to the Trp. In the unfolded form the heme and Trp are distant (~20 Å) compared to the native form

82

. Das et al. calculated the distance of Forster’s energy transfer for the medium lifetime

(τ2) may originate from the Trp residing around 16-22 Å from the center (figure 7) 81. Our results showing the increase in the medium lifetime (τ2) with refolding of h-cyt c originate from the same region from the center. Thus, increase in ordered structures of protein after refolding originates and shows a pattern that also followed by helical content of refolding protein shown in figure 5. The results corroborate with far-UV CD results which show increased helical content (secondary structure) of the unfolded protein that makes protein more compact. The Soret band in UV-vis results, figure 1, after the addition of [C10 mim]Cl show more blue shift compared to the [C8mim]Cl. The increase in alkyl chain of ILs from C8 to C10 generate more compact state of alkali-denatured h-cyt c which results in the structural changes in the microenvironment of the heme and restore nearby residues interactions. Figure 2 & 3, showed the quenching of fluorophore of the denatured h cyt c due to hydrophobic collapse. Moreover, the quenching of unfolded h-cyt c fluorescence by both of the ILs signifies the structural change within the heme region. Moreover, the quenching of unfolded h-cyt c fluorescence by both of the ILs signifies the structural change within the heme region. Interaction of unfolded h-cyt c with ILs, h-cyt c remains collapse after refolding observed by



Page 24 of 42

partial burial of Trp59 due to increase in hydrophobic interactions (due to the presence of the long hydrophobic alkyl chain as in necklace and bead model)

83-84

. The peak positions of

maximum emission intensity after refolding by [C8mim][Cl] and [C10mim][Cl] were distinct. The Ksv and Kb values in Table 1 and 2 observed for the refolding of denatured h-cyt c for [C8mim][Cl] and [C10mim][Cl]. The Ksv depicts the quenching of the fluorescence of the denatured h-cyt c by long alkyl chain of ILs, whereas the Kb shows strength of binding between alkyl chain of ILs to the denatured h-cyt c. The Ksv value for [C10mim][Cl] show increase of about 3 times than [C8mim][Cl]. Also, the Kb values for [C10mim][Cl] was observed 10 times higher than [C8mim][Cl] which shows the better refolding ability of [C10mim][Cl] and that can be serves as better refolding additive. The interaction mechanism of both the ILs show different behavior, the [C8mim][Cl] refold the denatured h-cyt c through dynamic quenching mechanism, while the [C10mim][Cl] refold it through static quenching mechanism. The higher Ksv and Kb values for [C10mim][Cl] is owing to longer alkyl chain of ILs which have more hydrophobic character, thus greater quenching and stronger binding with denatured h-cyt c is observed and therefore, the higher degree of interaction with the denatured h-cyt c and forms the MG state of h-cyt c. In other words, we can say more the hydrophobic content more effective is the refolding by ILs 38

. ANS studies also confirm the formation of refolded states by binding to the available

hydrophobic patches of refolded protein with increase in intensity and blue shift in the emission peak (figure 4). The tertiary structure of unfolded h-cyt c is dimensionally expanded, i.e. loss of well defined tertiary structure at high pH (pH 13), unlike native one having peaks at 282 nm and 289 nm 74-75. In presence of [C8mim][Cl] no peaks were observed although with [C10mim][Cl] small intensity


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negative peaks at 282 nm and 289 nm were observed. This peaks observed suggest the presence of the tertiary structure and packing of Trp & Tyr residues. Similar results by lifetime values and fluorescence spectra where the MG state formed by [C10mim][Cl] having similar native-like features were observed. Similar experiments illustrating NaCl induced role of hydrophobic interaction overriding electrostatic interactions for stabilization of B state were observed from base induced unfolding34. In the native state, the heme cleft of h-cyt c is buried inside the hydrophobic core. In unfolded state the heme shifts away with Soret band show a blue shift and the residues are exposed. This red shift in presence of ILs signifies the structural change at heme. The absorbance of the Soret band decrease and shift greater for [C10mim][Cl] is observed with the formation of MG state. The alkali-denatured h-cyt c possess high negative charge and the positively charged imidazolium head group [Cnmim]+ initiates first interaction as electrostatic. The presence of a hydrophobic alkyl chain of different length together with the electrostatic contribution of imidazolium head group [Cnmim]+ upon interaction produces the MG state. Ionic sites of negatively charged h-cyt c once saturated, the hydrophobic contribution predominates. The short range forces between nonpolar groups on h-cyt c and the nonpolar alkyl chian of [Cnmim][Cl] induces compact state of protein at low concentrations with native-like secondary structure and small tertiary structure

34

. At higher concentration (below CMC) in contrast to lower, the

hydrophobic interactions predominate relative to the electrostatic contribution. The formation of MG states in protein at alkaline pH with SDS provides information regarding the underline mechanism that suggests first neutralization of charges followed by hydrophobic effect 38. The alkyl chain of [C10mim][Cl] is greater compared to the [C8mim][Cl] in Figure 10 i.e. it induces more hydrophobic forces as a result of which the more compact refolded state were



formed similar reported by Chamani et al.

26-28

Page 26 of 42

. The hydrophobic content induced by the long

alkyl chain provides greater helical content, more compact structure and formation of MG state by [C10mim][Cl], while the [C8mim][Cl] shows slight refolding and forms different transition state. The m-value for a transition generally relates to the change in solvent exposure. This parameter is a measure of the cooperativity of unfolding transition, it is also proportional to the difference of solvent accessible area between both native and denatured states. The m-value for refolded state from unfolded h-cyt c by [C8mim][Cl] and [C10mim][Cl] tabulated in Table 3, is less for [C10mim][Cl] although it has greater chain length. The refolded states formed by both of the ILs are different discussed by techniques above, as [C8mim][Cl] refolds h-cyt c till 15 mM but [C10mim][Cl] shows efficient refolding upto 25 mM. The [C8mim][Cl] induced refolded state had lesser helical content and no gain in tertiary structure. Thus we conclude that [C10mim][Cl] overrides the predominant electrostatic interactions in alkali-denatured h-cyt c efficiently and refolds owing to its hydrophobic longer alkyl chain having positive charged imidazolium head group. Our results show main role of hydrophobicity towards the stability of the MG state. CONCLUSIONS This work reveals ability of the long chain imidazolium ILs to perform as refolding enhancers for denatured h-cyt c. The long chain IL having [C8mim]+ & [C10mim]+ cations promote the refolding upon addition into the alkali-denatured h-cyt c. The SAIL [C10mim][Cl] and [C8mim][Cl] interacts with alkali-denatured h-cyt c and refolds towards the formation of MG state. However, no refolding was observed in case of urea and guanidinium hydrochloride (GdnHCl) denatured h-cyt c by both of the ILs at reported concentrations. The [C10mim]+ cation bound to the unfolded h-cyt c trigger its refolding through electrostatic and hydrophobic interactions that stabilize the MG state. The [C10mim][Cl] acts as better refolding enhancer


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owing to its long alkyl chain and greater hydrophobic forces in the stabilization of the MG state. The discussed results relate the role of hydrophobic forces for the stability of the MG state. Supporting Information FT-IR and Time-Resolved Fluorescence results. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS Dr. Rajan Patel greatly acknowledges the financial support from Science and Engineering Research Board (EEQ/2016/000339) New Delhi, India. Authors also thank DST for providing the FIST grant with Sanction Order No. (SR/FIST/LS-541/2012). Upendra Kumar Singh is thankful to UGC, New Delhi for SRF fellowship.

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Figures & Tables

Figure 1. UV-Vis spectra of h-cyt c (5 µM) (8 M urea, 6 M GdnHCl and pH 13 buffer) with [C8mim][Cl] and [C10mim][Cl]. The D and N refer to the denatured and the native states of h-cyt c.



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Figure 2. Fluorescence of denatured h-cyt c (5 µM) (8 M urea, 6 M GdnHCl and pH 13 buffer) with [C8mim][Cl] and [C10mim][Cl]. The D and N refer to the denatured and the native states of h-cyt c.

Figure 3 (a). Stern-Volmer plot of h-cyt c (5 µM) (pH 13 buffer) with [C8mim][Cl] and [C10mim][Cl]. (b) Double logarithmic plots for the determination of the binding constant of h-cyt c (5 µM) with [C8mim][Cl] and [C10mim][Cl]. 36 ACS Paragon Plus Environment

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50

25

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native h-cyt c GdnHCl den h-cyt c 10 mM [C8mim][Cl] 50 mM [C8mim][Cl]

50

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ANS fluorscence Intensity


50 mM [C8mim][Cl]


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native h-cyt c alk den h-cyt c 10 mM [C8mim][Cl]

75

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native h-cyt c urea den h-cyt c 10 mM [C8mim][Cl]

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50 mM [C8mim][Cl]

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native h-cyt c alk den h-cyt c 50 mM [C10mim][Cl]

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10 mM [C10mim][Cl] 100 80 60 40 20

native h-cyt c GdnHCl den h-cyt c 10 mM [C10mim][Cl] 50 mM [C10mim][Cl]

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


native h-cyt c urea den h-cyt c 10 mM [C10mim][Cl]

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50 mM [C10mim][Cl]

20

0 400

450

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550

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Wavelength, nm

Figure 4. ANS fluorescence spectra of denatured h-cyt c (8 M urea, 6 M GdnHCl and pH 13 buffer) with [C8mim][Cl] and [C10mim][Cl].



Figure 5 (a & b). Far-UV CD spectra of h-cyt c (15 µM) (pH 13 buffer) with [C8mim][Cl] and [C10mim][Cl], respectively. 6

2

50 mM [C8mim][Cl]

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-2

-4

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h-cyt c Urea Den h-cyt c

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h-cyt c

10 mM [C8mim][Cl] 50 mM [C8mim][Cl]

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-2

GdmCl Den h-cyt c 10 mM [C8mim][Cl]

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Molar ellipticity (mdeg)

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h-cyt c Alk Den h-cyt c 10 mM [C8mim][Cl]



6

50 mM [C8mim][Cl]

2

0

-2

-4

-4

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h-cyt c Alk Den h-cyt c 10 mM [C10mim][Cl]

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h-cyt c


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h-cyt c Urea Den h-cyt c

Molar ellipticity(mdeg)

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Wavelength, nm 6


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Figure 6. Near-UV CD spectra of h-cyt c (18 µM) (8 M urea, 6 M GdnHCl and pH 13 buffer) with [C8mim][Cl] and [C10mim][Cl].


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Figure 7. Time-resolved fluorescence decay spectra of the denatured h-cyt c (8 M urea, 6 M GdnHCl and pH 13 buffer) with [C8mim][Cl] and [C10mim][Cl].

Figure 8. Fluorescence lifetime (τ2) of the refolded h-cyt c with [C8mim][Cl] and [C10mim][Cl]. 39 ACS Paragon Plus Environment


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Figure 9. Plot of absorbance at 400 nm versus concentrations of both [C8mim][Cl] and [C10mim][Cl].

Figure 10. Free energies values (∆GD) versus concentration for formation of MG-like state of hcyt c at pH 13 upon interaction with 1-alkyl-3-methylimidazolium chlorides. Plot of absorbance at 400 nm versus concentrations of both [C8mim][Cl] and [C10mim][Cl]. The error bars represent errors in ∆GD values.


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Table 1. (a) Stern-Volmer parameters obtained by plot of denatured h-cyt c (5 µM) with [C8mim][Cl] and [C10mim][Cl]. KSV [L mol-1]

kq [109 L mol-1 s-1]

R2

[C8mim][Cl]

28.8 ± 0.005

5.8 ± 0.008

0.9964

[C10mim][Cl].

63.8 ± 0.002

12.8 ± 0.012

0.9864

Table 1. (b) Binding Parameters from the Double logarithmic plot for the determination of the binding constant from Fluorescence Quenching Data

Kb * [ L mol-1]

(n)

R2

[C8mim][Cl]

37 ± 1.412

0.54 ± 0.060

0.9652

[C10mim][Cl].

337 ± 1.232

0.94 ± 0.112

0.9786

Table 2: The h-cyt c secondary structures determined by Chen et al. of denatured h-cyt c in absence & presence of [C8mim][Cl] and [C10mim][Cl].

Native h-cyt c

Mean Residue Ellipticity at θ222 (deg cm2 dmol-1) 11514 ± 345

% α-helical content at θ222 a 30

5340 ± 240 Alk Den h-cyt c 10 6285 ± 326 6 mM [C8mim][Cl] 13 8280 ± 412 6 mM [C10mim][Cl] 20 a α-helical content at θ222 were calculated by the Equation (2) Table 3. ∆G D ( H 2 O ) , m-values and inflection transition points for the MG-like state of h-cyt c at pH 13 upon interaction with 1-alkyl-3-methylimidazolium chlorides. [Cnmim][Cl]

∆G D ( H 2O ) (kJ mol−1)

a

mb (kJ mol−1 M−1)

[C8mim][Cl] 10.81 ± 0.36 1.21 ± 0.04 [C10mim][Cl] 7.63 ± 0.27 0.65 ± 0.02 a ∆G D ( H 2 O ) was calculated by the linear extrapolation method from Eq. (6) b Measure of hydrophobicity of the transition state. c Midpoint concentration of transition.

Cmc (mM) 8.93 ± 0.28 11.74 ± 0.32



Graphical Abstract:

Significant refolding of h-cyt c

2.0

Helical content increases

Lifetime remains almost constant after maximum refolding of alkaline denatured protein

20 of alkaline denatured protein

[C10mim][Cl]

1.8 1.6 1.4

[C8mim][Cl] 1.2

% Helical content

2.2

Fluorescence lifetime ()

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Slight refolding of h-cyt c

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Dynamics of Ionic Liquids Assisted Refolding of Denatured

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