Mechanism and Dynamics of Long-Term Stability of Cytochrome c

Research Article pubs.acs.org/journal/ascecg

Mechanism and Dynamics of Long-Term Stability of Cytochrome c Conferred by Long-Chain Imidazolium Ionic Liquids at Low Concentration Upendra K. Singh,† Meena Kumari,† Sabab H. Khan,† Himadri B. Bohidar,‡ and Rajan Patel*,† †

Biophysical Chemistry Laboratory, Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (A Central University), New Delhi 110025, India ‡ Polymer and Biophysics Laboratory, School of Physical Sciences, Jawaharlal Nehru University, New Delhi 110067, India S Supporting Information *

ABSTRACT: Protein stability has been a concern for researchers for a long time as they are sensitive toward their environment. Mostly proteins during experiments require medium that keep them stable at room temperature (RT). Recent research utilizing ionic liquids (ILs) to stabilize protein has gained much importance. Although a few ILs have been claimed to suit this requirement, reported studies employ IL concentrations that might produce irreversible denaturation and aggregation. This study demonstrates the first report for long-term stabilization of horse heart cytochrome c (h-cyt c) by longchain imidazolium ILs at far low concentration (1 mM) of IL when stored at RT. Long-chain imidazolium ILs until now were less familiar for their stabilizing nature toward protein. A significant increase in the helical content of h-cyt c (dissolved state) was observed with prolonged structural stability (secondary and tertiary) for about 6 months in aqueous solutions of 1-methyl3-octyl imidazolium chloride [C8mim][Cl] and 1-decyl-3-methylimidazolium chloride [C10mim][Cl]. The in-depth mechanism discussed suggests interaction of ILs with amino acid residues of h-cyt c, which rigidifies the loop regions with reduced mobility; hence, prolonged stability is achieved. The study firmly advocates the use of long-chain imidazolium ILs as the potent inhibitor against denaturation during storage of h-cyt c at RT. KEYWORDS: Cytochrome c, Ionic liquid, Long-term stability, Time-resolved fluorescence spectroscopy, Peroxidase activity

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INTRODUCTION Protein stability has been a muse for years, and controlling factors that influence stability and folding have been a key challenge. An altered environment around proteins affects their stability and hence their function. Therefore, ineffective preservation can lead to instability of proteins and severe damage to protein-based pharmaceuticals. Hence, there exists a strong need for solvents that confine these molecules and confer stability by influencing their physical and chemical properties of the proteins at room temperature (RT). Use of ionic liquids (ILs) as solvents for protein stabilization has been reported at much higher concentrations (>1 M),1−3 although not all stabilizing ILs support long-term stability.4 There exists a need for preservation of proteins in native-like environment to resist aggregation and denaturation for maintaining stability at very low concentration of ILs (micro- to millimolar). The most important factor is the reusability of protein after storage for different purposes, this also remains an issue. Hence, it is quite fascinating to study the long-term stability and interaction of the proteins with cosolvents, as stability and activity of proteins are largely dependent on the nature of co-solvents in which they are stored because they are influenced by their physical and chemical properties. Also, understanding about the © 2017 American Chemical Society

mechanism of their bimolecular interactions on structural basis causing stabilization/destabilization is fundamental to our accumulating knowledge. The dissolution of proteins in a stable medium is important for advances in liquid formulation for protein pharmaceuticals and various other applications.5 Several challenges confront scientists involved in the development of protein-based biotechnological medicine. First, the stability of proteins (physical and chemical) is confronted if they have to deal with external factors (pH, impurities, thermal conditions, etc.) during handling and processing at RT as many of them are dramatically prone to aggregation and denaturation upon exposure to RT which raises seriuos doubts about their longterm stabilization (protein-based drugs like Aldesleukin, Insulin, Enbrel, Refacto, etc.).5,6 The development of protein-based drugs is a challenge due to need of maintaining proteins in its native state, from processing until the end point of use. Second, to mitigate the expense of liquid formulations, it is challenging to develop protein drugs that can ease the complexity of administration through desired approach.5 Additionally, the use Received: September 8, 2017 Revised: October 28, 2017 Published: November 3, 2017 803

DOI: 10.1021/acssuschemeng.7b03168 ACS Sustainable Chem. Eng. 2018, 6, 803−815

ACS Sustainable Chemistry & Engineering

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of long-chain ILs is an advancement for stabilizing protein and other biomolecules in various sectors of material research and other biotechnological applications. Hence, long-term stability at processing temperature of biomolecules, which is preferably RT, makes ILs a convenient and sustainable medium to preserve protein for applications. These ILs provide an extra advantage of extending the shelf life of protein-based drugs without any issues of aggregation and maintain easy reusability. The quest for liquids that possess such fascinating properties of molecular liquids and provide a unique possibility to modulate protein stability, a reason for this burst of interest includes their widely tunable properties.3,4,6−11 The different interaction behavior of ILs with proteins has long been reported in neat IL and ILs in aqueous medium.4,10 Significant advances in the ability of ILs to impart stability to proteins and ultimately conserve their structure have been made.7,9,10,12−16 A few ILs have been reported as having an outstanding ability to stabilize proteins over time and high temperature.3,6,10,17 A study by Bihari et al. showed that the presence of neat IL alters the tertiary structure of h-cyt c; however, it was not accompanied by aggregation of the protein.4 Recently, a fascinating study of newly synthesized protein−polymer surfactant nanoconstructs reported by Alex et al. imparts high thermal stability to protein.18 However, in existing literatures the use of high concentrations of ILs have been shown that raise question about the reusability of protein for different purposes. The impact of these chemicals on the environment raises concerns about environmental safety. A constant use of neat ILs as reaction media for heme proteins has been seen to monitor activity and stability of IL at high temperatures,1,10 but such a high concentration of ILs is quite not necessary, as it perturbs the tertiary structure of the heme proteins by interfering with charge transfer band.4,19 Thus, it is desirable to sprung up with ILs which at low concentration (micro- to millimolar) possess the ability to sustain the stability and activity of proteins in the dissolved state for long-term storage periods and that can be easily removed by dialysis before use.1 Thus, such biomolecular interactions causing stabilization− destabilization have been an active subject of considerable interest. Horse heart cytochrome c (h-cyt c) is the most thoroughly studied model metalloprotein, commonly used for folding/ unfolding experiments.20 It contains a heme prosthetic group which has been exploited as probe to monitor structural changes during folding and possesses a separable nature of cooperative folding/unfolding units (foldons).21 We have used the difference in alkyl chain length of the imidazolium cation containing two ILs and consecutively studied the effect of chain length of ILs on long-term stability and interaction at RT, they offer dramatically improved protein stability with respect to time. Reports for h-cyt c with ILs stability and solubility were reported that showed the enhanced stability of h-cyt c.1,4,18,22−24 This encouraged us to check into the utility of long-chain imidazolium ILs, 1-methyl-3-octylimidazolium chloride ([C8mim][Cl]) and 1-decyl-3-methylimidazolium chloride ([C10mim][Cl]), to preserve structure and stability of h-cyt c for long periods at RT. Fortunately, we obtained surprising results in which both ILs not only stabilized h-cyt c but also increased their shelf life for a long period of time at RT at low concentration (1 mM) which is sufficient to meet the requirements for in vitro applications of protein.

Research Article

MATERIALS AND METHODS

Equine heart cytochrome c (h-cyt c, type IV), sodium monobasic dihydrate, sodium dibasic dihydrate, 1-methyl-3-octylimidazolium chloride [C 8 mim][Cl], 1-decyl-3-methylimidazolium chloride [C10mim][Cl], and sodium chloride were purchased from SigmaAldrich. All chemicals and reagents used were of analytical grade. The structures of h-cyt c, [C8mim][Cl], and [C10mim][Cl] are depicted in Scheme 1. Millipore water was used throughout the experiments.

Scheme 1. Structures of h-cyt c, [C8mim][Cl], and [C10mim][Cl]a

a

(a) Showing helices and loops (PDB ID: 1HRC). Two axial ligands His18 and Met80 are covalently linked to heme and the single Trp residue at its position. (b) 1-Methyl-3-octylimidazolium chloride [C8mim][Cl], having positively charged imidazolium and negatively charged chloride (green). (c) 1-Decyl-3-methylimidazolium chloride [C10mim][Cl], having positively charged imidazolium and negatively charged chloride (green).

Preparation of Stock Solution. h-cyt c was oxidized completely to its ferric form by adding 0.1% potassium ferriccyanide to a solution of h-cyt c, then dialyzed against several changes of 0.1 M NaCl, at pH 7.0.25 The oxidized h-cyt c solutions were filtered by using a 0.22 μm Millipore filter. Concentration of the dialyzed solution of h-cyt c was determined experimentally by using molar absorbance coefficient (ε) values of 1.06 × 105 M−1 cm−1 at 410 nm.26 All the measurements were carried out in the degassed sodium phosphate buffer at pH 7.0. The samples were incubated in the dark for a period of 4 h before each experiment. The stock solutions of [C8mim][Cl] and [C10mim][Cl] ILs were made in analytical-grade Millipore water and stored in glass vials. The critical micelle concentrations (cmc) of [C8mim][Cl] and [C10mim][Cl] ILs were 220 and 55 mM, respectively.27 804


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The mean fluorescence lifetimes ⟨τ⟩ for triexponential iterative fittings and pre-exponential factors were calculated using the following relation:33

Circular Dichroism Measurements. Far-UV CD spectra of h-cyt c were carried out through Jasco spectropolarimeter (J-1500), equipped with Peltier type temperature controller (PTC-100). CD spectra of native h-cyt c were taken in the absence and presence of [C8mim][Cl] and [C10mim][Cl] at 25.0 ± 0.1 °C and pH 7.0. For near-UV and Soret spectral measurement, a cuvette with a 1 cm path length was utilized, while a 0.1 cm path length was used for far-UV measurements. The raw CD data were converted into [θ]λ; the mean residue ellipticity (MRE) (deg cm2 dmol−1) at a given wavelength λ using the relation28 [θ ]λ = θλM 0 /10lc

⟨τ ⟩ =

[θ ]222 − 2340 × 100 30300

(3)

where ai and τi are the relative contribution and lifetime of different components to the total decay. A fit was considered acceptable when plots of the weighted residuals, and the autocorrelation function showed random deviation of about 0 with minimum χ2 values. Steady-State Fluorescence Measurements. Steady-state fluorescence measurements were performed on a Cary Eclipse spectrofluorimeter (Varian, USA) equipped with a 150W xenon lamp using 1 cm path length quartz cuvette at 25 °C. Temperature was controlled during experiments using constant-temperature cell holder connected to constant-temperature water circulator (Varian,USA). The excitation and emission slits with a band pass of 5 nm for all experiments. Fluorescence spectra of the h-cyt c was measured at an excitation wavelength of 280 nm, and the emission spectra were recorded in the wavelength range of 300−400 nm. The 8-Anilino-1napthalenesulfonic acid (ANS) is a extrinsic fluorescent dye and was used to detect the presence of partially folded intermediates. The excitation wavelength was set at 360 nm for ANS fluorescence measurements and the protein to ANS molar ratio of 1:20 was used for fluorescence measurements. The concentration for fluorescence measurements of h-cyt c was kept between 5 and 7 μM. Differential Scanning Calorimetry. Differential scanning calorimetry (DSC) measurements of pure h-cyt c and in the presence of 1 mM [C8mim][Cl] and [C10mim][Cl] were performed in MicroCal (USA) VP differential scanning calorimeter having a cell volume of 0.5 mL under a constant pressure of 34 psi in order to avoid the formation of gas bubbles during the experiment. The scanning rates of 1 °C per minute in the temperature range of 20−110 °C for all experiments were used. The protein and buffer solutions were degassed for 20 min prior to loading into the DSC cells. The protein concentration used was 1 mg/mL. The data was analyzed using the Origin computer program supplied by MicroCal, Inc. The instrumental baseline was subtracted from the sample data, and the data were normalized from calories per degree Celsius to calories per mole per degree Celsius. The baselines were created with the software progress baseline function. Peroxidase Activity of h-cyt c. The peroxidase activity of the hcyt c in absence and presence of [C8mim][Cl] and [C10mim][Cl] at 1 mM were investigated. The samples of h-cyt c (2 μM) were preincubated for 5 min with guaiacol in 50 mM potassium phosphate buffer, pH 7.0, and we used the catalytic activity of guaiacol as substrate and H2O2 as an oxygen donor at 25 °C. h-cyt c catalyzes the oxidation of guaiacol to tetraguaiacol and produces an orange color that shows absorption band at 470 nm.34 The rate of formation of tetraguaiacol was observed from the appearance of orange color and qualitatively measured using molar absorption coefficients of 26,678 mM−1 cm−1.35,36 H2O2 solution was prepared from its 30% stock solution (9.79M), and a working solution was prepared form it of 100 mM. The concentrations of guaiacol and H2O2 used for the activity studies were 2 and 10 mM, respectively. Molecular Docking. The docking of ILs with h-cyt c was performed by using molecular docking software AutoDock4.2.37 The 3-D structure of h-cyt c (PDB ID: 1HRC) was downloaded from the Protein Data Bank. The binding structures of [C8mim][Cl] and [C10mim][Cl] were generated on Chem3D Ultra 8.0. Blind docking was performed for docking of both [C8mim][Cl] and [C10mim][Cl] with h-cyt c. The possible binding conformation of the ILs with h-cyt c was computed by using Lamarckian Genetic Algorithm (LGA) implemented in AutoDock4.2. For blind docking, the grid volume of the protein was set in such a way that it covers the whole surface of the h-cyt c. Grid volume was set as 86 × 82 × 98 points with a grid spacing of 0.375 Å and grid center of 46.775, 22.633, 4.362. In the genetic algorithm parameters file, 10 GA runs, 2.5 × 106 maximum number of energy evaluations, 27 000 maximum numbers of generations, and a

(1)

where θλ is the observed ellipticity (millidegrees) at wavelength λ, M0 is the mean residue weight of the protein, c is the protein concentration (mg/cm3), and l is the path length (centimeters). The α-helical content of h-cyt c was calculated from the [θ]λ value at 222 nm using following equation.29

%α =

∑ aiτi 2 ∑ aiτi

(2)

FT-IR Spectroscopy Measurements. 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 pure h-cyt c and with [C8mim][Cl] and [C10mim][Cl]. The concentration of h-cyt c used was 5 mg/mL, and the ILs concentration utilized for the experiments was 10 × 10−4 M. In addition, the FT-IR spectra were also utilized to estimate the secondary structure compositions of pure h-cyt c and with [C8mim][Cl] and [C10mim][Cl] by curve fitting results of the amide I band.41 The subtraction of the reference spectrum was carried out in accord with the criteria described by Dong et al.30 Absorbance Measurements. Soret absorption spectra of h-cyt c in the absence and presence of [C8mim][Cl] and [C10mim][Cl] were obtained with the help of Specord 210 plus spectrophotometer (Analytik Jena, Jena, Germany) equipped with a Peltier controlled temperature controller JUMO dTRON 308 to maintain the constant temperature at 25.0 ± 0.05 °C throughout the experiments. Dynamic Surface Tension Measurements. Dynamic surface tension measurements were performed using Du Nouy−Padday method on DeltaPi-4 (Kibron, Helsinki, Finland), equipped with four parallel microbalances and having a small diameter (0.51 mm) special alloy wire (Dyne Probes, cleaned by Blazer piezo micro torch). The dynamic surface tension of h-cyt c was monitored in buffer when spread at air/water interface.31 The temperature control plate was attached with an external circulating water bath (Grant GD120 water thermostat) to control the subphase temperature at 25.0 ± 0.1 °C. The Delta Graph software was utilized to record the data on the computer. Time-Resolved Fluorescence Measurements. Fluorescence lifetimes were calculated from time-resolved 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 °C. Lamp profiles were measured at the excitation wavelength using Ludox (colloidal silica) as a scatterer. The fluorescence lifetime data were measured to 10 000 counts in the peak, otherwise indicated. The instrumental response function was recorded sequentially using a scattering solution and a time calibration of 114 ps/channel. All experiments were performed using excitation and emission slits with a band pass of 8 nm. The emission wavelength was set to 340 nm and the goodness of fit was judged in terms of both a chi-squared (χ2) value and weighted residuals. Data was analyzed using a sum of exponentials, employing a nonlinear least-squares reconvolution analysis from Horiba (Jobin Yvon, IBH Ltd.). The impulse response functions (IBH DAS6 software) were used to analyze decay curves.32 805


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Figure 1. (a and b) Far-UV CD spectra of h-cyt c (18 × 10−6 M) with [C8mim][Cl] and [C10mim][Cl] of freshly prepared samples. (c and d) NearUV CD spectra of h-cyt c (18 × 10−6 M) with [C8mim][Cl] and [C10mim][Cl] of freshly prepared samples. (e and f) Soret UV-CD spectra of h-cyt c (18 × 10−6 M) with [C8mim][Cl] and [C10mim][Cl] of freshly prepared samples.

Table 1. Far UV CD data of h-cyt c with ILs Shows the Mean Residue Ellipticity at θ222 and α-helical Content (%) at Respective Concentration concentration (× 10‑6 M) 0 5 10 50 100 1000

[C10mim][Cl] θ222 (deg cm2 dmol‑1) −11444 −11507 −12083 −12622 −13395 −14123

± ± ± ± ± ±

343 345 423 379 536 381

[C8mim][Cl] θ222 (deg cm2 dmol‑1) −11444 −12635 −13032 −12939 −13682 −13859

± ± ± ± ± ±

343 379 456 388 547 374

150 population size were set for docking of both ILs. After completion of the docking experiment, 10 different conformers were obtained for binding of ILs with h-cyt c. Out of 10 conformers, the one with the lowest energy (selected on the basis of moldock rank scores based on the lowest binding energy and highest H-bonding score) was used for analysis of the results.

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[C10mim][Cl] α-helical content (%)

[C8mim][Cl] α-helical content (%)

30 30 32 34 37 39

30 34 35 35 37 38

the interaction of residues of h-cyt c with the alkyl chain of ILs and stability induced as a result of the interaction, with respect to the techniques performed. The effect of interaction on structure, surface activity, and thermal behavior of protein with ILs ([C8mim][Cl] and [C10mim][Cl]) was probed through techniques mentioned. The second section presents the timedependent stability of protein at the structural level of protein (secondary and tertiary) and activity of h-cyt c, when stored for 6 months at RT, and discuss its aspects. This is followed by a brief discussion of comparative long-term stability due to the

RESULTS

The following study of h-cyt c interaction and stability with [C8mim][Cl] and [C10mim][Cl] ILs were divided and presented in two separate sections. The first section dictates 806


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change in the spectrum reflects change in heme pocket with distinct heme environment. The Soret region is mainly observed due to the coupling of heme π−π* electric dipole transition moments with those of nearby aromatic residues in the protein. The h-cyt c spectrum shows a bisignate pattern, i.e., one negative at 416 and the other positive at 405 nm. The positive peak at 405 nm quantifies the heme−globin interaction and the spin state of heme−Fe, which under progressive disruption increases and shifts toward a lower wavelength. The 416 nm negative band corresponds to Met80−Fe and Phe82− heme interaction.43,44 The band also corresponds to the distance and orientation of the Met80 side of heme plane. The Soret CD spectra of both ILs show no change in spin state in heme Fe and Met80−Fe as shown in Figure 1e,f, respectively. This shows the ILs both maintains heme−globin interaction and the spin state of heme−Fe and stabilizes it. FT-IR Spectroscopy. FT-IR spectroscopy serves well for investigating the structural characteristic of proteins that range from gross aspects to subtle rearrangement upon ligand binding. The amide band I peak is located in the 1600−1700 cm−1 region is associated with mainly CO stretching and is quite sensitive to study the secondary structure changes of protein. The amide peaks in Figures 2 and S2a of h-cyt c with

difference in alkyl chain of ILs and probable mechanism involved. Interaction of Long-Chain ILs with h-cyt c shows Stabilizing Nature. Circular Dichroism Spectroscopy. FarUV spectroscopy investigates the alterations in the secondary structure of proteins in the range of 200−250 nm. Any change in the backbone orientation will affect the optical transition and hence indicate the changes in the secondary structure of the protein. Helical proteins give characteristic negative peaks at 208 nm (π−π* transition) and 222 nm (n−π* transition). The secondary structure of h-cyt c was first estimated by the far-UV CD upon interaction with [C8mim][Cl] and [C10mim][Cl] shown in Figure 1a,b, respectively. In native conformation, hcyt c possess dominant α-helical structure with minimal sheet content. The secondary structural content of h-cyt c at different concentrations of [C8mim][Cl] and [C10mim][Cl] calculated by eq 2 are listed in Table 1.38 The variation of helical content as a function of [C8mim][Cl] and [C10mim][Cl] was complementary to the results observed in UV for 409 nm peak, time-resolved studies as well as those of near-UV and Soret UV CD (discussed later). In the far-UV range at high concentrations (10 and 50 × 10−3 M), spectra were not recorded owing to high tension voltage. The observed increase in helical content of the hydrophobic ILs alkyl chain interact with the side chains of the hydrophobic amino acid around the heme pocket of h-cyt c and play important role in its stabilization. The interaction of ILs were located through the help of docking at respective sites of h-cyt c. Figure S1a,b, shows the image of the docked sites of the h-cyt c at respective domains bound to long-chain ILs ([C8mim][Cl] and [C10mim][Cl], respectively) as a result of interaction. The residues involved are the mostly placed around the loops of h-cyt c lying adjacent to the heme crevice. Results obtained after docking were analyzed and interpreted in order to understand the effect of interaction with different chain length ILs. The near-UV spectrum has been used for studying the change in tertiary structure of h-cyt c in the (270−300 nm). The structure is maintained by nonspecific interactions of side chains of amino acid in peptide backbone by hydrophobic forces. The near-UV spectrum of the native h-cyt c gives two sharp minima near 282 and 289 nm39 which resemble tight tertiary structural packing of tryptophan residue at position 59 (Trp59) and tyrosine residues (Tyr).39,40 Figure 1c,d shows the stability of tertiary structure upon addition of ILs, respectively. The bands at 282 and 289 nm in the presence of ILs increase, and the tertiary structure of h-cyt c is preserved by tight packing of side chains.39 The tertiary structure is preserved even at high concentration of ILs due to the conserved interaction of Trp59 with the heme propoinate39 that suggest the increased stability in the presence of alkyl chain of ILs to h-cyt c which maintains its tertiary structure. The ILs having a long alkyl chain are hydrophobic and contain positively charged imidazolium ring that interacts with the hydrophobic side chains of various amino acids in protein backbone residues, like lysine having hydrophobic side chain and isoleucine that is itself hydrophobic.41,42 Thus, the interaction increases the stability of the tertiary structure and also maintains the heme crevice, hence preserves the structure of h-cyt c. The Soret region of CD spectra provides insight into the integrity of heme crevice, i.e., heme−polypeptide interactions. The Soret spectrum gives information regarding the immediate conformational environment of the heme group, and any

Figure 2. FT-IR spectra of h-cyt c in absence and presence of 10 × 10−4 M [C8mim][Cl] and [C10mim][Cl].

ILs show the effect of ILs on interaction with h-cyt c on the secondary structure of h cyt c. The strong band appears at the 1652 cm−1; the dominant peak shows the presence of major αhelix with the presence of some different structures represented by small peak.45 The major peak at 1652 cm−1 shifted slightly toward 1654 cm−1 with increased intensity, which shows an increase in content of helix with the addition of ILs to it. [C10mim][Cl] showed a greater increase in the intensity than [C8mim][Cl]. Thus, upon ILs interaction with h-cyt c the increase in structure occurs (helical content). The curve fitted spectra shown in Figure S2b−d depict the changes in peak position of bands with the respective ILs ([C8mim][Cl] and [C10mim][Cl]). The peaks in spectral region of 1700−1600 cm−1 were resolved, and the range in which the peak lies and corresponds to the their respective secondary structural content are reported elsewhere.46 The free h-cyt c spectra obtained were consistent with the far-UV CD data and earlier reported data.6,47 The square of the correlation coefficient (R2) shows good fitting, and the data is in Table S1. These results conclude that the [C10mim][Cl] shows greater interaction and serves as a better additive to maintain the enhance structural content of the h-cyt c than the [C8mim][Cl]. 807


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Figure 3. (a and b) UV−vis spectra of h-cyt c (18 × 10−6 M) with [C8mim][Cl] and [C10mim][Cl] for fresh prepared samples. Insets in (a) and (b) shows plots of the presence of charge transfer band (Met80−Fe axial bond) at 695 nm.

Table 2. Fluorescence Lifetime Measurements and the Population Distribution of h-cyt c with [C8mim][Cl] and [C10mim][Cl] of Freshly Prepared Samples at Room Temperature τ1 (ns)a

τ2 (ns)a

τ3 (ns)a

h-cyt c

0.39

1.98

6.87

5 × 10−6 M 10 × 10−6 M 5 × 10−5 M 10 × 10−5 M 10 × 10−4 M 10 × 10−3 M 50 × 10−3 M

0.39 0.35 0.37 0.32 0.36 0.37 1.63

2.02 2.01 2.10 1.94 1.98 1.93 2.76

6.19 6.17 6.70 6.12 5.93 5.64 5.19

5 × 10−6 M 10 × 10−6 M 5 × 10−5 M 10 × 10−5 M 10 × 10−4 M 10 × 10−3 M 50 × 10−3 M

0.39 0.31 0.41 0.35 0.37 0.34 0.43

2.31 1.90 2.22 1.91 1.94 1.19 2.02

6.86 6.09 5.89 5.66 5.94 14.44 12.39

samples

a

α1 (%) 29 [C8mim][Cl] 29 29 30 28 30 29 53 [C10mim][Cl] 26 27 25 29 33 37 39

α2 (%)

α3 (%)

τ

46

25

4.99

1.12

46 46 45 45 43 48 29

25 26 25 27 27 24 18

4.43 4.49 4.84 4.55 4.37 3.96 3.26

1.12 1.04 1.20 1.16 1.15 1.08 1.06

50 45 45 43 44 58 50

24 28 30 28 23 5 11

4.83 4.57 4.41 4.22 4.19 7.19 7.44

1.14 1.19 0.90 1.07 1.13 1.47 1.21

av

(ns)a

χ2

The mean error estimated for the lifetime parameters: τ1 is ±0.02 ns, τ2 is ±0.07 ns, τ3 is ±0.21 ns, and τav is ±0.15.

UV−Vis Spectroscopy. The h-cyt c in its native conformation shows absorption bands at 280 nm (n−π* transition of aromatic amino acids) and a Soret band at 409 nm (π−π* transition, a position reflecting low spin state iron, a form imposed by the heme’s axial ligands, His18 and Met80 of the heme group). Figure 3a,b gives the absorbance spectra for pure h-cyt c and h-cyt c in the presence of increasing concentrations of both ILs. The absorbance of the peak at 409 nm shows an increase with no peak shift that suggest no changes in the ground state properties of the protein in the presence of ILs and that the heme group in the protein remains intact. The heme of h-cyt c is buried inside the packed hydrophobic core of hydrophobic amino acids, with some lysine residues at the adjacent position. The increase in the absorbance of the Soret peak indicates a transition toward nonpolar environment around the heme cleft as the hypochromic shifts in UV−vis spectra are observed due to the lowering in the energy of the π* orbital. The slight unfolding of h-cyt c has been reported with decreasing absorbance around 409 nm having peak at the same position.48 The inset in the Figure 3a,b shows the 695 nm peak (charge transfer band). This peak corresponds to the Met80− Fe interactions and measures local stability of protein (appears

due to axial ligation of heme with Met80 that yields a sixcoordinated low-spin form); it is enhanced in the presence of ILs. This depicts the stability conferred by the ILs to the protein structure in solution. The UV−vis spectra at relatively high concentration of ILs (2 × 10−1 M), in Figure S3, show no significant change in spectral characteristics, i.e., the heme crevice remained intact and the spin state of metal ion in heme group remains the same with no perturbation of existing bonds (also the charge transfer band at 695 nm). The heme−globin interactions are not perturbed i.e the 409 nm peak is maintained even at such higher ILs concentration, this shows maintained structure of protein. The DSC thermograms for h-cyt c alone and with both ILs at 10 × 10−4 M are shown in Figure S4. The graph shows the midpoint of denaturation temperature (Tm) of 82.6 °C for h-cyt c in agreement with that reported in literature.11 The h-cyt c with [C8mim][Cl] and [C10mim][Cl] at 10 × 10−4 M do not perturb the Tm values of the h-cyt c, which are 82.2 and 82.6 °C, respectively. This suggests that both ILs maintains stability of h-cyt c at 10 × 10−4 M. The respective Tm values are listed in the Table S2. 808


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Figure 4. (a and b) Time-resolved fluorescence decay spectra of the h-cyt c (18 × 10−6 M) with [C8mim][Cl] and [C10mim][Cl] of freshly prepared samples, respectively.

Figure 5. Far-UV CD spectra of h-cyt c (18 × 10−6 M) with [C8mim][Cl] and [C10mim][Cl] of fresh and sample stored 3 and 6 months at room temperature.

Time-Resolved Spectroscopy. Fluorescence lifetime is a very sensitive technique utilized to monitor molecular interactions and motions useful in analyzing the structure and dynamics of biological macromolecules. Here we have measured the decay of the discrete lifetimes of h-cyt c upon interaction with ILs. The h-cyt c is a single Trp containing protein, and it shows a multimodal form of distribution due to the existence of different rotameric conformations of tryptophan side chain that interacts differently with the environment.12 The lifetime h-cyt c is believed to be dependent on the relative orientation and distance between tryptophan and the heme.33 The lifetime experiments of h-cyt c with [C8mim][Cl] and [C10mim][Cl] fitted triple exponential decay at pH 7.0 shown in Table 2. The average lifetime (τav) shows decrease accompanied by decrease in longer lifetime of the h-cyt c with addition of increasing concentration of ILs shown in decay curve spectra in Figure 4a,b, respectively. Earlier studies of protein with ILs11,49 have also shown similar trend in which a decrease in longer lifetime (τ3) relates to a change in polarity. The results corroborate with those of CD and FTIR in which an increase in the helical content of the protein in its secondary structure is observed and the protein becomes more compact. Precisely, the fast and medium components are from the vicinity of heme region, so a change in polarity around heme and the conformation can easily be monitored.50 The results obtained for both ILs at 50 × 10−3 M showed a greater increase of fast and medium components with their pre-exponential factors. The decrease

in longer lifetime correlates to the increase in hydrophobicity around the fluorophore. The overall contributions of the individual components show a greater pre-exponential factor (αi) for shorter lifetimes (τ1). The parameter αi relates to the individual rotamer population and corresponds to the secondary structure of the tryptophan residue by main chain on rotamer populations. A very interesting observation was noted at relatively high concentration (10 and 50 × 10−3 M) of [C10mim][Cl], where a drastic increase in longer lifetime (τ3) with an exponential decrease in its contribution occurs (α3). It was assumed that due to the increase in hydrophobicity around Trp59 it becomes part of regular secondary structure.51 Trp59 resides around the hydrophobic core placed in the loop region of the h-cyt c, and at this concentration, it becomes part of the secondary structure due to greater increase in helical content with IL concentration. Here, the longer lifetime probably correlates to the increase of hydrophobicity around Trp59 that is placed in the hydrophobic core around the heme region, so the decrease in longer lifetime is mainly attributed to the increase in hydrophobicity around the fluorophore, corresponding to the secondary structural increase that makes protein become more compact (the decrease relates to the optimum packing and efficient electron transfer).11,50 This confirms greater interaction of [C10mim][Cl] to nearby residues of Trp59 that might help long-term stability and decrease the mobility of the loops. Moreover, dynamic surface tension (DST) of proteins shows different adsorption behavior on air/water interface dependent 809


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Figure 6. (a and b) UV−vis spectra of h-cyt c (18 × 10−6 M) with [C8mim][Cl] and [C10mim][Cl] of samples stored for 6 months at RT. Inset in (a) and (b) shows plots of the presence of charge transfer band (Met80−Fe axial bond) at 695 nm. (c and d) Near UV CD spectra of h-cyt c (18 × 10−6 M) with 10 × 10−4 M [C8mim][Cl] and [C10mim][Cl] of samples stored for 6 months at RT. (e) Soret CD spectra of h-cyt c (18 × 10−6 M) in 10 × 10−4 M [C8mim][Cl] and [C10mim][Cl] stored samples for 6 months at RT. (f and g) Fluorescence and ANS Fluorescence spectra of h-cyt c in 10 × 10−4 M [C8mim][Cl] and [C10mim][Cl] stored samples for 6 months at RT.

checked through far-UV CD to analyze the structural stability maintained by the ILs when the h-cyt c was stored at RT for 6 months. The loss of helical content for fresh sample and for sample stored for 3 and 6 months at RT for both ILs are shown in Figure 5, respectively. Most of the aliquots of different concentrations of [C8mim][Cl] and [C10mim][Cl] stored for 3 months retained helical content when measured at [θ]222. The long-chain ILs preserve the secondary structure of h-cyt c from unfolding, as presented in Figure S6a,b and Table S3. Our observation for samples stored 6 months reveal unprecedented results. Although the secondary structural

on important factors as conformational stability and hydrophobicity.52 Results shown in Figure S5 suggest that pure h-cyt c has an equilibration time much higher than that of h-cyt c with [C8mim][Cl] and [C10mim][Cl], which clearly suggests the interaction of ILs with h-cyt c as a result of faster adsorption occurs with conformational changes. The greater surface activity shown by the complex (more by [C10mim][Cl]) confirms the interaction of h-cyt c with the ILs. Influence of [C8mim][Cl] and [C10mim][Cl] on TimeDependent Stability of h-cyt c. Secondary Structural Stability. The secondary structural features of protein were 810


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Table 3. Fluorescence Lifetime Measurements and the Population Distribution of h-cyt c with [C8mim][Cl] and [C10mim][Cl] of Samples Stored 3 and 6 Months at Room Temperature

a

samples

τ1 (ns)a

τ2 (ns)a

h-cyt c 10 × 10−4 M [C8mim][Cl] 10 × 10−4 M [C10mim][Cl]

0.36 0.36 0.42

1.82 2.05 2.22

h-cyt c 10 × 10−4 M [C8mim][Cl] 10 × 10−4 M [C10mim][Cl]

0.18 0.47 0.48

1.00 2.46 2.29

τ3 (ns)a 3 Months 6.39 5.84 6.09 6 Months 4.44 7.18 7.12

α1 (%)

α2 (%)

α3 (%)

τ

24 19 19

47 44 48

29 37 33

4.81 4.62 4.64

1.26 1.30 1.27

60 25 24

33 49 52

7 26 24

2.43 5.12 4.98

0.99 1.10 1.05

av

(ns)a

χ2

The mean error estimated for the lifetime parameters: τ1 is ±0.02 ns, τ2 is ±0.07 ns, τ3 is ±0.1 ns, and τav is ±0.15.

Dhp ILs). The near-UV CD spectra showed distinct bands of 282 and 289 nm for samples having relatively higher concentrations of ILs (10 × 10−4 M and 10 × 10−5 M for [C8mim][Cl] and [C10mim][Cl], respectively, same as UV−vis spectra. Figure 6c,d shows stable heme−Trp59 interactions (compact structure) due to maintained packing of residues around heme. Again, the Soret CD spectra shown in Figure 6e, depicts conserved bisignate pattern of peaks at respective positions of both the ILs. This showed conserved structure of h-cyt c by both imidazolium ILs at 1 mM after 6 months of storage at RT. The heme−globin interaction at 405 nm shows no change in the spin state of heme Fe and Met80−Fe (also shown by 695 nm in UV−vis), and Phe82−heme interaction at negative peak at 416 nm was preserved which signifies the stability of h-cyt c. Fluorescence emission was utilized to probe the structural changes in aqueous environment of h-cyt c strored with longchain imidazolium ILs. The emission spectra of native h-cyt c is characteristic of aromatic Trp59, which is quenched by heme group through foster energy transfer, so it produces no fluorescence. Upon protein unfolding, the distance between Trp59 and the heme moiety increases which further reduces the extent of quenching, and an increase in tryptophan fluorescence is observed which goes in hand with loss in protein structure. To further warrant the conformational changes, we recorded the fluorescence spectra of stored samples. The fluorescence spectra of h-cyt c in Figure 6f shows distinguished emission maximum at 340 nm having higher intensity. This hints toward the loss of tertiary structure of the protein during storage at RT with exposure of Trp59. The samples with ILs at 1 mM showed preserved structure and produced almost no fluorescence. 8Anilino-1-naphthalene sulfonic acid (ANS) is a hydrophobic fluorescent molecule that is commonly used for the characterization of partially folded intermediates.55,56 They are minimally fluorescent in polar environments, but upon exposure to a nonpolar environment their fluorescence emission increases dramatically.57 ANS fluorescence was also utilized as probe to inspect the presence of exposed hydrophobic patches on the protein.58 The presence of these exposed hydrophobic patches are confirmed by the binding of ANS to it, producing a blue shift in its emission maximum, different from when ANS is present in aqueous solution.55 The spectra of h-cyt c stored for 6 months (denatured h-cyt c) shown in Figure 6g have almost same emission maximum as native h-cyt c, i.e., no hydrophobic patch of proteins are exposed for binding to ANS. The results obtained confirm that no shift in emission maximum of stored sample stored sample denotes preserved structure of h-cyt c by ILs that restrain unfolding and provide stabilization at RT for long storage periods. The lifetime results augmented the above

content is not preserved at lower concentrations of both ILs (up to 50 × 10−6 M), high concentration of the [C8mim][Cl] (100 × 10−6 M) preserved little helical content when compared to h-cyt c without IL. Most amino acid residues involved in the interaction with [C8mim][Cl] are located in the helix. Striking consequences were obtained from h-cyt c with [C10mim][Cl] that shows the preserved secondary structure even when stored for a long-term period at RT (helical content was maintained corresponding to increase) shown in Figure S6c,d and Table S3. This was probably due to the interaction pattern of both hydrophobic ILs having long-chain with the side chains of the hydrophobic amino acids around the heme pocket53 (docking results Figure S1a,b). Tertiary Structure Stability. The h-cyt c stored for 3 months showed loss of structure by absence of charge transfer band at 695 nm in UV−vis results with decreased intensity around 409 nm. It was due to disrupted heme−globin interaction (reduction in efficiency of electronic transition and decreased stability of heme) but consisting of the Q-band (530 nm) shown in Figure S7a,b. The optical transitions are quite sensitive regarding ligand exchange reactions.6,44 Samples stored with both ILs for 3 months show almost no change in spectral characteristics. The Soret CD spectra of samples showed loss of negative peak at 416 nm of h-cyt c (data not shown), while stored samples having 1 mM [C8mim][Cl] and [C10mim][Cl] showed bisignate pattern with both peaks conserved. Lifetime results in Figure S7c,d and Table S4a show stable protein samples with minimal change in lifetimes at several concentrations of both ILs. Little perturbation in fast and medium lifetimes was observed. However, extending our observations to 6 months in Figure 6a,b, UV−vis results show the h-cyt c without any IL showed broad Soret band; this is due to exposure of the heme to aqueous solution after dissociation (disrupted heme−globin interaction moves the heme out of its pocket). The loss in the structure of the h-cyt c is due to the loss of axial heme ligand after peptide chain shows extended configuration due to denaturation.54 Samples with lower concentrations of both ILs (up to 50 × 10−6 M) showed similar trends (Table S4a,b). To our surprise, at concentrations beyond these, as shown Figure 6a,b, the structural integrity of h-cyt c with both ILs was maintained with noted 409, 530, and 695 nm distinctly visible bands in the spectra shown. The stabilization effect of the h-cyt c by ILs has been strongly evident. The maintenance of tertiary structure and heme packing (heme stability) of h-cyt c for a long time at RT was perhaps due to lower water content in direct interaction with the protein. Concomitant results have been shown by Fujita et al.6 by storing protein in short chain ILs but at comparatively very high concentrations (80 wt % 811


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Figure 7. (a and b) Time-resolved fluorescence decay spectra of h-cyt c (18 × 10−6 M) with 10 × 10−4 M [C8mim][Cl] and [C10mim][Cl of samples stored for 6 months at RT, respectively.

surface, which restrains the unfolding of polypeptide chain, disallows the disruption of hydrogen bonds, and thus holds back the loops to cooperatively unfold by excluding excessive water content and maintaining the structural integrity.53 The primary unfolding hotspots reported in literature which trigger sequential unfolding of h-cyt c are concentrated in the loop region;59,60 thus, interaction of ILs confers greater stability to the native oxidized form of h-cyt c.

results as the average lifetimes listed in Table 3 also differ from the stored h-cyt c without IL due to the different conformational fluctuations, shown in Figure 7a,b. Influence of [C8mim][Cl] and [C10mim][Cl] on Peroxidase Activity of h-cyt c. The activity of h-cyt c is useful to study its unfolding and was also utilized as an indicator to monitor the conformational changes. The spectra at 470 nm by UV−vis spectroscopy were recorded to monitor formation of tetraguaiacol from guaiacol in the presence of H2O2 with increase in absorbance.35,36 The activity of fresh samples of hcyt c as well as those with [C8mim][Cl] and [C10mim][Cl] were recorded. The h-cyt c showed peroxidase activity slight higher in the presence of both ILs as in Figure 8. The samples

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DISCUSSION Why do ILs stabilize the protein for a longer period without perturbing the heme crevice even at higher concentrations? We analyzed the results in accordance to the specificity of IL ions since the protein stabilization by ILs is the result of a subtle balance between ion−protein and ion−solvent interaction.53 The anionic moiety of ILs play important role, as in Hofmeister series Cl− is placed in the middle of the range for stabilizing/ destabilizing effect on proteins, so they have smaller tendency toward protein denaturation. The Cl− anion is excluded from the protein surface that equips the cation to interact and promote stability. The Cl− anion is strongly hydrated; thus, it has limited interaction with the cationic moiety in aqueous solution and with the charged or polar residues of the protein backbone which restrains dehydration of the protein surface. The internal mobility of h-cyt c (mostly in loop region) in the presence of IL is also more restricted than in aqueous solutions that rigidify the segments. Cl− ion can show weak interactions in area with positively charged patches at the protein surface,61 so Cl− enhances the stability by preserving structure of h-cyt c and indirectly the heme packing (heme stability) for longer period of time. Thus, availing the advantage over Cl− by the imidazolium IL cation ([C10mim]+ and [C8mim]+) is that it interacts at the protein surface (Figure S1a,b). The cationic part with the imidazolium headgroup is amphiphillic in nature as it consist of a high charge density part (polar headgroup) and low charge density (nonpolar tail). The accumulation of imidazolium cation on surface is useful in removing the surrounding water molecules that reduce the probable hydrogen bonding of water from the protein surface.9,53,62 Owing to the different chain lengths of both ILs, [C10mim]+ and [C8mim]+, different specificity to protein residues is shown. The [C10mim]+ interacts with accessible residues of 60’s α-helix and with unstructured loop regions, infrared loop and red loop (Met-80 and considered “hotspot” for unfolding)59,60 at the protein surface; thus, the affinity of imidazolium IL cation for protein surface depends upon the hydrophobicity and accessibility (size of cation). In results, the presence of charge transfer band at

Figure 8. Peroxidase activity of h-cyt c in the presence of 10 × 10−4 M [C8mim][Cl] and [C10mim][Cl]. The graph shows data for samples freshly prepared and those stored h-cyt c for 6 months at RT.

subjected to storage for 6 months were also checked for their activity. The stored h-cyt c peroxidase activity was enhanced due to altered conformation. The h-cyt c preserved with ILs for over 6 months showed slightly reduced activity compared to that of fresh sample. The formation of tetraguaiacol has been shown for formation of product during reaction shown in Figure S8. The results show that the structure of h-cyt c is preserved by ILs for 6 months at RT. The ILs have hydrophobic chain through which they tend to interact with amino acid residue which increases the hydrophobicity around heme. The ILs stabilize the tertiary structure of h-cyt c by interacting around the flexible structures at protein 812


ACS Sustainable Chemistry & Engineering 695 nm (Figure 6a,b) of h-cyt c with both ILs preserves the structural integrity of h-cyt c and restrains denaturation. The charged residues at pH 7.0 show cation binding and thus more electrostatic contribution after interaction, but this is limited with the case of longer alkyl chain. [C10mim][Cl] interacts at structures located on the protein surface and show prolonged effect for maintaining structural integrity (tertiary and secondary) of h-cyt c. This shows the significant role of both anion and cation in determining the stability of protein. Recent literature suggests the important function of Lys73 along with other lysine residues (Lys72 and Lys79) required for stabilizing axial coordination of Met80-heme as well as the tertiary structure.41 It maintains two salt bridges with Glu66 and Glu69, and in the absence of Lys73 both are lost.41 It is also involved in molecular recognition when binding with cardiolipin.41 Another study provides hydrophobic binding pocket for long-chain ligands that binds creating small shifts in position of lys55, val57 and tyr74 (least stable infrared loop).63 The accommodation of hydrophobic chain slightly affects the tertiary structure of h-cyt c, and Tyr67 is nearest to it lying adjacent to the heme.63 These findings corroborate docking results obtained for [C10mim][Cl] after interaction with h-cyt c (Figure S1b) and demonstrate its long-term stability in aqueous solution of IL.

ACKNOWLEDGMENTS

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REFERENCES

(1) Fujita, K.; MacFarlane, D. R.; Forsyth, M.; Yoshizawa-Fujita, M.; Murata, K.; Nakamura, N.; Ohno, H. Solubility and Stability of Cytochrome c in Hydrated Ionic Liquids: Effect of Oxo Acid Residues and Kosmotropicity. Biomacromolecules 2007, 8 (7), 2080−2086. (2) Byrne, N.; Angell, C. A. Protein Unfolding, and the “Tuning In” of Reversible Intermediate States, in Protic Ionic Liquid Media. J. Mol. Biol. 2008, 378 (3), 707−714. (3) Byrne, N.; Wang, L. M.; Belieres, J.-P.; Angell, C. A. Reversible folding-unfolding, aggregation protection, and multi-year stabilization, in high concentration protein solutions, using ionic liquids. Chem. Commun. 2007, 26, 2714−2716. (4) Bihari, M.; Russell, T. P.; Hoagland, D. A. Dissolution and Dissolved State of Cytochrome c in a Neat, Hydrophilic Ionic Liquid. Biomacromolecules 2010, 11 (11), 2944−2948. (5) Frokjaer, S.; Otzen, D. E. Protein drug stability: a formulation challenge. Nat. Rev. Drug Discovery 2005, 4 (4), 298−306. (6) Fujita, K.; Forsyth, M.; MacFarlane, D. R.; Reid, R. W.; Elliott, G. D. Unexpected improvement in stability and utility of cytochrome c by solution in biocompatible ionic liquids. Biotechnol. Bioeng. 2006, 94 (6), 1209−1213. (7) Jha, I.; Attri, P.; Venkatesu, P. Unexpected effects of the alteration of structure and stability of myoglobin and hemoglobin in ammoniumbased ionic liquids. Phys. Chem. Chem. Phys. 2014, 16 (12), 5514− 5526. (8) Patel, R.; Kumari, M.; Khan, A. B. Recent advances in the applications of ionic liquids in protein stability and activity: a review. Appl. Biochem. Biotechnol. 2014, 172 (8), 3701−3720. (9) Jha, I.; Venkatesu, P. Unprecedented Improvement in the Stability of Hemoglobin in the Presence of Promising Green Solvent 1-Allyl-3-methylimidazolium Chloride. ACS Sustainable Chem. Eng. 2016, 4 (2), 413−421. (10) Bharmoria, P.; Kumar, A. Unusually high thermal stability and peroxidase activity of cytochrome c in ionic liquid colloidal formulation. Chem. Commun. 2016, 52 (3), 497−500. (11) Jaganathan, M.; Ramakrishnan, C.; Velmurugan, D.; Dhathathreyan, A. Understanding ethylammonium nitrate stabilized cytochrome c − Molecular dynamics and experimental approach. J. Mol. Struct. 2015, 1081, 334−341. (12) Kumari, M.; Singh, U. K.; Singh, P.; Patel, R. Effect of N-ButylN-Methyl-Morpholinium Bromide Ionic Liquid on the Conformation Stability of Human Serum Albumin. ChemistrySelect 2017, 2 (3), 1241−1249. (13) Kumari, M.; Dohare, N.; Maurya, N.; Dohare, R.; Patel, R. Effect of 1-methyl-3-octyleimmidazolium chloride on the stability and activity of lysozyme: a spectroscopic and molecular dynamics studies. J. Biomol. Struct. Dyn. 2017, 35, 2016−2030. (14) Patel, R.; Parray, M. u. d.; Singh, U. K.; Islam, A.; Venkatesu, P.; Singh, S.; Bohidar, H. B. Effect of 1,4-bis(3-dodecylimidazolium-1-yl) butane bromide on channel form of gramicidin vesicles. Colloids Surf., A 2016, 508, 150−158. (15) Singh, U. K.; Dohare, N.; Mishra, P.; Singh, P.; Bohidar, H. B.; Patel, R. Effect of pyrrolidinium based ionic liquid on the channel form of gramicidin in lipid vesicles. J. Photochem. Photobiol., B 2015, 149, 1− 8. (16) Kumari, M.; Maurya, J. K.; Singh, U. K.; Khan, A. B.; Ali, M.; Singh, P.; Patel, R. Spectroscopic and docking studies on the interaction between pyrrolidinium based ionic liquid and bovine serum albumin. Spectrochim. Acta, Part A 2014, 124, 349−356.

CONCLUSION This work reveals the unusual potential of the long-chain imidazolium ILs to maintain the stability of h-cyt c for longer periods at RT at low concentrations (1 mM) never reported before. Long-chain IL [C10mim][Cl] interacts around the 60’s helix and unstructured loops present at protein surface with Met-80 that reduces mobility of loop region and excludes excessive water content. This enhances stability to local protein regions which are first prone to unfolding during denaturation, so the study provides a promising and compatible solvent for maintaining protein structural stability and prompts us to further evaluate parameters controlling stability. We propose that development of long-chain imidazolium ILs can cause major advances in maintenance of the cyt c stability for longterm periods. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b03168.

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Dr. Rajan Patel greatly acknowledges the financial supports from Science and Engineering Research Board (EEQ/2016/ 000339 and SR/S1/PC-19/2011) 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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Research Article

Docking figures, FT-IR, UV−vis, DSC, far-UV CD, timeresolved fluorescence spectra, surface tension, peroxidase activity (long-term stability) results (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel.: +91-8860634100. Fax: +91-11-26983409. E-mail: [email protected], [email protected]. ORCID

Rajan Patel: 0000-0002-3811-2898 Notes

The authors declare no competing financial interest. 813


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Mechanism and Dynamics of Long-Term Stability of Cytochrome c

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