Molecular investigation of the interaction between ionic liquid type gemini surfactant and lysozyme: A spectroscopic and computational approach

Materials

Lysozyme was purchased from Sigma Aldrich and the [C₁₂−4-C₁₂im]Br₂ was synthesised in our lab according to the literature.39 The structure of [C₁₂−4-C₁₂im]Br₂ is shown in Scheme 1. The stock solution of lysozyme (5 × 10⁻⁶M L⁻¹) was prepared in phosphate buffer at pH 7.40. The concentration was calculated by dividing absorbance at 280 nm by the molar extinction coefficients of the lysozyme ε₂₈₀ = 2.64 mL mg⁻¹ cm⁻¹.40 All other reagents were of analytical reagent grade and doubly distilled water was used throughout the experiments.

Steady-State Spectra

The steady state fluorescence spectra were obtained at fixed concentration of lysozyme (5 × 10⁻⁶M) and varied concentration of [C₁₂−4-C₁₂im]Br₂ (1.1 × 10⁻⁵−4.5 × 10⁻⁵M) by spectrofluorimeter (Varian Cary Eclipse) with the wavelength range of 290 to 500 nm. All fluorescence spectra were collected using 1.0 cm quartz cuvette with both excitation and emission band width set at 5 nm. The intrinsic fluorescence of the lysozyme was measured at an excitation wavelength of 280 nm to ensuring the overall fluorescence of the protein i.e. excitation of both Trp and Tyr residues. However, Phe is not excited in most cases and its quantum yield in protein is rather low, so the emission from this residue can be ignored.28 The samples were carefully degassed using pure nitrogen gas before the experiment.

UV-Vis Spectra

The absorption spectra were carried out on Analytik Jena specord-250 spectrophotometer with the wavelength range of 230 to 400 nm. Quartz cuvettes having 1 cm path length were used for the measurements. The absorbance of lyzozyme was measured by keeping constant concentration of lysozyme (5 × 10⁻⁶M) and varying the concentrations of the [C₁₂−4-C₁₂im] Br₂. The experiments were recorded at 298 K.

Time-Resolved Measurements

Fluorescence lifetime measurements were carried out in a picosecond time correlated single photon counting (TCSPC) spectrometer equipped with pulsed nanosecond LED excitation heads at 280 nm (Horiba, JobinYvon, IBH Ltd, Glasgow, UK). The fluorescence lifetime data were measured to 10,000 counts in the peak, unless otherwise indicated. The excitation of the sample is carried out by a nanosecond diode laser at 280 nm as a light source. The data analysis was carried out by the software provided by IBH (DAS-6).

Fourier Transform-Infrared Spectroscopy (FT-IR) Measurements

FT-IR spectra were recorded with a Spectrum BX-II Perkin-Elmer Spectrometer (Perkin-Elmer). The FT-IR spectra of lysozyme in the absence and presence of [C₁₂−4-C₁₂im]Br₂ were recorded at room temperature in the range of 1700 to 1500 cm⁻¹. Lysozyme was prepared using a deuterated buffer to prevent interference by water in the amide region of the protein spectra. In addition, the FT-IR spectra was also utilized to estimate the secondary structure compositions of pure lysozyme and lysozyme-[C₁₂−4-C₁₂im]Br₂ complex by curve fitting results of the amide I band.41

Molecular Docking

MGL tools 1.5.4 with AutoGrid 4 and AutoDock 4 were used to set up and perform blind docking calculations between the [C₁₂−4-C₁₂im]Br₂ and lysozyme. The crystal structure co-ordinates for lysozyme (PDB ID: 6LYZ) were obtained from Protein Data Bank (http://www.rcsb.org/pdb.). The three-dimensional structure of ligand [C₁₂−4-C₁₂im]Br₂ was created by CORINA software. Receptor (lysozyme) and ligand ([C₁₂−4-C₁₂im]Br₂) files were prepared using AutoDock Tools. The lysozyme was enclosed in a box with number of grid points in x _ y _ z directions, 98 _ 84 _ 112 and a grid spacing of 0.375 Å. The center of the grid set to 0.082, 20.969, and 19.816 Å. Lamarckian genetic algorithm (LGA) implemented in AutoDock was applied to calculate the possible conformation of the [C₁₂−4-C₁₂im]Br₂ that binds to the lysozyme. During docking process, a maximum of 10 conformers was considered for the lysozyme. The conformer with the lowest binding free energy was used for further analysis. The output from AutoDock was rendered with PyMol and python molecular viewer 1.5.6.

Molecular Dynamic Simulation

Molecular dynamics (MD) simulation is an important tool to understand the binding mode of the ligand to the protein. Moreover, MD simulation also provides the information about the function of biological molecules as well as the physical basis of the structure.42 The MD simulations were performed using the GROMACS 5.0 package, in conjugation with the GROMOS96 43a (I/II) force field.43, 44 The GROMACS and Prodrg program were used to create the topology parameter of lysozyme and [C₁₂−4-C₁₂im]Br₂, respectively. The lysozyme-[C₁₂−4-C₁₂im]Br₂ complex was immersed in the cubic box of simple point charge (SPC) water molecule.45 Counterions were added to neutralize the entire solvated system. The steepest decent method of 500 steps was used for energy minimization to release the conflict contacts. Additionally, it was followed by a conjugate gradient method for 500 steps. Generally, there are two phase in MD simulation studies, equilibration and production phase. In equilibration phase the counter ions, lysozyme and [C₁₂−4-C₁₂im]Br₂ were fixed and the position controlled the dynamic simulation of the system, wherein the atom position of lysozyme was controlled at 300 K for 100 ps. In the last step, the full system was subjected to 4000 ps MD simulation at 300 K and 1 bar pressure. During simulation process, the leaf frog algorithm was carried out with a time step of 2 fs. Atomic coordinates were also recorded at every 0.5 ps for latter analysis.

Fluorescence Quenching of Lysozyme by [C₁₂−4-C₁₂im]Br₂

The fluorescence measurements provide useful information for the binding mechanism between ligand and protein.46 There are three intrinsic fluorophore molecules, tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) present in protein which provides information about protein conformation and intermolecular interactions. Figure 1 shows the fluorescence spectra of lysozyme in the absence and presence of [C₁₂−4-C₁₂im]Br₂ in buffer (pH = 7.40) when excited at 280 nm at 298 K. The solution of lysozyme (5 μM) was titrated by the gradual addition of [C₁₂−4-C₁₂im]Br₂ solution (stock solution 8 mM) to give the concentration range from 1.1 × 10⁻⁵ to 4.5 × 10⁻⁵M. It is evident from the Figure 1 that the emission intensity of lysozyme decreases with increasing concentrations of [C₁₂−4-C₁₂im]Br₂, however, there is no shift in the maximum emission wavelength suggesting that [C₁₂−4-C₁₂im]Br₂ binds to lysozyme and quenches its intrinsic fluorescence.47

The fluorescence quenching method has been extensively used to investigate the protein-surfactant interaction and quenching mechanism. For any process the decrease in fluorescence intensity of a fluorophore refers to fluorescence quenching. Usually the fluorescence quenching is classified as dynamic quenching and static quenching. Dynamic quenching results from collision between the fluorophore and quencher, while static quenching results from the formation of a ground-state complex between the fluorophore and quencher.48

Fluorescence intensities of lysozyme samples were corrected for respective absorbances of the [C₁₂−4-C₁₂im]Br₂ at emission and excitation wavelengths of the fluorescence data. Corrections for inner filter effect were made using the equation49

(1)

where F_cor is the corrected fluorescence intensity and F_obsd is the background subtracted observed fluorescence intensity. The values A_ex and A_em are the measured absorbances at the excitation and emission wavelengths. In order to determine the quenching mechanism, the fluorescence data was analysed by the Stern-Volmer equation50

(2)

where F₀ and F are the fluorescence intensities in the absence and presence of quencher, K_SV is the Stern-Volmer quenching constant, [Q] is the concentration of quencher, k_q is the bimolecular quenching rate constant and τ₀ is the average life time of molecules in the absence of [C₁₂−4-C₁₂im]Br₂ and its value is about 10⁻⁸ s.50 Figure 2 shows the Stern–Volmer plot of [C₁₂−4-C₁₂im]Br₂-lysozyme system at 298 K. From the slopes of the liner plot, the K_SV was calculated and found 3.058 × 10¹¹ (L M⁻¹) for lysozyme. The k_q for lysozyme-[C₁₂−4-C₁₂im]Br₂ system are found to be 3.058 × 10¹¹ (L M⁻¹ s⁻¹) which is higher than that of scatter procedure (2 × 10¹⁰ L mol⁻¹ s⁻¹).51 This results shows that the quenching of lysozyme by [C₁₂−4-C₁₂im]Br₂ is initiated by static quenching mechanism, resulting from the ground state complex formation between [C₁₂−4-C₁₂im]Br₂ and lysozyme.

UV-Visible Absorbance Study

The absorption spectra were recorded to explore the structural change in protein and quenching processes.52 The lysozyme exhibits a characteristic strong absorption band at 220 nm and a week band near 280 nm, which corresponds to n-π* and π-π* transition of the aromatic amino acid residues, Tyr and Trp.53 The change in peak near 220 nm explores the change in secondary structure of protein while peak at 280 nm indicates the change in chromophore microenvironment. The absorption spectra of lysozyme in the presence and absence of [C₁₂−4-C₁₂im]Br₂ are shown in Figure 3. The increase in absorbance at 280 nm by increase in [C₁₂−4-C₁₂im]Br₂ concentration indicates the formation of a ground state complex, since in dynamic quenching the absorption spectrum of quenching molecule remains unaffected, as it only affects the excited state of quenching molecule. The observed changes in lysozyme absorbance in the presence of different concentrations of [C₁₂−4-C₁₂im]Br₂ suggests the occurrence of static quenching interaction between lysozyme and [C₁₂−4-C₁₂im]Br2.54, 55

Determination of Binding Constant

The absorption relationship between the lysozyme and [C₁₂−4-C₁₂im]Br₂ is expressed by double reciprocal equations56

(3)

where A₀ and A are the absorbance of lysozyme in the absence and presence of [C₁₂−4-C₁₂im]Br₂ and C_F is the analytical concentration of [C₁₂−4-C₁₂im]Br₂, ɛ_D and ɛ_DF are the molar extinction coefficients of lysozyme and lysozyme-[C₁₂−4-C₁₂im]Br₂ complex respectively at 280 nm and K is the binding constant. The double reciprocal plot of 1/(A − A₀) versus 1/C_F (Figure 4) is linear and the binding constant (K) was estimated by calculating the ratio of the intercept to the slope along with the values of correlation coefficient (R = 0.9914) that justifies the excellence of the linear fit. The value of K was found to be 2.541 × 10⁵ M⁻¹. The high K value indicates that there is a strong interaction between lysozyme and [C₁₂−4-C₁₂im]Br₂. The free energy change (ΔG) for this process of complexation was obtained at room temperature by employing the following relation:57

(4)

The negative value of ΔG (−31.35 k J mol⁻¹) show that the lysozyme-[C₁₂−4-C₁₂im] Br₂ complexation is energetically favorable.

Time-Resolved Measurements

Time-resolved fluorescence is a very precise technique to describe the quenching mechanism.58 Therefore, the quenching mechanism for the interaction between [C₁₂−4-C₁₂im]Br₂ and lysozyme was further confirmed through life time measurements. Figure 5 shows the fluorescence lifetime decay profile of lysozyme in absence and presence of [C₁₂−4-C₁₂im]Br₂. The fluorescence decay profile curves are well fitted to biexponential function with relative fluorescence lifetime 1.20 ns (τ₁) and 3.06 ns (τ₂) which is typical for tryptophan in proteins.59 On increasing the concentration of [C₁₂−4-C₁₂im]Br₂, the value of τ₁ and τ₂ did not vary significantly. Therefore, average fluorescence lifetime (τ_av) was used for exploring these results.60 From Table 1 it was observed that there is no significant change in average lifetime of lysozyme with the different concentration of [C₁₂−4-C₁₂im]Br₂. It was reported that for a static quenching the fluorescence life time of fluorophore will not disturb during complex formation.58 Thus, the result clearly confirmed that the static quenching was dominant in this interaction system. These results are in good agreement with the results of steady state fluorescence and UV-visible study.

FT-IR Analysis

FT-IR spectroscopy is a highly sensitive technique to study the secondary structural characteristics of proteins.61 Wu et al has employed FT-IR spectroscopy to analysis the secondary changes in BSA upon binding with EQ14-2-14.62 Similarly, Wang et al studied the quantitative analysis of secondary structure of BSA on interacting with NAEn-s-n.63 The infrared spectra of protein give nine characteristic infrared absorption bands. Out of these, the amide-I and amide-II are the most important absorption bands.64-66 The amide-I (1600–1700 cm⁻¹) absorption band is characteristic to the CO stretching vibrations of the peptide linkages which is the characteristic of hydrogen bonding having significantly higher signal intensity whereas amide-II (1500–1600 cm⁻¹) band is in-plane NH bending and CN stretching vibration of peptide group and is quiet as not sensitive to the protein conformation. The important factors that govern the conformational sensitivity of amide bands are the hydrogen bonding and coupling between transition dipoles67. From Figure 6, a peak at 1643 cm⁻¹ was observed in the FT-IR spectra of lysozyme. Also, a decrease in the intensity of amide bands with a small shift in the position of amide I band was observed (from 1643 cm⁻¹ to1640 cm⁻¹) in presence of [C₁₂−4-C₁₂im]Br₂. These changes in the infrared spectrum of lysozyme confirmed the conformation change in lysozyme by the addition of [C₁₂−4-C₁₂im]Br₂.

The protein secondary structure is determined from the shape of amide I band (Figures 7A and 7B) which reveals the quantitative analysis of the protein secondary structure of lysozyme before and after the interaction with [C₁₂−4-C₁₂im]Br₂. The FT-IR spectra were smoothed and their baselines were corrected. The peak positions in spectral region 1700 to 1600 cm⁻¹ of amide I bands are α-helix (1660–1650 cm⁻¹), β-sheet (1638–1610 cm⁻¹), turn (1680–1660 cm⁻¹), random coil (1648–1638 cm⁻¹), and β-antiparallel (1692–1680 cm⁻¹) respectively.

The spectral region was deconvoluted by the curve fitting method using the Levenberg-Marquadt algorithm and the major peaks and the protein and the complexes in the region mentioned were resolved. Gaussian function fitting curve was used to calculate the deconvoluted area in the spectral region. The square of the correlation coefficient (R²) for all fittings is 0.9999 for lysozyme and 0.9998 for lysozyme-[C₁₂−4-C₁₂im]Br₂ complex. After the interaction of lysozyme with [C₁₂−4-C₁₂im]Br₂, a decrease of α-helix was observed from 34% to 29% and β-sheet structure from 28% to 9%, respectively. The random coil structure increases from 16% to 34% and unordered structure from 21% to 28%. The decreased in α-helix and β-sheet structure and increased in the random coil and unordered structure, suggests a conformational change of lysozyme induce by [C₁₂−4-C₁₂im]Br₂.29

Molecular Docking Study

Molecular docking methods have been used to find out the various binding modes and binding sites for lyosozyme-[C₁₂−4-C₁₂im]Br₂ interaction system. Lysozyme has two domain α-helices and β-strand domain. The α-domain contains several α-helices (A, B, C, D, and 3₁₀). The β domain comprised by β-strands β1 (43–46), β2 (51–54), and β3 (58–60).68, 69 The principal region of [C₁₂−4-C₁₂im]Br₂ binding sites of lysozyme is located in loop and β strand domain. The amino acid residues lining this binding site were composed of Lys 33, Phe 34, Glu 35, Ser 36, Asn 37, Asn 52, Gln 57, Asn 59, Trp 62, Trp 63, Ile 98, Ala 107, Trp 108, and Val 109 (Figure 8A). All the Trp residues (Trp 62, Trp 63, and Trp 108) are located at the active site and are important for the enzymatic activity of lysozyme.70 The involvement of Trp residues in the protein ligand complex indicates that [C₁₂−4-C₁₂im]Br₂ binds to the active site of lysozyme. The conformation with the lowest binding energy (−3.49 kcal/mol) was used for analysis. The more negative the relative binding energy, the more potent the binding of ligand to protein. The essential driving force of [C₁₂−4-C₁₂im]Br₂ binding to this site was hydrophobic interaction (Figure 8B). Additionally, docking results reveal that there is one hydrogen bond between [C₁₂−4-C₁₂im]Br₂ and Asn 52 amino acid residue of lysozyme with the bond length of 3.005 Å (Figure 8C).

Molecular Dynamic Simulation Study

The lowest docked energy conformation of lysozyme-[C₁₂−4-C₁₂im]Br₂ complex was selected for MD simulation. To estimate the stability of this conformer the properties, root mean square deviation (RMSD), root mean square fluctuation (RMSF) and radius of gyration (R_g) were determined. The RMSD, R_g, and RMSF values of the atoms in the lysozyme with and without [C₁₂−4-C₁₂im]Br₂ were determined against the simulation time scale of 4000 ps. The RMSD values of the atoms in pure lysozyme and the lysozyme-[C₁₂−4-C₁₂im]Br₂ complex was plotted from 0 to 4000 ps, as shown in Figure 9A. From the figure it can be seen that the RMSD values for free lysozyme and the lysozyme-[C₁₂−4-C₁₂im]Br₂ complex, reached equilibrium and oscillates around an average value after 3000 ps time and remained stable until the end of the simulation. The observed values of RMSD for free and bound lysozyme are found to be 0.184 ± 0.01 and 0.132 ± 0.01 nm respectively. The lesser value of RMSD of bound lysozyme as compared to that of free lysozyme suggests the conformational change in the lysozyme after the binding with [C₁₂−4-C₁₂im]Br₂.71 In addition, the R_g values were also measured for overall structural dimension of the protein.72 The calculated R_g values for free lysozyme and the lyosozyme-[C₁₂−4-C₁₂im]Br₂ complex were found to be 1.3436 nm and 1.3493 nm, respectively and presented in Figure 9B. The R_g values for free and bound lysozyme are highly closer to each other, suggesting that there is no such structural unfolding is induced by [C₁₂−4-C₁₂im]Br₂ after binding with the protein 72 Therefore, to confirm the actual conformational change in lysozyme the solvent accessible surface area (SASA) variation in the time interval of 4000 ps in presence and absence of [C₁₂−4-C₁₂im]Br₂ were analyzed and is shown in Figure 10A. It is obvious from the figure that SASA of lysozyme when bound to [C₁₂−4-C₁₂im]Br₂ is more and can be attributed to the reduction of intra molecular hydrogen bonds of lysozyme. This again confirms the unfolding of lysozyme after interacting with [C₁₂−4-C₁₂im]Br₂.73 This result is in good agreement with FT-IR results.

The values of RMSF of free lysozyme and the lysozyme-[C₁₂−4-C₁₂im]Br₂ complex was also observed from MD simulation and presented in Figure 10B. The RMSF values enables to analyze the local protein mobility which were plotted against the residue numbers based on the 4000 ps trajectory data. Our result clearly shows that α helices domain and their subdomain (A, C and D) have highest while β strand domain and subdomain B of α helix shows lowest fluctuation, suggesting that the [C₁₂−4-C₁₂im]Br₂ bind to residues Lys 33, Phe 34, Glu 35, Ser 36, Asn 37, Asn 52, Gln 57, Asn 59, Trp 62, Trp 63, Ile 98, Ala 107, Trp 108, and Val 109. Thus, the principal region of surfactant binding sites of lysozyme is located in loop and β strand domain and mainly bind to Trp63, Trp62, and Trp108, which is in good agreement with docking results.