A Spectroscopic and Molecular Simulation Approach toward the

Article pubs.acs.org/JPCB

A Spectroscopic and Molecular Simulation Approach toward the Binding Affinity between Lysozyme and Phenazinium Dyes: An Effect on Protein Conformation Sabera Millan,† Lakkoji Satish,† Krishnendu Bera,‡ B. Susrisweta,† Durg Vijay Singh,‡ and Harekrushna Sahoo*,† †

Department of Chemistry, National Institute of Technology (NIT), Rourkela, Odisha, India Department of Bioinformatics, Central University of South Bihar, Patna, India

‡

S Supporting Information *

ABSTRACT: A comparative study of binding interaction between Safranin O (SO) and Neutral Red (NR) with lysozyme (Lyz) has been reported using several spectroscopic methods along with computational approaches. Steady-state fluorescence measurements revealed static quenching as the major quenching mechanism in Lyz−SO and Lyz−NR interaction, which is further supported by time-resolved fluorescence and UV−vis measurements. Additionally, binding and thermodynamic parameters of these interactions are calculated from temperature dependent fluorescence data. Moreover, conformational changes of protein upon binding with SO and NR are provided by synchronous and circular dichroism (CD) measurements. Molecular docking study provided the exact binding location of SO and NR in lysozyme. Along with this study, molecular dynamics simulation is carried out to measure the stability of Lyz, Lyz−SO, and Lyz−NR complex. The present study revealed the strong binding affinity of dyes with lysozyme, and this study would be helpful toward medical and environmental science.

1. INTRODUCTION The binding studies of proteins to a number of small molecules (including natural original molecules and synthetic organic molecules) have been extensively investigated in the field of chemical biology and medical science for decades.1 Furthermore, such binding can efficiently regulate biological properties of protein by alter its conformation and also significantly affects distribution, metabolism, and excretion properties of small molecules at the molecular level.2−4 Consequently, the detailed characterization of interaction between small molecules and proteins will be helpful to understand its fundamental aspects as well as to explore the nature of their activity in the body. Small synthetic dye molecules have shown both positive and negative impact on human as well as entire ecosystems. Previous studies reported that these dyes not only behave as hazardous pollutants because of their toxic, carcinogenic, and mutagenic properties but also performed a wide range of biological applications like vital staining.3,5,6 Safranin O (SO) and Neutral Red (NR) are the phenazinium dyes, are selected from various types of synthetic dyes from structural point of view (a planar phenazine ring is found to be common in both dyes). Safranin O (3,7-diamino-2,8-dimethyl5-phenylphenazinium chloride) (as shown in Figure 1A) is a cationic phenazinium dye, which is widely used as dying agent in textile, paper, cosmetic, and pharmaceutical field and also © 2017 American Chemical Society

shows a large number biological and photophysical applications.7,8 Similarly, Neutral Red (3-amino-7-dimethylamino-2methylphenazine) (Figure 1B) is a kind of neutral phenazinium dye and extensively used as a textile dye, an intracellular pH indicator, fluorescent probe, and marker for detection of biological systems.9,10 Furthermore, understanding the interaction of SO and NR with biomacromolecules may be helpful to dig out their toxicological and therapeutic impact on living systems. Recently, the binding of phenazinium dyes to nucleic acids and serum albumins has been studied in terms of spectroscopic, thermodynamic, or electrochemical methods.3,7,8,10 But as per our knowledge, the interaction between lysozyme (Lyz) with SO and NR has not been investigated. Therefore, the present study is designed to gain deeper insights into the interaction of Lyz with SO and NR. Lysozyme (Lyz, also known as muramidase) (crystal structure is represented in Figure 1C) is a small globular protein with 129 amino acids, which contains six tryptophan residues, three tyrosine residues, and four disulfide bonds in its monomeric unit.11 Out of these six tryptophan residues in lysozyme (28, 62, 63, 108, 111, and 128), Trp 62, 63, and 108 Received: November 1, 2016 Revised: January 20, 2017 Published: February 1, 2017 1475

DOI: 10.1021/acs.jpcb.6b10991 J. Phys. Chem. B 2017, 121, 1475−1484

Article

The Journal of Physical Chemistry B

Figure 1. Chemical structure of (A) Safranin O (SO) and (B) Neutral Red (NR). (C) Crystal structure of lysozyme (Lyz) showing the position of tryptophan in red (PDB ID: 2CDS).

2. MATERIALS AND METHODS 2.1. Materials. Chicken egg white lysozyme (product code: RM074), NR (product code: GRM122), and SO (product code: GRM129) are purchased from Himedia, India, and are used without further purification. The concentration of lysozyme is measured spectrophotometrically using ε280 = 36 500 L mol−1 cm−1.19 All samples are prepared in 0.1 M sodium phosphate buffer (pH 7.4) throughout the experiment. 2.2. Methods. All the experiments are done in triplicates, and further, the averages of the experimental results are considered for analyses. 2.2.1. Fluorescence Measurements. All fluorescence measurements (including emission spectra and synchronous experiment) are carried out with a Fluoromax-4 spectrofluorometer (Horiba Scientific, Japan) with a quartz cell having path lengths of 1.0 cm. For emission spectra, the excitation wavelength is fixed at 295 nm (selectively excites tryptophan residues), and both excitation and emission slit width are maintained at 5 nm. The fluorescence intensities are corrected for absorption of the exciting light and/or reabsorption of the emitted light by the molecules present in the solution to eliminate the inner filter effect using the relationship20

are found at the substrate binding sites. Trp 28, 108, 111, and 123 are present in the α-domain, and Trp 62 and 63 are located at the active site hinge region between the α and β-domains. From steady state fluorescence studies, it has been observed that Trp 62 and 108 are mostly responsible for lysozyme emission.12,13 Lysozyme is widely found in saliva, milk, tears, skin, blood, and mucus, etc.14 Because of its pharmaceutical and physiological functions, for example antibacterial, antiviral, antitumor, and anti-inflammatory activities, it is extensively used in food preservation and pharmaceutical fields. Lysozyme is selected as a model protein because of its natural abundance, small size, higher thermostability, and their ability to carry drugs.3,15 In recent years, the interactions of some of the synthetic dye molecules like rhodamine B, thionine, bromophenol blue, and phenosafranine with Lyz have been reported by some research groups, and they explored the binding mechanism by spectroscopic and docking methods.3,16−18 In this paper, we reported the binding affinities of lysozyme with SO and NR using both spectroscopic and computational methods. The quenching mechanism, the specific binding site, the mode of interaction force, and the impact of SO and NR on the conformation of lysozyme are analyzed by using different spectroscopic methods (including absorption, fluorescence, and circular dichroism (CD) spectroscopy). Computational approaches like docking and simulation studies are used to find out probable binding site and binding forces. The overall study will be helpful in understanding the binding properties of dyes and their toxicological action on proteins in a detailed manner.

Icor = Iobse(Aex + Aem)/2

(1)

where Icor and Iobs are the corrected and observed fluorescence intensities, respectively, and Aex and Aem are the absorption values of SO and NR at the excitation and emission wavelengths, respectively. The corrected fluorescence intensities are used in the present study. Synchronous fluorescence is recorded on same fluorometer, and the Δλ values are set to 15 nm (for tyrosine residue) and 60 nm (for tryptophan residue). 1476


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Figure 2. Emission spectra of lysozyme (Lyz) in the absence and presence of SO (A) and NR (B). The concentration of Lyz is fixed at 12 μM while the SO and NR concentration corresponding to 0, 4, 8, 12, 16, 20, 24, and 28 μM (from a to h); T = 298 K; pH = 7.4.

A circular dichroism spectrometer (J-1500, JASCO, Japan) in fluorescence mode is used to record temperature-dependent fluorescence spectra at 25, 29, 33, and 37 °C in the range of 305−450 nm upon excitation at 295 nm. Lysozyme concentration is kept at 12 μM, and the concentration of SO and NR is varied from 4 to 28 μM with an increment of 4 μM for all required measurements. For fluorescence lifetime (or time-resolved) measurement of lysozyme−SO and NR system, the protein sample (12 μM) is excited with a LED source (λex = 290 ± 5 nm and λem = 344 nm). The experiments are carried out by using a Horiba Delta Flex Lifetime instrument. The measured data are fitted to biexponential decay with good χ2 values. The average lifetime (τ) is calculated using the equation21

α‐helix (%) =

∑ αiτi i=1

(2)

where αi is the pre-exponential factor corresponding to the ith decay time constant τi. 2.2.2. Absorption Measurements. The absorption spectra are performed on a double beam Cary 100 UV−vis spectrophotometer (Agilent Technologies, USA) at room temperature, and 1.0 cm quartz cells are used for the measurement. 2.2.3. Circular Dichroism (CD) Spectroscopy. The far-UV (secondary structure) and near-UV (tertiary structure) CD measurements are carried out with J-1500 CD spectrometer (JASCO, Japan) using a 0.1 cm cuvette with three scans averaged for each CD spectrum. The far-UV CD measurements of lysozyme in the absence and presence of SO and NR are taken in the range of 200−250 nm. The CD results are expressed as mean residue ellipticity (MRE) in deg cm2 dmol−1, which is defined as22 MRE =

θobs 10nlCP

(4)

where 4000 is the MRE value of the β-form and random coil conformation cross at 208 nm and 33000 is the MRE value of a pure α-helix at 208 nm. Similarly, the near-UV CD spectra are recorded in the range of 260−300 nm with same instrumental parameters setup. The lysozyme concentration is fixed at 12 and 80 μM for the far and near-UV CD measurements, respectively. 2.2.4. Molecular Docking Studies. The AutoDock4.223 program is used to perform the molecular docking study of the lysozyme and Safranin O (SO), Neutral Red (NR) system. The crystal structure of lysozyme (PDB ID: 2CDS) is taken from the Protein Data Bank, and the structures of SO (PubChem ID: 2723800) and NR (PubChem ID: 11105) are used. The identification of binding site is performed using CASTp, MetaPocket 2.0. The top 20 ranked poses with maximum number of interactions and consistency in the binding pattern are considered. The putative binding site is confirmed by combining the results from these tools. The molecules are prepared for molecular docking by merging nonpolar hydrogens, assigning Gastegier charges. Grid is calculated using 0.375 Å spacing in a cubic box which size is set to 48 Å × 48 Å × 48 Å (x, y, and z). The grid center along the x-, y-, and z-axis is set to −4.328, 56.956, and 3.6 Å. To search the best conformational space of the ligand, the 100 genetic algorithm experiments are performed with a population size 300, 2.5 billion evolutions, and 270000 generations, while other parameters kept as default. Ten independent docking runs are performed, and complexes, Lyz−SO and Lyz−NR, with lowest binding free energy (ΔG) from the largest cluster are considered. The docking results are visualized using PyMOL and Discovery studio visualizer software. 2.2.5. Molecular Dynamics (MD) Simulation Studies. The MD simulations for Lyz-SO and Lyz-NR complex and only lysozyme systems are done using GROMACS 5.0.424 in the periodic boundary conditions with GROMOS96 53a625 force fields. The topology files, force field parameters, and charges for the SO and NR are generated by the online program PRODRG.26 The Lyz−apo (without dye), Lyz−NR, and Lyz−SO systems are solvated with 5882, 5773, and 5820 explicit simple point charge (SPC)27 waters molecules, respectively, and all the systems are embedded in the simulation

n

⟨τ ⟩ =

−MRE208 − 4000 × 100 33000 − 4000

(3)

where θobs is the CD in millidegrees, n is the number of amino acid residues (129), l is the path length of the cell, and CP is the molar concentration of lysozyme. Then the α-helical content of lysozyme is calculated on the basis of molar ellipticity value at 208 nm using the equation22 1477


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F0 = 1 + KSV[Q] = 1 + kqτ0[Q] (5) F where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. KSV is the Stern− Volmer quenching constant, τ0 is the fluorescence lifetime of biomolecule (here value of τ0 for lysozyme is equal to 1.87 ns) without any quencher, kq is the bimolecular quenching rate constant (usually equal to KSV/τ0), and [Q] is the quencher concentration.35,36 The calculated KSV and kq values for Lyz− SO and NR complex at different temperatures are presented in Table 1 (representative Stern−Volmer plots are shown in

boxes where the protein is at center and at least 1.0 nm from the edge. In order to neutralize the systems, eight chloride ions are added to both the apo and Lyz−NR complex systems, and nine chloride ions are added to Lyz−SO complex. The longrange electrostatics were calculated using particle mesh Ewald (PME) method28 with a real space cutoff of 1 nm. The van der Waals interaction cutoff was 1 nm taken for all the three systems. The systems are energy minimized using 50 000 steps of steepest descent method with restraining the heavy atoms of the protein by 100 kJ mol−1 Å followed by 50 000 steps of steepest descent method without position restrained. Further the systems are equilibrated with temperature up to 300 K using the Berendsen thermostat29 in NVT with coupling constant of 0.1 ps and 1 bar pressure using Parrinello−Rahman barostat30 in NPT with an isotropic coupling constant of 0.1 ps for 1 ns, respectively. The heavy atoms including hydrogens are constrained by LINCS algorithm.31 Finally, 100 ns MD simulations are performed for each of the three systems with 2 fs time step.

Table 1. Stern−Volmer Quenching Constants (KSV) and Bimolecular Quenching Rate Constants (kq) for Lysozyme− SO and NR Systems at Different Temperatures

3. RESULTS AND DISCUSSION 3.1. Fluorescence Measurement. Fluorescence measurements have been widely used to study the interaction between the ligand molecule and protein, which provides the information about the structural changes and the microenvironment in the vicinity of the fluorophore molecules along with the binding properties through the analysis of the emission peak, the lifetime, the transfer efficiency of energy, and fluorescence polarization, etc.32 3.1.1. Fluorescence Quenching of Lysozyme by SO and NR. In general, fluorescence quenching is a process that decreases the fluorescence intensity of a fluorophore by a variety of molecular interactions including excited-state reaction, molecular rearrangement, energy transfer, groundstate complex formation, and collisional quenching.33 Thus, this study reveals the mechanism of protein−ligand binding and gives an idea about nature of the binding phenomenon.18 As shown in the Figure 2, the addition of SO and NR causes a progressive quenching of the intrinsic tryptophanyl fluorescence of lysozyme without any significant shift in the maximum of emission wavelength.16,34 Hence, this result suggests that both SO and NR are interacting with Lyz, and this interaction also caused changes in the microenvironment around tryptophan residues within the protein. The quenching mechanisms are mainly of two types including static quenching and dynamic quenching. The static quenching arises due to the formation of nonfluorescent fluorophore−quencher compound whereas dynamic quenching initiates from collision between fluorophore and quencher at excited state. Both quenching process can be differentiated by their dependence on temperature, viscosity, and excited state lifetime. On the basis of temperature-dependent measurements, it is expected that the bimolecular quenching constants are increasing with increasing in temperature and higher temperature results in faster diffusion and larger amounts of dynamic quenching. The reversed effect is observed for the static quenching where the quenching constants are decreased with increasing temperature. Therefore, temperature-dependent fluorescence measurements are carried out at different temperatures to differentiate static and dynamic quenching. The observed results are analyzed in terms of the following Stern−Volmer equation:33

phenazinium dyes

temp (K)

SO

298 302 306 310 298 302 306 310

NR

a

KSV (104 M−1)

kq (1013 M−1 s−1)

Ra

± ± ± ± ± ± ± ±

1.89 1.74 1.62 1.53 1.65 1.33 1.18 0.94

0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99

3.54 3.25 3.03 2.86 3.08 2.49 2.20 1.76

0.10 0.09 0.09 0.10 0.07 0.07 0.08 0.07

R is the correlation coefficient.

Figure S1). From Table 1, it is observed that calculated KSV and kq values are decreased with increase in temperature, and the values of bimolecular quenching rate constants are much larger than 2.0 × 1010 M−1 s−1. This suggests that major quenching mechanism observed in the lysozyme−SO and NR interaction is probably happened by static quenching rather than dynamic quenching.37,38 Therefore, it is revealed that the ground state complex between lysozyme with SO and NR plays an important role in quenching of protein by SO and NR. To reveal the conclusive confirmation supporting the static quenching mechanism, absorption as well as lifetime fluorescence measurements are carried out. Absorption spectroscopy is widely used to investigate the structural changes in proteins as well as to detect the complex formation in protein− ligand interaction. From our observation, the absorption profile of the protein shows two absorption bands (Figure S2): one is between 200 and 230 nm due to the π−π* transition of polypeptide backbone structure CO of protein molecule, and other band between 260 and 300 nm provides information about the aromatic amino acid residues (tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe)).38,39 Figure S2 shows the variation of absorption spectrum of lysozyme in the presence of SO and NR at pH 7.4. The observed variation in absorption spectra confirmed the involvement of static quenching instead of dynamic quenching as the major contributor as dynamic quenching has no effect on absorption profile, because dynamic quenching only affects the excited state of quenching substances. 4,39 In other words, the absorbance of lysozyme at ∼280 nm increased after the addition of SO and NR, which indicates a specific type of binding is involved in the protein−ligand adduct. Time-resolved (lifetime) fluorescence measurement serves as another conclusive method in differentiating static and dynamic quenching processes because its sensitivity toward excited-state interactions.40,41 Therefore, this study is carried out to 1478


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Table 2. Binding Parameters and Thermodynamic Parameters for the Binding of Lysozyme with SO and NR at Different Temperatures phenazinium dye

temp (K)

SO

298 302 306 310 298 302 306 310

NR

a

K (104 M−1)

n

Ra

ΔG (kJ mol−1)

ΔH (kJ mol−1)

± ± ± ± ± ± ± ±

0.919 0.921 0.922 0.916 0.929 0.938 0.914 0.911

0.997 0.996 0.998 0.998 0.996 0.997 0.997 0.996

−23.8 −23.9 −24.1 −24.2 −23.8 −23.4 −23.1 −22.7

−15.5

27.9

−49.8

−87.3

1.49 1.39 1.32 1.16 1.41 1.28 0.87 0.67

0.16 0.01 0.08 0.06 0.03 0.10 0.10 0.48

ΔS (J mol−1 K−1)

R is the correlation coefficient.

Figure 3. van’t Hoff plots for lysozyme−SO (A) and lysozyme−NR (B) system at different temperatures.

understand the quenching mechanism of protein fluorescence by SO and NR. From the time-resolved fluorescence decay curve of Lyz in the absence and presence of SO and NR (as represented in Figure S3), the fluorescence lifetime values and their amplitudes are calculated and summarized in Table S2. These results reveals that the fluorescence lifetime of protein does not vary significantly in the presence of increasing concentration of SO and NR. In principle, the fluorescence lifetime values remain unperturbed in the case of static quenching mechanism while in dynamic quenching these values change significantly. So, the constancy of the fluorescence lifetime of Lyz in the presence of SO and NR clearly confirmed that the fluorescence quenching is static in nature.16,38 3.1.2. Determination of Binding Parameters. When small molecules bind independently to a set of equivalent sites on a protein molecule, binding parameters including binding constant (K) and number of binding sites (n) can be evaluated by using following equation:3,17,28,42 F −F log 0 = log K + n log[Q] F

3.1.3. Evaluation of Thermodynamic Parameters and Mode of Interaction. The interactions between small binding molecules and biomolecules are mainly regulated by four types of major binding forces including van der Waals interaction, electrostatic forces, hydrogen bonding, and hydrophobic interactions.18 The magnitudes and signs (positive and negative) of thermodynamic parameters such as enthalpy change (ΔH), entropy change (ΔS), and free energy change (ΔG), etc., of interaction play the major role to find out principal binding forces in protein−ligand interaction. It is assumed that enthalpy change (ΔH) does not show significant variation within the temperature range studied; both the enthalpy change (ΔH) and entropy change (ΔS) for the protein−SO and NR binding process can be elucidated by using the van’t Hoff equation. ln K =

−ΔH ΔS + RT R

ΔG = ΔH − T ΔS

(7) (8)

where R is the gas constant and T is the temperature in K. ΔH and ΔS can be evaluated from the slope and intercept of the linear plot of ln K vs 1/T as shown in the Figure 3. Equation 8 is used to find out free energy change (ΔG). The calculated thermodynamic parameters for the interaction of SO and NR with lysozyme are given in Table 2. The negative value of free energy change (ΔG) indicates that the binding of SO and NR with lysozyme is exothermic and spontaneous. In the case of the lysozyme−SO system, a negative enthalpy change suggests the contribution of van der

(6)

The representative plot of log[(F0 − F)/F] vs log[Q] (as shown in Figure S4) resulted a straight line, and the corresponding calculated results are summarized in Table 2. The obtained values of K decrease with the increasing of temperature, which indicates that Lyz−SO and Lyz−NR complex partially dissociates with temperature rising. There is one binding site for SO and NR available on the lysozyme molecule because the values of n approximately equal unity. 1479


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Figure 4. Far-UV CD spectra of lysozyme in the absence and presence of SO (A) and NR (B). [Lyz] = 12 μM, [SO] and [NR] = 0, 15, and 30 μM (from a to c), respectively.

Figure 5. Synchronous fluorescence spectra of lysozyme in the absence and presence of SO (A) and NR (B) at Δλ = 60 nm. [Lyz] = 12 μM; [SO], [NR] = 0, 4, 8, 12, 16, 20, 24, and 28 μM (from a to h); pH = 7.4.

are similar in shape, which indicates that the structure of lysozyme after dye binding to protein is predominantly αhelical. To know further the effect of dyes on the α-helicity content of protein, eqs 3 and 4 are used to calculate the percentage of α-helicity. Figure 4A shows that the CD signal of lysozyme is increased with increasing the concentration of SO, and the secondary structure of protein is stabilized after the binding with SO. The α-helical content is increased from 31.92% (in free lysozyme) to 43.23% when SO (having concentration is 30 μM) is added into the lysozyme solution, which reveals that the binding of SO to lysozyme greatly affects the conformation of protein.46 In the Lyz−NR system (Figure 4B), the band intensity of curves B−D decreased regularly with increasing addition of NR to protein that indicates the conformational changes of lysozyme. The α-helix content, 31.92% (in free lysozyme), is reduced to 25.02% when the 30 μM NR is added to the lysozyme solution. It suggests that the binding of NR to lysozyme prompted destabilization and partial unfolding of protein.38 The near-UV CD spectra of lysozyme in the absence and presence of SO and NR are recorded to explore the tertiary structure of protein. It is observed from Figure S5 that the nearUV CD spectra (250−300 nm) of lysozyme exhibit two minima at 261 and 268 nm (which are characteristic of disulfide bonds)

Waals force and hydrogen bonding as the major binding force whereas positive entropy change indicates the involvement of electrostatic interactions as well as hydrophobic interactions. But the negative values of ΔH and ΔS in the case of the lysozyme−NR system indicate that van der Waals forces and hydrogen bonding are involved in the binding between Lysozyme and NR. Therefore, it is clear from the obtained thermodynamic parameters (ΔH and ΔS) that all the four above-discussed binding forces are significantly involved in lysozyme−SO interactions while only van der Waals force and hydrogen bonding play as major binding forces in the case of the lysozyme−NR interaction.43−45 3.2. Conformational Investigation. 3.2.1. Circular Dichroism (CD) Spectroscopy. Circular dichroism (CD) spectroscopy is employed as a powerful technique to investigate the secondary and tertiary structure of protein. The far-UV CD spectrum of α-helical structure in protein exhibits two negative peaks: ∼208 nm (due to π−π* transition) and ∼222 nm (due to n−π* transition).38,46 The far-UV CD measurements of lysozyme in the presence of increasing concentration of SO and NR is carried out to reveal the impact of binding interaction of the protein with SO and NR on the secondary structure of the lysozyme (as shown in Figure 4). It is observed that the CD spectra of lysozyme in the presence and absence of SO and NR 1480


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Figure 6. Surface diagram of lysozyme (sphere representation) with SO (A) and NR (B) (stick representation). The 2D detailed view shows the interaction between SO (C) and NR (D) with neighboring residues of Lyz. The different color circles represent the residues participating in various types of interaction.

the tryptophan residues are responsible for intrinsic fluorescence of lysozyme. It is observed that there is no significant change in maximum emission wavelength at Δλ = 15 nm, which suggests that the tyrosine environment of protein is not affected in the presence of SO and NR (results are not shown). From Figure 5, it is explored that the maximum emission wavelength has a red-shift at Δλ = 60 nm. The red-shift indicates the polarity around tryptophan residues is increased, which means the tryptophan residues are exposed to polar environment in the presence of different concentration of SO and NR respectively.15,50 Thus, synchronous result confirmed the conformational occurred in lysozyme after the binding with these dyes, which is shown consistent with CD results. 3.3. Computational Investigation. 3.3.1. Molecular Docking Studies. Molecular docking study is carried out to determine the binding energy of protein−ligand complex and also identify a putative binding site for ligands on protein.16,39 As shown in Figure 6, the molecular docking studies for Safranin O and Neutral Red on the lysozyme protein provide evidence for its binding sites on the protein. Docking results revealed that Asp 52, Ile 58, Trp 63, Ile 98, Ala 107, and Trp 108 residues of Lyz are involved in the interaction with SO, whereas NR binds with Asn 46, Asp 52, Ile 98, Ala 107, Trp 108, and Val 109 amino acid residues. Additionally, these binding residues suggest that the tryptophan residues play a prominent role in the interaction with SO and NR.

as well as shoulders at 283, 289, and 295 nm (for aromatic chromophores (Trp, Tyr, and Phe) and the asymmetric environment) respectively.46 It is clear from Figure S5 that the addition of SO and NR does not cause significant change in the positions of the peaks in the near-UV regions. Thus, the obtained results suggest that the tertiary structure, especially the asymmetric environment of the aromatic amino acid residues and disulfide bonds, is not undergoing any drastic changes upon the addition of SO and NR. 3.2.2. Synchronous Fluorescence Spectroscopy (SFS). Synchronous fluorescence spectroscopy (SFS) is a simple and efficient fluorescence technique, which is employed to investigate fluorescence quenching and conformational change of protein. It involves simultaneous scanning of the excitation and the emission monochromators while keeping a constant wavelength interval between them. When the wavelength interval (Δλ) is stabilized at Δλ= 15 nm and Δλ = 60 nm, synchronous fluorescence offers characteristics of tyrosine (Tyr) and tryptophan (Trp) residues of the protein molecules.47−49 Synchronous fluorescence intensity of lysozyme decreases with increasing concentration of SO and NR with respect to both tyrosine and tryptophan environment, which confirms the occurrence of fluorescence quenching in the binding process. Fluorescence intensity of lysozyme for tryptophan environment is more than that for tyrosine environment, which suggests that 1481


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Figure 7. (A) Backbone RMSD plot, (B) RMSF plot, and (C) radius of gyration plot for apo, Lyz−NR, and Lyz−SO bound states.

Figure 8. Conformational changes of active sites in Lyz associated with SO and NR. Observed conformational change of binding pattern of Lyz on apo form of Lyz (A), and on SO (B) and NR (C) over the course of 100 ns simulations. Time 0 ns is representing the starting of production phase while 100 ns represents the ending.

stability of the docked complexes is compared with that of the apo form of Lyz to find the differences in structural stability by the root-mean-square deviation (RMSD) plot. The RMSD plot of each snapshot with respect to the initial structure over the course of MD simulation has shown in Figure 7A. A jump in RMSD is shown within the 0.5 ns, which occurs as a consequence of relaxation of the initial structures. One can observe the RMSD pattern is not all most similar; hence, the

Furthermore, the obtained results are in consistent with the synchronous fluorescence findings where the conformational changes happened around the tryptophan environment. The binding free energy of Safranin O and Neutral Red to lysozyme protein is found to be −5.87 and −5.89 kcal/mol. 3.3.2. Molecular Dynamics Simulations Studies. The stability and conformational changes of static protein and docked complexes are evaluated by MD simulations.4,50 The 1482


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static interactions) whereas van der Waals force and hydrogen bonding play as major binding forces between Lyz−NR interactions. The synchronous and far-UV CD studies reveal the binding of SO and NR induced the conformational changes in the protein structure. Along with these experimental methods, computational investigation (including docking and simulation studies) provided deep insights regarding the binding sites, binding energy, and structural stability of the Lyz−SO and Lyz−NR complex, respectively. On the basis of both experimental and computational measurements, it is clearly observed that tryptophan is one of the residues causing the interaction of protein with ligand. Moreover, moderate structural variations in Lyz are observed after binding with dyes.

binding of stains affects much the stability. The plots also revealed that the RMSD values in all the three systems remained within 4 Å. Up to 44 ns all most similar pattern of RMSD found for all the three systems, suggesting the systems having similar kind of stability, but later we can observe higher RMSD for the two ligand binded complexes, the ligands binding triggers the maximum possible Lyz structural changes. The RMSD of apo form is comparatively lower; hence, it is more stable. We found Lyz−SO complex having higher RMSD, which indicates higher structural perturbation after its binding. The fluctuation of each residue in each system is also analyzed by root-mean-square fluctuation (RMSF). The RMSF values of apo form of Lyz is considered as baseline to other two studied ligands (SO and NR) binding with Lyz. The higher RMSF values being observed within residues 65−77 in the each complex (Figure 7B). The levels of compactness of the proteins are monitored by radius of gyration (Rg), which is shown in Figure 7C. The Rg values for all three systems lie within 1.37−1.47 nm. Almost a similar pattern of Rg values is found for the unbound Lyz and Lyz−NR complex, and little higher Rg values are found for the Lyz−SO complex. This indicates that the Lyz structure for both the unbound and bound states remain largely the same without any significant conformational changes along the entire trajectory. To investigate the overall structural and binding sites for each of the three systems (Lyz−apo, Lyz−SO, and Lyz−NR), we have visualized the snapshots at 0, 50, and 100 ns simulation. The Lyz snapshots from the molecular dynamics trajectories (shown in Figure 8) are captured to observe the conformational changes in their binding sites. The snapshots clearly indicate that the α-helix (residue 102−106) is lost in the case of Lyz− SO and Lyz−NR systems. But at the same time, a new secondary structure (α-helix) formed between residues 61−63 in the Lyz−SO system, which suggests that the lysozyme structure is comparatively more stabilized by SO as compared to NR. These results are in accordance with the CD results where higher α-helix content observed in the case of Lyz−SO and loss of structure in the case of the Lyz−NR system. These snapshots also indicate that the dyes are not diffused away from its binding sites through the course of simulation (Figure 8). The binding free energy of the protein−ligand complex is investigated by linear interaction energy (LIE) calculations and represented in Figure S6. The binding free energy of Lyz−SO and Lyz−NR complexes are found to be −23.905 and −11.95 kcal/mol, respectively, over the course of simulations. The lower binding energy and all other analyses show that SO is strongly interacting with Lyz and thus stabilizing protein compared to that of NR.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b10991. Figures S1−S6 and Tables S1 and S2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(H.S.) E-mail [email protected]; Ph +91 6612462665. ORCID

Harekrushna Sahoo: 0000-0003-2655-1196 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.M. acknowledges DST-INSPIRE Fellowship (IF130984) for financial assistance. The authors acknowledge Dr. Hirak Chakraborty, School of Chemistry, Sambalpur University, Burla, for his assistance in fluorescence lifetime measurements and Dr. Madhurima Jana for her valuable suggestions. The authors also thank NIT Rourkela, India, for providing the facility to carry out the experiments.

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REFERENCES

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4. CONCLUSIONS In this paper, the binding affinity between SO and NR with Lyz and its effect on protein conformation has been investigated in term of spectroscopic methods (including fluorescence, UV− vis, and CD) as well as molecular docking and simulation studies. Fluorescence (steady-state and time-resolved mode) analysis along with absorption measurement showed that static quenching behaves as major quenching process over dynamic quenching. The negative value of free energy change (ΔG) elucidate that the interaction process is spontaneous. Thermodynamic parameter measurements reveal the binding of SO to Lyz is driven mainly by all four forces (including van der Waals force, hydrogen bonding, hydrophobic and electro1483


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