Insights into the binding mechanism of a model protein with fomesafen: Spectroscopic studies, thermodynamics and molecular modeling exploration

Journal of Molecular Structure 1195 (2019) 892e903

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Journal of Molecular Structure journal homepage: http://www.elsevier.com/locate/molstruc

Insights into the binding mechanism of a model protein with fomesafen: Spectroscopic studies, thermodynamics and molecular modeling exploration Xue Dong, Leng Wang, Ruirui Feng, Zhaoyang Ren, Li Zhang**, Huizhe Lu* Department of Applied Chemistry, College of Science, China Agricultural University, 100193, Beijing, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 April 2019 Received in revised form 29 May 2019 Accepted 29 May 2019 Available online 1 June 2019

The long residual period and toxicity of fomesafen makes it accumulating in the environment as environmental pollutant. Bovine serum albumin (BSA) plays an indispensable role in the binding, transmission, distribution and efficacy of fomesafen in vivo. Thereby, we studied the interaction of fomesafen and BSA by spectroscopic methods and molecular simulation. Spectroscopic measurements reveal that static quenching is the predominant quenching mechanism, resulting in the forming of BSA-fomesafen. The thermodynamic results manifest that hydrophobic force is the major factor. We have found an active pocket around Trp-213 based on competitive binding of fomesafen and warfarin. The model optimized by MD is the ultimately identified model reflecting real situation, from which we can find that the interactions of BSA-fomesafen depend mainly on the contribution of hydrophobic residues. Compared with the models before and after MD, we have found that aqueous solution is an indispensable condition for the emergence of significant hydrophobic interaction in this system. The free energy and energy decomposition measured by the MD simulations indicate that the interaction is spontaneous and Trp-213 has great contributions to the formation of BSA-fomesafen system. In summary, our results have provided meaningful reference to assess the biological toxicity risk of fomesafen. © 2019 Published by Elsevier B.V.

Keywords: BSA Fomesafen Spectroscopic measurements Molecular simulation

1. Introduction Fomesafen (Fig. 1), a diphenyl ether, has been widely applied as a kind of post-emergence herbicide in bean and soybean crops [1]. The anionic character and water-solubility of fomesafen result in a potential risk to groundwater and runoff [2]. In addition, it has been reported that the death of fish in runoff is related to fomesafen [3]. Besides, there has been reported that fomesafen has the reproductive toxicity in freshwater snails (Lymnaea stagnalis) and hepatic uroporphyria in mice [4,5]. In addition, fomesafen has a slow natural decomposition rate in soil with a half-life varying from 45 days to 12 months, meaning that there will be plenty of fomesafen as pollutants accumulating in the environment [6e8]. Consequently, it is easily ingested by people and animals, which indicates one of its potential toxicities. Moreover, it is reported that the binding interactions of pollutants in vivo can initiate most

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (L. Zhang), [email protected] (H. Lu). https://doi.org/10.1016/j.molstruc.2019.05.128 0022-2860/© 2019 Published by Elsevier B.V.

biochemical reactions [9]. Together with the reasons for the high likelihood of the existence of fomesafen in people and animals, it is extremely urgent and primary to ascertain the initial response of fomesafen in vivo. Bovine serum albumin (BSA) is the most widely studied bioactive model protein, which can play a crucial role in combining and transporting exogenous and endogenous compounds [10]. For these characteristics of BSA, interactions between drugs and BSA are often studied to provide information on the absorption of drugs in vivo. BSA is a 66.4 kDa transporter with three homologous domains (I, II, III) and 583 amino acids including 2 tryptophan and 20 tyrosine [11,12]. When the ligand binds to BSA, there are two main regions, sites I and II, which have their own markers: warfarin, phenylbutazone, etc. correspond to site I and ibuprofen, flufenamic acid etc. correspond to site II [13]. There are two tryptophan (Trp) in BSA, Trp-134 and Trp-213, locating in subdomains I B and II A respectively [14]. Because of the functional characteristics of BSA, BSA will certainly play an indispensable role in the combining, transmission, distribution and efficacy of fomesafen in vivo. It is important to explore the interaction of drugs-serum proteins in pharmacology

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(PerkinElmer LS 55) with a thermostat bath. Both the excitation and emission slit widths were set to 2.5 nm. For emission spectra, the excitation wavelength was fixed at 281 nm, the emission wavelength range was from 300 nm to 400 nm. We carried out emission spectra at 298 K, 303 K, 308 K, 313 K. The synchronous fluorescence spectra were measured at △l ¼ 15 nm (for tyrosine (Tyr) residue) and △l ¼ 60 nm (for Trp) at 298 K [25,26]. EEMS of BSA in the presence and absence of fomesafen were recorded over that the excitation wavelength range was from 300 to 400 nm and the emission wavelength range was from 200 to 300 nm.

Fig. 1. Structure of fomesafen.

and pharmacodynamics for which affinities between the two determine the free form of drugs [15]. For those reasons, it is significant for toxicity evaluation of fomesafen to probe the interaction between fomesafen and BSA. A variety of physicochemical methods and theoretical simulation have been used to explore the interaction between drugs and BSA, such as equilibrium dialysis [16], raman spectroscopy [17], spectroscopic techniques (such as UV absorption, fluorescence and circular dichroism (CD) spectroscopies, fluorescence polarization measurements) [18e20], Zeta potentials which can explain the electrostatic effects and ascertain the existence of binding [21,22], Resonance light scattering (RLS) which can be used in nonfluorescent molecules [15,21,23], molecular modeling [15,21], etc. In our work, spectroscopy measurements provided abundant information for the binding mechanism and active site, in which the former was studied by varies of spectral measurements and the latter was determined by synchronous fluorescence spectroscopic and competition experiments [24]. Based on the experimental results, the atomistic modeling embodying molecular interaction was built by molecular docking to study a variety of molecular forces in this system. Beyond that, we explored the influence of water molecules on this interaction by comparing the models before and after MD. MD simulations were carried out to measure the free energy and the energy decomposition of this complex, which can explain some experimental results. In summary, our results have provided meaningful reference to assess the biological toxicity risk of fomesafen. 2. Materials and methods 2.1. Materials Fomesafen (purity is 99.5%) was purchased from Aladdin. Bovine serum albumin (BSA, purity
98%) was purchased from Sigma Chemical Co., USA, and warfarin (purity >98.0%) from AccuStandard Inc. All other chemical substances were of analytical grade. The water used was deionized. All samples were prepared in Tris-HCl buffer (0.1 M Tris, 0.15 M NaCl, pH 7.4) throughout the experiments. 2.2. Methods 2.2.1. Fluorescence spectroscopy All fluorescence measurements including emission spectra, synchronous experiment and excitation-emission matrix spectroscopy (EEMS), were performed on fluorescence spectrophotometer

2.2.2. Circular dichroism (CD) spectroscopy Circular dichroism (CD) spectra of BSA in the presence and absence of fomesafen were recorded over the range of 200e260 nm on a Circular British Applied Photonics Chirascan spectropolarimeter. 2.2.3. The UVeVis absorption spectral measurement The UVeVis absorption spectra were monitored on UVevis spectrophotometer (PerkinElmer Lambda 660 S) at room temperature. Tris-HCl buffer was used for background correction in this section. 2.2.4. In silico analysis It shows in Fig. S7 that the structures of BSA (PDB ID: 4f5s) and HSA complexed with warfarin (PDB ID: 1HA2) were received from the Protein Data Bank (PDB: http://www.pdb.org/pdb/home/home. do). We extracted ligand substructures, removed unwanted substructure such as water from BSA, analyzed selected structure and added hydrogens using sybyl 2.0. Due to the deep homology between BSA and HSA, the structure of HSA was used to help determine the binding site of warfarin in BSA. The structure of fomesafen was optimized by sybyl 2.0. Molecular docking was performed using sybyl 2.0. According to the competitive binding of fomesafen and warfarin, a reasonable pocket around Trp-213 was found. The preprocessed fomesafen conformer was docked to the site in BSA considering ring flexibility. Afterwards, for exploring electrostatic interaction of fomesafen, we obtained the electrostatic potential distributions of ligand and residues within 5 Å of ligand using sybyl 2.0. The prepared BSA-fomesafem from the docking study was subjected to MD using Amber 14 program to obtain the thermodynamic parameters [27]. Nonpolar hydrogen and water in proteins were removed using Antechamber modules in AmberTools15 and fomesafen molecular was optimized at standard 3-21G basis set using Gaussian 16 program [28]. Afterwards, the fomesafen was prepared under GAFF force field and BSA was prepared under AMBER03 force field. The complex was placed in TIP3PBOX water environment with 10 Å and Naþ ions were added into the system to neutralize the system. The steepest descent method and the conjugate gradient method were used to optimize the system. Water molecules were optimized with a force constant of 200 kcal/mol$ Å for 2000 steps of steepest descent and conjugate gradient; water and side chain atoms were optimized with a force constant of 200 kcal/mol$ Å for 5000 steps of steepest descent and conjugate gradient; all the atoms were subjected to unrestricted energy minimization for 10000 steps meaning 5000 steps of steepest descent and conjugate gradient. Then, the simulation entered the stage of heating and equilibration. The system was gradually heated from 0 K to 310 K by 40 ps, then the simulation run for 30 ns at NPT ensemble (T ¼ 310 K and p ¼ 1atm) without limits. The particle mesh Ewald (PME)

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algorithm was utilized to obtain the full electrostatic energy of a periodic box [29], and hydrogen atoms that form of hydrogen bonds were restrained by the SHAKE algorithm [30].

3. Results and discussion 3.1. Type of fluorescence quenching In this work, it is fundamental to figure out whether the interaction between fomesafen and BSA causes fluorescence quenching. In a general sense, fluorescence quenching signifies that the fluorescence intensity of fluorophores is weakened by molecular interactions such as excited-state reaction, energy transfer, groundstate complex formation [31e33]. Fig. S1 depicts the effects of fomesafen on the fluorescence of BSA revealing that a gradual quenching of BSA is caused by the incremental fomesafen concentration. There are several different types of fluorescence quenching, including static quenching, dynamic quenching and energy transfer quenching [31]. UV/vis absorption spectra, a simple and powerful method, can be used to probe into structural changes and the formation of complex. Here, UV/vis absorption spectra were used as a preliminary approach to study the types of fluorescence quenching. The changes in the absorption spectrum of BSA caused by fomesafen are shown in Fig. 2. The information of three amino acid residues viz., Trp, Tyr and phenylalanine (Phe) can be obtained at the absorption peak at ~280 nm [34e37]. The inset shows the UV/ vis of BSA and the difference spectrum have a large difference in ~300 nm, which cannot be explained completely by the experimental error. It suggests that there are new compounds without ultraviolet absorption forming, in other words, the static quenching procedure is the major fluorescence quenching mechanism of BSA with fomesafen. Fluorescence spectroscopy is an extremely useful and accurate way to explore the type of fluorescence quenching. Fluorescence quenching studies were used to discuss the mechanism of the binding of protein with ligand for further demonstrating the

Table 1 SternVolmer Quenching Constants (Ksv) and Bimolecular Quenching Rate Constants (kq) for BSA-fomesafen systems at different temperatures. Herbicide

Temp (K)

Ksv (104 M1)

kq (1012 M1)

R2*

fomesafen

298 303 308 313

5.83 5.84 6.25 6.66

5.83 5.84 6.25 6.66

0.9981 0.9993 0.9947 0.9905

conclusion of UV/vis absorption spectra [38]. The fluorescence quenching data were analyzed by SternVolmer equation [39,40]:

F0 = F ¼ 1 þ Ksv ½Q  ¼ 1 þ kq t0 ½Q  where F0 and F are the fluorescence intensities with and without fomesafen respectively. Ksv is the SternVolmer quenching constant, [Q] is the fomesafen concentration, kq is the bimolecular quenching rate constant and t0 is the fluorescence lifetime of biomolecule (for most biomolecules, the value of t0 for BSA is about equal to 108 s [41]) without quencher. When the interferer absorbs the absorption wavelength or emission wavelength of the protein, the inner filter effect in fluorescence spectroscopy is generated [42]. And the internal filter effect will be obvious at high concentration of the interference [43]. According to the absorption spectra of fomesafen, the absorbance of fomesafen at ~280 and ~340 nm so low that the internal filter effect can be ignored in BSA-fomesafen. Fig S2 shows the plots of F0/F vs [Q]. Moreover, Table 1 summarizes the calculated Ksv and kq values of BSA-fomesafen complex at different temperatures. In static quenching, the values of Ksv can reflect the stability of the complex [44]. The value of Ksv raises with increasing temperature, indicating that the BSA-fomesafen complex is stable over the experimental temperature range (from 298 K to 313 K). This means that the complex is stable at physiological temperature and can be formed at situation in vivo. The values of kq are all much larger than 2.0  1010 M1 s1 (the upper limit value reported for various kinds of quenchers of biopolymers [45]), suggesting that static quenching is the predominant quenching

Fig. 2. UV/vis of BSA-fomesafen at room temperature, c (BSA) ¼ 10 mM, c (fomesafen) ¼ 0, 6, 12, 18, 24 mM (1 / 5). UV/vis of fomesafen, c (fomesafen) ¼ 6, 12, 18, 24 mM (a/d).

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mechanism in the BSA-fomesafen interaction rather than dynamic quenching and the binding is quite strong [44]. Therefore, the quenching happens because of forming the BSA-fomesafen complex [46]. Overall, static quenching is the predominant quenching mechanism in the BSA-fomesafen interaction.

factors to evaluate the interaction forces lie in DH and DS. For determining the major force in the interaction, thermodynamic parameters were calculated. It is assumed that enthalpy change (DH) was not changed at the temperature range studied. The Van't Hoff equation is used to calculate the enthalpy change (DH) and entropy change (DS) for the BSA-fomesafen binding process.

3.2. Determination of binding parameters

ln K ¼  DH=RT þ DS=R

The determination of binding parameters is important for the establishment of the interaction model. The quenching of BSA caused by fomesafen is a static process, thus the following equation was used for calculating the apparent binding constant (Ka) and the number of binding sites (n) [47].

Where R is the gas constant and T is the temperature in K. DH and DS can be evaluated from the slope and intercept of the linear plot of ln K vs 1/T as shown in Fig. 3. DG is counted from the following equation. The calculation results are similar at the experimental temperature range: DH＞0, DS > 0 and DG < 0 (shown in Table S1).

lgððF0  FÞ=FÞ ¼ lgKa þ n lg½Q  The values of Ka and n were estimated by the intercept and slopes of the representative plot of lg [(F0  F)/F] vs lg[Q] (in Fig. S3). Kq reflects the accessibility of the fluorophores to the quencher, whereas Ka reflects the affinity of the protein binding to the drugs [44]. The results in Table 2 show that the Ka increases with temperature increasing, indicating that the binding ability for fomesafen to BSA is strengthened with the temperature increasing. It is because the increase of temperature enhances the fomesafen molecular movement ability, which is beneficial for fomesafen to go deeper inside the active pocket of BSA. n can be considered as the maximum number of binding sites per macromolecule [18,48]. At each temperature, the slope n is approximately equal to one, indicating that there is only one active site on BSA for fomesafen without cooperativity in vivo [18]. Although there is only one active site in BSA-fomesafen, the value of n increases (from 0.96 to ~0.11) with increasing temperature (from 298 K to 313 K), indicating that the fomesafen has the largest occupancy rate of the active site and the best binding efficiency at physiological temperature (between 308 and 313 K). This will be explained using the force models. This result provides a basis for the establishment of interaction model. 3.3. Thermodynamic parameters and binding modes There are four types of major interaction forces to regulate the interactions between the drug and biomolecules, including van der Waals interaction, electrostatic forces, hydrogen bonding and hydrophobic interactions [49]. Thermodynamic method is an important method to determine the binding mode of molecule - protein. In this work, we used this method to study the specific binding mode of BSA-fomesafen. Considering Ka is temperature dependent, it is realistic to estimate the thermodynamic parameters for describing the mode of binding. Here, the change in free energy DG indicates the tendency of the interaction of protein and inhibitor and includes the changes in enthalpy DH (the complex combination of binding events) and entropy DS (randomness of system, including solvent). The main Table 2 The values of Ka and n for BSA-fomesafen Systems at Different Temperatures. Herbicide

Temp (K)

Ka (104 M1)

n

R2*

fomesafen

298 303 308 313

3.85 8.20 19.91 19.91

0.96 1.03 1.11 1.10

0.9986 0.9993 0.9949 0.9949

a R2 is the correlation coefficient.

DG ¼ DH  T DS ¼ RT ln K Ross and Subramanian [50] have spread the thermodynamic contributions and their relationship to confirm the principal interaction force between a small molecule and macromolecules: if DH < 0 and DS > 0, electrostatic/ionic interactions play a leading role; DH < 0 and DS ＜0 correspond to van der Waals interaction, hydrogen bond formation; and DH＞0 and DS > 0 means that hydrophobic forces are the most important power. Therefore, the positive values of DH and DS indicate that hydrophobic interactions are the major force. The negative value of DG suggests that the interaction between fomesafen and BSA is spontaneous. The results show that the change of entropy is the main driving force in this system. A positive enthalpy value indicates an endothermic process, and the situation in vivo with a constant temperature favors the interaction [51]. Moreover, the positive DS is a manifestation of the increase of randomness coming from the release of water molecules from the pocket. 3.4. Changes of protein conformation Excitation-Emission Matrix Spectroscopy (EEMS) has been used in different fields [52e54]. Through a display of the fluorescence, characteristics of the sample both as a function of excitation and emission wavelengths at the same time, the technique used as a common way can give information of the change of protein conformation in the process of the interaction of drug and protein [55]. Fig. 4 displays the EEMS of BSA in the absence and presence of fomesafen. After the addition of fomesafen, the changes in the EEMS indicate the presence of interaction between the protein and the fomesafen. There are two detectable peaks in the EEMS of BSA (Fig. 4a), Peak 1 and Peak 2. After the fluorescence of the phenylalanine (Phe) residue being negligible, it can be thought Peak 1 (lex ~ 280 nm, lem ~ 340 nm) is on account of both the Trp and Tyr residues of BSA. Hence, the fluorescence spectra of protein have the property of intrinsic fluorescence of both Trp and Tyr at lex ~280 nm [52, 56e58]. BSA has another fluorescence peak (peak 2) in EEMS, at (lex ~ 240 nm, lem ~ 340 nm), due to the polypeptide backbone structures. Fig. 4a and b reveals the changes of BSA clearly. The decreases of peak 1 and peak 2 are due to the interaction with fomesafen. The intensity ratio of peak 1 of BSA alone and the BSA-fomesafen system changes to 1:0.62, while the same for peak 2 changes to 1:0.60. Although the two fluorescence peaks of BSA (Fig. 7a) come from different types of residues, there is almost no difference in the degree of perturbation under a certain concentration of fomesafen. It suggests that fomesafen has fluorescence quenching on both the Trp, Tyr residues and the polypeptide backbone structures,

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Fig. 3. Van't Hoff plots for BSA-fomesafen.

indicating there are some differences on protein conformation in the presence and absence of fomesafen. There is no change in the maximum emission wavelength (lex ¼ 240 nm and 280 nm) suggesting fomesafen has no obvious influence on the environment of Tyr and Trp in BSA. As a powerful technology, CD spectroscopy can explore the secondary structure of protein. In order to further probe into the change of protein conformation, CD technology was carried out to study the secondary and tertiary structure of protein. The CD spectrum of BSA (Fig. 8) shows that there are two negative bands in the UV region at 208 and 220 nm representing p/p* and n/p* transitions being characteristics of the a-helical structure of proteins [59]. In order to study the impact of fomesafen on the secondary structure of the BSA, the CD measurements of BSA in the absence and presence of fomesafen were performed. The experimental results are illustrated in Fig. 5, in which the shape of the CD spectrum is similar in the absence and presence of fomesafen. It indicates that the structure of BSA is mainly a-helical after the binding of fomesafen and protein. The comparison between the line of BSA and BSA-fomesafen in Fig. 5 demonstrates fomesafen have no change significantly on the CD signal. There is correlation between the content of the a-helix and signal [60], hence fomesafen have little effect on the content of a-helix, suggesting that the binding of fomesafen to BSA has no effect on the secondary structure of proteins. Combined with the results of EEMS, the interaction of BSA and fomesafen can weaken the fluorescence of polypeptide backbone while cannot destroy the secondary structure of BSA. The parameters of the secondary structure could be affected by the hydrophobic interaction and the hydrogen bonding [18]. Despite the strong hydrophobic force in BSA-fomesafen, the secondary structure of BSA did not change significantly from the results of CD spectra, which is because the fomesafen molecules are too small relative to the hydrophobic cavity to change the secondary structure of BSA. Synchronous fluorescence can monitor the conformational

changes of proteins when proteins bind to ligands [25]. To further investigate the polarity around the fluorophore, we used synchronous fluorescence spectroscopy to study the structural changes in BSA caused by fomesafen. Synchronous fluorescence spectra of BSA-fomesafen systems were studied at two different scanning intervals (Dl ¼ 15 nm and Dl ¼ 60 nm). From Fig. 6, it is easy to get the following information: (1) In the case of Dl ¼ 15 nm or Dl ¼ 60 nm, the fluorescence intensity decreases with the increase of fomesafen. It suggests that fluorescence quenching occurred during the binding process. (2) The fluorescence intensity of BSA for Tyr is weaker than that for Trp indicating that the Trp residues play a major role in the endogenous fluorescence of the BSA. (3) The absence of changes in the maximum emission wavelength at Dl ¼ 15 nm and 60 nm reveals that fomesafen has no obvious influence on the environment of Tyr and Trp in BSA. It is consistent with the conclusion of EEMS. (4) Fig. 6c shows the relative variation (I/I0) of synchronous fluorescence intensity of BSA with varying drug concentration for Dl ¼ 60 nm and Dl ¼ 15 nm (Here I0 designates the intensity in the absence of fomesafen and I denotes the same in the presence of varying concentrations of fomesafen). It has shown from Fig. 6 that for both the cases, the synchronous fluorescence intensity of BSA reduces obviously with the increase of fomesafen concentration. However, the origin for Dl ¼ 60 nm is higher, suggesting that the fomesafen has greater impact on Trp than Tyr when drug combines with BSA.

3.5. Competitive binding of fomesafen and warfarin The binding parameter calculated previously shows that there is only one active site on BSA for fomesafen and we know that hydrophobic interaction is the major force and fomesafen is close to

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Fig. 4. EEMS of BSA (a) and BSA-fomesafen (b) at room temperature. c (BSA) ¼ 10 mM, c (fomesafen) ¼ 9 mM.

tryptophan in BSA. Warfarin is a site-specific marker for the subdomain IIA cavity that is a typical hydrophobic pocket and is close to Trp. Therefore, we conjectured that warfarin and fomesafen have a common binding site. To confirm our conjecture, we performed competitive binding of fomesafen and warfarin. The fluorescence spectra of the mixture of warfarin-BSA and that of the mixture warfarin-BSA-fomesafen are exhibited in Fig. 7. It is obvious that the fluorescence intensity of warfarin in the BSA environment is much stronger than that in buffer. The fluorescence intensity of warfarin in the mixtures of warfarin-BSA-fomesafen is

less than that in the mixtures of warfarin-BSA, and the intensity of warfarin is reduced with the increase of fomesafen. It is because warfarin and fomesafen have a common binding site, so when the concentration of fomesafen increases, some warfarin leaves BSA to enter the buffer. 3.6. In silico analysis 3.6.1. Molecular docking The atomistic modeling is conducive to intuitive understanding

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Fig. 5. CD spectra of BSA, fomesafen and BSA-fomesafen. c (BSA) ¼ 1 mM, c (fomesafen) ¼ 0.6 mM.

of molecular interactions and atomic details. By molecular docking, an interaction model was built to decipher the atomic details of the binding system according to the above experimental results (the conclusion of synchronous fluorescence experiment and competitive binding of fomesafen and warfarin). There are only two Trp in BSA, of which Trp-134 is near the surface of the BSA and Trp-213 close to site I is within BSA [61]. Based on above points, a binding site around Try-213 has been found. Supporting information has the specific description of the processes of finding active pockets. Fig. 8a shows the interaction mode of fomesafen with BSA (totalscoring is 6.491). Fomesafen is surrounded by Arg-435, Lys-187, Thr-190, Tyr-451, Arg-194, Glu-152, His 287, Ala-290, Arg-256, Arg-198, Trp-213, His-217, Tyr-149, Ser-286,Glu-291, Tyr156, Ser191,Asp450, Glu-186 within 5 Å, there Trp accounts for 50% of total Trp and Tyr accounts for 15% of total Tyr in BSA. It is in consensus with the experimental conclusion of synchronous fluorescence experiment that the fomesafen has greater impact on Trp than Tyr. It is shown that the model exhibit light hydrophobic interaction through the fact that only 11% of the hydrophobic residues is around ligand. Fig. 8b shows that there are 6 H-bonds within ligand-protein complex, 4 H-bonds of which exist in ligand and Arg-198, indicating that hydrogen bonding interactions play a positive role and Arg-198 is one of the key residues to help stabilizing the complex in this model. Fig. 8c shows that electrostatic interaction contributes to the interaction of fomesafen and BSA (especially between part a, b, c of ligand and protein). In addition to classical noncovalent interaction, there is a non-classical noncovalent interaction, cation-p interaction, between an electron-rich p system and a positive charged [62,63]. Calculating the distance and graphing to inspect the angle (Fig. S5) proves there is cation-p interaction between the nitrogen atoms of Arg-194 (217) and the aromatic rings in the fomesafen in Fig. 8d (Basic criteria: the distance is less than 6 Å, and the angle is between 60 and 90 [63]; the specific conditions are listed in Table 3). In summary, the docking model shows that the fomesafen has greater impact on Trp than Tyr, which is in consensus with the experimental conclusion. The characteristics of interaction in this

model: strong hydrogen bond, certain extent of electrostatic interaction and cation-p interaction and slight hydrophobic forces, is contrary to the experimental results. Therefore, we speculate on the reasons for this situation that the effect of aqueous solution is neglected, justified in the following molecular dynamics simulations.

3.6.2. Molecular dynamics simulations The value of RMSD [64] describes that the difference between initial structures and current structures of complex and ligand at each time point. The stability of ligand and complex can be expressed by the trend of RMSD vs time. Fig. 9a shows that the RMSD vs time function is balanced in the last time of the 25 ns, declaring the structure of the complex and ligand have stabilized in a simulated environment. In order to explore the flexibility of different regions, the RMSF [65] of the last 4 ns have been analyzed. The flexibility of amino acid residues can be determined by the magnitude of disturbance in Fig. 9b. We think the RMSF of residues around ligand are low, caused by that the fomesafen containing a rigid benzene ring can hinder the movement of residues around it. The model processed by MD was built to explore the influence of water on this interaction and to prove the hypothesis presented above. In the model after MD, the position of the fomesafen in the pocket is deeper inside the pocket. In this model, the fomesafen is surrounded by Glu-290, Ser-285, Ile-288, Ala-289, Arg-255, Ala259, Leu-258, Ile-262, Arg-220, Phe-221, Val-239, Leu-236, Leu232, Leu-217, HIe-240, Arg-216, Ala-213, Lys-197, Trp-212, Leu201, Phe-209, Lys-210, Ser-200, Leu-196, Leu-479 within 5 Å, where there are 64% of hydrophobic residues of the 25 residues (the residual number listed here is the number after MD). It indicates that the model exhibits a prominent hydrophobic interaction. Moreover, there is one Trp and no Tyr within 5 Å of fomesafen, consistent with the conclusion of synchronous fluorescence. No cation satisfying conditions around ligand is found in Fig. 10, so there is no cation-p interaction in the model. There is also no hydrogen bond in this model between fomesafen and BSA. However, p-p stacking has been found between ring1and ring 2, ring 3 and electrostatic

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experimental conclusion. The characteristics of interaction in this model: prominent hydrophobic interaction, certain extent of electrostatic interaction and p-p interaction, no cation-p interaction and hydrogen bond, are consistent with experiments. Therefore, this is the ultimately identified model reflecting the real situation, from which we can find that the interactions between fomesafen and BSA depend mainly on the contribution of hydrophobic residues. Compared with the models before and after MD, aqueous solution is an indispensable condition for the emergence of significant hydrophobic interaction in this system. The model confirms our conjecture that water can promote fomesafen penetration into BSA and surround fomesafen with hydrophobic residues. In the force model without water molecules, fomesafen tend to bind to BSA with hydrogen bonding, and the binding site is closer to the surface. In the force model with water molecule, fomesafen and BSA mainly bind with hydrophobic interaction without hydrogen bonding, and the binding site is inside the BSA. Therefore, the binding of fomesafen to BSA can be divided into two steps: fomesafen and BSA bind in the first mode; but the first mode is unstable in the water environment, so the water molecules force fomesafen into the interior of BSA to bind in the second mode. This explains the experimental phenomenon that Ksv rises with increasing temperature. Because the increasing temperature can help fomesafen to get rid of hydrogen bonds and enter the internal cavity to bind with BSA. The binding free energy of BSA-fomesafen complex was calculated separately using MM-GBSA [65] and MM-PBSA [66] within the last 4 ns of the MD trajectories at 310 K. The calculated DG value from MM-GBSA is 123.20 kJ/mol, and that from MM-PBSA is 113.86 kJ/mol (see Table S2). The binding energy from MD simulation and that from fluorescence spectrum are consistent in nature that they are both negative. The negative DG value indicates that this interaction is spontaneous in theory. However, the two values of binding energy are not identical because experiment conditions and the status of molecules in two techniques are different [67,68]. The results of energy decomposition can reflect the contribution of each residue and ligand to the formation of the complex. Energy decomposition was calculated at the last 4 ns to figure out the contribution of every amino acid within 5 Å to the overall binding free energy. From Table S3, it is seen that Trp-213 and fomesafen molecule are the two most important contributions to the formation of complex, explaining that the fomesafen has a greater impact on Trp than Tyr when fromesafen is combined with BSA. 4. Conclusions

Fig. 6. Synchronous fluorescence spectra of BSA-fomesafen while (a) Dl ¼ 15 and (b) Dl ¼ 60 nm at room temperature. c (BSA) ¼ 10 mM, c (fomesafen) ¼ 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 mM (a / j). (c) I/I0 of BSA-fomesafen concentration.

interactions still exist mainly in a, b and c. Fig. 10e shows the distribution of water molecules within 5 Å of the ligand that there are few water molecules between ligands and pockets, which results from the release of water from pocket by fomesafen. It cause the higher randomness of this system resulting in the positive DS. In summary, this model explains that the fomesafen has greater impact on Trp than Tyr, which is in consensus with the

In our research, the interaction of BSA and fomesafen has been explored utilizing in silico analysis and physicochemical methods. The values of bimolecular quenching rate constants calculated by SternVolmer equation and the results of the UV/Vis absorption spectral measurement suggest that static quenching is the main quenching mechanism. The thermodynamic constants from the fluorescence quenching studies indicate hydrophobic force plays a major role of the interaction of fomesafen and BSA. Combined with the results of EEMS and CD spectroscopy, the interaction of BSA and fomesafen can weaken the fluorescence of Trp, Tyr and polypeptide backbone while cannot destroy the secondary structure of BSA. In view of the results of synchronous fluorescence studies and competitive binding of fomesafen and warfarin, it appears that fomesafen and warfarin bonded to an identical binding site around Trp-213. The models after MD is the ultimately identified model reflecting the real situation, from which we can find that the interactions between fomesafen and BSA depend mainly on the contribution of hydrophobic residues. Moreover, this model

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Fig. 7. Fluorescence spectra of warfarin and warfarin-BSA-fomesafen (a/j), c (fomesafen) ¼ 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 mM (a / j), c (BSA) ¼ 10 mM, c (warfarin) ¼ 5 mM. The excitation at 310 nm and slit widths set to 5 nm.

Fig. 8. Putative binding mode of fomesafen after docking calculations. (a) (I) Stereo view of the docked conformation of the drug fomesafen with the protein BSA; (II) A magnified view at the site of interaction of the drug. (b) Hydrogen bonding interactions of fomesafen with the surrounding protein. (c) (I) The electrostatic potential distributions of residues within 5 Å of ligand; (II) The electrostatic potential distributions of ligand. (III) The electrostatic potential distributions of ligand and protein. Red to blue represents positive to negative (see Fig. S4) (d) The model of p-cation interaction in the BSA-famesafen complex.

explains that the fomesafen has greater impact on Trp than Tyr, which is in consensus with the experimental conclusion. Compared with the models before and after MD, aqueous solution is an indispensable condition for the emergence of significant hydrophobic interaction in this system. The free energy and the energy decomposition measured by the MD simulations indicate that the interaction is spontaneous and Trp-213 has great contributions to

the formation of BSA-fomesafen system. In summary, the results show that fomesafen has binding force with BSA, which makes it possible to transport and distribute fomesafen in organism. Besides, our work points to the potential risk of fomesafen and provides a basis for further study on biochemical reactions of fomesafen.

X. Dong et al. / Journal of Molecular Structure 1195 (2019) 892e903

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Table 3 Cation-p interaction between BSA and fomesafen. nitrogen atom

aromatic nucleus

Distance(Å)

if there is p-cation interaction

N N N N N N

Ring1 Ring2 Ring1 Ring2 Ring2 Ring2

4.2 4.1 6.3 4.3 3.7 4.9

Yes Yes Weak Yes Yes Yes

a a b b c d

Fig. 9. (a) RMSD values for the fomesafen and for the complexes (Ca) during 25 ns simulations at 310 K. (b) RMSF values at each residue position for the BSA-fomesafen complexes at 310 K.

Fig. 10. Binding mode of fomesafen after MD. (a) (I) Stereo view of the docked conformation of fomesafen with BSA; (II) A magnified view at the site of interaction of fomesafen. (b) (I) and (II) The electro-static potential distributions of residues within 5 Å of fomesafen in different angle; (III) The electrostatic potential distributions of fomesafen. Red to blue represents positive to negative (see Fig. S4) (c) The model of p-p stacking in the complex. (d) The model of p-cation interaction in the BSA-famesafen complex. (e) The distribution of water molecules within 5 Å of the ligand.

Funding

Appendix A. Supplementary data

This work was supported by the National Natural Science Foundation of China (grant number 21873115); National Training Program of Innovation and Entrepreneurship for Undergraduates (grant number 201810019156).

Supplementary data to this article can be found online at https://doi.org/10.1016/j.molstruc.2019.05.128.

Acknowledgements The authors would like to thank K. Q. Fan Researcher [Institute of Microbiology Chinese Academy of Science] for his help in CD spec-trum measurements.

References [1] P.H. Sikkema, C. Shropshire, S. Nader, Response of dry bean to preplant incorporated and preemergence applications of Smetolachlor and fomesafen, Crop Protect. 28 (2009) 744e748. [2] M.S. Khorram, Y. Wang, X.X. Jin, H. Fang, Y.L. Yu, Reduced mobility of fomesafen through enhanced adsorption in biochar-amended soil, Environ. Toxicol. Chem. 34 (2015) 1258e1266. [3] EPA-HQ-OPP-2006-0239, Mar. 2007.

902

X. Dong et al. / Journal of Molecular Structure 1195 (2019) 892e903

[4] J. Krijt, I.V. Holsteijn, I. Hassing, M. Vokurka, B.J. Blaauboer, Effect of diphenyl ether herbicides and oxadiazon on porphyrin biosynthesis in mouse liver, rat primary hepatocyte culture and HepG2 cells, Arch. Toxicol. 67 (1993) 255e261. [5] A. Jumel, M.A. Coutellec, J.P. Cravedi, L. Lagadic, Nonylphenol polyethoxylate adjuvant mitigates the reproductive toxicity of fomesafen on the freshwater snail Lymnaea stagnalis in outdoor experimental ponds, Environ. Toxicol. Chem. 21 (2002) 1876e1888. [6] M.S. Khorram, Y. Zheng, D. Lin, Q. Zhang, H. Fang, Y.L. Yu, Dissipation of fomesafen in biochar-amended soil and its availability to corn (Zea mays L.) and earthworm (Eisenia fetida), J. Soils Sediments (2016), https://doi.org/ 10.1007/s11368-016-1407-4. [7] X. Wu, J. Xu, F. Dong, X. Liu, Y. Zheng, Responses of microbial community to different concentration of fomesafen, J. Hazard Mater. 273 (2014) 155e164. [8] T.C. Mueller, B.W. Boswell, S.S. Mueller, L.E. Steckel, Dissipation of fomesafen, saflufenacil, sulfentrazone, and flumioxazin from a Tennessee soil under field conditions, Weed Sci. 62 (2014) 664e671. [9] R.A. Copeland, Enzymes, A practical introduc-tion to structure, mechanism, and data analysis, Sov. Appl. Mech. 26 (2000) 515e523. [10] H. Xu, Q. Liu, Y. Zuo, Y. Bi, S. Gao, Spectroscopic studies on the interaction of vitamin C with bovine serum albumin, J. Solut. Chem. 38 (2009) 15e25. [11] J.M. Dixon, S. Egusa, Conformational change-induced fluorescence of bovine serum albuminegold complexes, J. Am. Chem. Soc. 140 (2018) 2265e2271. [12] U. Kragh-Hansen, Molecular aspects of ligand binding to serum albumin, Pharmacol. Rev. 33 (1981) 17e53. [13] T. Peters, All about Albumin; Biochemistry, Genetics and Medical Applications, Academic, San diego, CA, 1995. [14] E. Guercia, C. Forzato, L. Navarini, F. Berti, Interaction of coffee compounds with serum albumins. Part II: Diterpenes, Food Chem. 199 (2016) 502e508. [15] H. Vahedian-Movahed, M.R. Saberi, J. Chamani, Comparison of binding interactions of lomefloxacin to serum albumin and serum transferrin by resonance light scattering and fluorescence quenching methods, J. Biomol. Struct. Dyn. 28 (2011) 483e502. [16] Y.Q. Zhou, Y.W. Wang, X.Y. Hu, J.S. Huang, Y.Q. Hao, H. Liu, P.W. Shen, Equilibrium dialysis of metal-serum albumin. I. Successive stability constants of Zn(II)-serum albumin and the Zn2þ-induced cross-linking self-association, Biophys. Chem. 51 (1994) 81e87. [17] X.C. Shen, H. Liang, J.H. Guo, C. Song, X.W. He, Y.Z. Yuan, Studies on the interaction between Agþ and human serum albumin, J. Inorg. Biochem. 95 (2003) 124e130. [18] Z. Sharif-Barfeh, S. Beigoli, S. Marouzi, A.S. Rad, A. Asoodeh, J. Chamani, Multispectroscopic and HPLC studies of the interaction between estradiol and cyclophosphamide with human serum albumin: binary and ternary systems, J. Solut. Chem. 46 (2017) 488e504. [19] M. Chen, Y. Liu, H. Cao, L. Song, Q.Q. Zhang, The secondary and aggregation structural changes of BSA induced by trivalent chromium: a biophysical study, J. Lumin. 158 (2015) 116e124. [20] J. Chamani, M.R. Saberi, H.A. Tousi, Comparing the interaction of cyclophosphamide monohydrate to human serum albumin as opposed to holotransferrin by spectroscopic and molecular modeling methods: evidence for allocating the binding site, Protein Pept. Lett. 17 (2010) 116e124. [21] Z. Mahboobeh, P. Maliheh, A. Asoodeh, M.R. Saberi, J. Chamani, A comparison investigation of DNP-binding effects to HSA and HTF by spectroscopic and molecular modeling techniques, J. Biomol. Struct. Dyn. 32 (2014) 1936e1952. [22] P. Schlieper, R. Steiner, The effect of different surface chemical groups on drug binding to liposomes, Chem. Phys. Lipids 34 (1983) 81e92. [23] R. Pasternack, P. Collings, Resonance light scattering: a new technique for studying chromophore aggregation, Science (Wash. D C) 269 (1995) 935e939. [24] O.K. Abou-Zied, N. Al-Lawatia, M. Elstner, T.B. Steinbrecher, Binding of hydroxyquinoline probes to human serum albumin: combining molecular €rster's resonance energy transfer spectroscopy to understand modeling and Fo flexible ligand binding, J. Phys. Chem. B 117 (2013) 1062e1074. [25] J.B.F. Lloyd, Synchronized excitation of fluorescence emission spectra nat, Phys. Sci. 231 (1971) 64e65. [26] A.P.C. Ribeiro, S. Anbu, E.C.B.A. Alegria, A.R. Fernandes, P.V. Baptista, R. Mendes, A.S. Matias, M. Mendes, M.F.C. Guedes da Silva, A.J.L. Pombeiro, Evaluation of cell toxicity and DNA and protein binding of green synthesized silver nanoparticles, Biomed. Pharmacother. 101 (2018) 137e144. [27] G. Vettoretti, E. Moroni, S. Sattin, J. Tao, A.D. Agard, A. Bernardi, G. Colombo, Molecular dynamics simulations reveal the mechanisms of allosteric activation of hsp 90 by designed ligands, Sci. Rep. 6 (2016) 23830. [28] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, G.A. Petersson, H. Nakatsuji, et al., Gaussian 16, Revision A.03, Gaussian, Inc., Wallingford CT, 2016. [29] T. Darden, D. York, L. Pedersen, Particle mesh Ewald: an N,log(N) method for Ewald sums in large systems, J. Chem. Phys. 98 (1993) 10089e10092. [30] J.P. Ryckaert, G. Ciccotti, H.J.C. Berendsen, Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n -alkanes, J. Comput. Phys. 23 (1977) 327e341. [31] D. Gao, Y. Tian, S. Bi, Y. Chen, A. Yu, H. Zhang, Studies on the interaction of colloidal gold and serum albumins by spectral methods, Spectrochim. Acta Mol. Biomol. Spectrosc. 62 (2005) 1203e1208. [32] D. Silva, C.M. Cortez, S.R.W. Louro, Chlorpromazine interactions to sera albumins A study by the quenching of fluorescence, Spectrochim. Acta, Part A 60 (2004) 1215.

[33] J.R. Lakowicz, Principles of Fluoresncence Spectroscopy, Plenum Press, New York, London, 1983. [34] M.M. Chen, Y. Liu, H. Cao, L. Song, Q. Zhang, The secondary and aggregation structural changes of BSA induced by trivalent chromium: a biophysical study, J. Lumin. 15 (2015) 116e124. [35] T. Mohammadi, Y. Ghayeb, T. Sharifi, T. Khayamian, The effect of dichlorvos on the structural alteration of serum albumins: a combined spectroscopic and molecular dynamic simulation approach, Monatshefte für Chemie - Chemical Monthly 148 (2017) 1141e1151. [36] Z. Chi, R. Liu, Phenotypic characterization of the binding of tetracycline to human serum albumin, Biomacromolecules 12 (2011) 203e209. [37] X.C. Zhao, R.T. Liu, Z.X. Chi, Y. Teng, P.F. Qin, New insights into the behavior of bovine serum albumin adsorbed onto carbon nanotubes: comprehensive spectroscopic studies, J. Phys. Chem. B 114 (2010) 5625e5631. [38] S. Millan, L. Satish, S. Kesh, Y.S. Chaudhary, H. Sahoo, Interaction of lysozyme with rhodamine B: a combined analysis of spectroscopic & molecular docking, J. Photochem. Photobiol., B 162 (2016) 248e257. [39] C. Albrecht, J.R. Lakowicz, Principles of fluorescence spectroscopy, Anal. Bioanal. Chem. 390 (2008) 1223e1224, third ed. [40] J. Kucera, P. Lubal, S. Lis, P. Taborsky, Determination of deuterium oxide content in water based on luminescence quenching, Talanta 184 (2018) 364e368. [41] J.K. Lakowicz, G. Weber, Quenching of Protein fluorescence by oxygen detection of structural fluctuation in proteins on the nanosecond time scale, Biochemistry 12 (1973) 4171e4179. [42] Z. Chi, R. Liu, Phenotypic Characterization of the binding of tetracycline to human serum albumin, Biomacromoleules 12 (2011) 203e209. [43] J.J. Mobed, S.L. Hemmingsen, J.L. Autry, L.B. Mcgown, Fluorescence characterization of IHSS humic Substances: total luminescence spectra with absorbance correction, Environ. Sci. Technol. 30 (1996) 3061e3065. [44] H. Sanei, A. Asoodeh, S. Hamedakbari-Tusi, J. Chamani, Multi-spectroscopic investigations of aspirin and colchicine interactions with human hemoglobin: binary and ternary systems, J. Solut. Chem. 40 (2011) 1905e1931. [45] Y.P. Wang, Y.L. Wei, C. Dong, Study on the interaction of 3,3-bis(4-hydroxy-1naphthyl)-phthalide with bovine serum albumin by fluorescence spectroscopy, J. Photochem. Photobiol. A Chem. 177 (2006) 6e11. [46] M.X. Xie, X.Y. Xu, Y.D. Wang, Interaction between hesperetin and human serum albumin revealed by spectroscopic methods, Biochim. Biophys. Acta 1724 (2005) 215e224. lio, Spectral [47] C.R. Camargo, I.P. Caruso, S.J.C. Gutierrez, J.M.B. Filho, M.L. Corne and computational features of the binding between riparins and human serum albumin, Spectrochim. Acta Mol. Biomol. Spectrosc. 190 (2017) 81e88. [48] F.W. Dahlquist, The meaning of Scatchard and Hill plots, Methods Enzymol. 48 (1978) 270e299. [49] S. Millan, L. Satish, S. Kesh, Y.S. Chaudhary, H. Sahoo, Interaction of lysozyme with rhodamine B: a combined analysis of spectroscopic & molecular docking, J. Photochem. Photobiol., B 162 (2016) 248e257. [50] P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions: forces contributing to stability, Biochemistry 20 (1981) 3096e3102. [51] A.A. Moosavi-Movahedi, A.R. Golchin, K. Nazari, Microcalorimetry, energetics and binding studies of DNAedimethyltin dichloride complexes, Thermochim. Acta 414 (2004) 233e241. [52] B.K. Paul, N. Guchhait, A spectral deciphering of the binding interaction of an intramolecular charge transfer fluorescence probe with a cationic protein: thermodynamic analysis of the binding phenomenon combined with blind docking study, Photochem. Photobiol. Sci. 10 (2011) 980e991. [53] K. Vuignier, J. Schappler, J.L. Veuthey, P.A. Carrupt, S. Martel, Drug-protein binding: a critical review of analytical tools, Anal. Bioanal. Chem. 398 (2010) 53e66. [54] Y. Liu, J. He, C.W. Song, Y. Li, S. Wang, Y.L. Han, H.X. Wang, Oil fingerprinting by three-dimensional (3D) fluorescence spectroscopy and gas Chromatogra^Mass spectrometry (GCa ^MS), Environ. Forensics 10 (2009) 324e330. phya  s, C. Víctor, Environmental applications of excitation[55] A.E. Aurea, C. Moise emission spectrofluorimetry: an in-depth review II, Appl. Spectrosc. Rev. 48 (2013) 77e141. [56] M.D.C. Pinto, A.L. Duque, P. Macías, Fluorescence quenching study on the interaction between quercetin and lipoxygenase, J. Fluoresc. 21 (2011) 1311e1318. [57] F.F. Tian, F.L. Jiang, X.L. Han, C. Xiang, Y.S. Ge, J.H. Li, Y. Zhang, R. Li, X.L. Ding, Y. Liu, Synthesis of a novel hydrazone derivative and biophysical studies of its interactions with bovine serum albumin by spectroscopic, electrochemical, and molecular docking methods, J. Phys. Chem. B 114 (2010) 14842e14853. [58] F. Ding, W. Liu, F. Liu, Z.Y. Li, Y. Sun, A study of the interaction between malachite green and lysozyme by steady-state fluorescence, J. Fluoresc. 19 (2009) 783e791. [59] B.K. Paul, K. Bhattacharjee, S. Bose, N. Guchhait, A spectroscopic investigation on the interaction of a magnetic ferrofluid with a model plasma protein: effect on the conformation and activity of the protein, Phys. Chem. Chem. Phys. 14 (2012) 15482e15493. [60] Y.H. Chen, J.T. Yang, H.M. Martinez, Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion, Biochemistry 11 (1972) 4120e4131.   [61] N.M. Nunes, A.F.C. Pacheco, A.J.P. Agudelo, A.J.P. Agudelo, M.S. Pinto, M.D.C. Hespanhol, A.C.D.S. Pires, Interaction of cinnamic acid and methyl cinnamate with bovine serum albumin: a thermodynamic approach, Food

X. Dong et al. / Journal of Molecular Structure 1195 (2019) 892e903 Chem. 237 (2017) 525e531. [62] S. Gao, G. Shi, H. Fang, Impact of cation-p interactions on the cell voltage of carbon nanotube-based Li batteries, Nanoscale 8 (2015) 1451e1455. [63] Z. Liang, Q.X. Li, p-Cation interactions in molecular recognition: perspectives on pharmaceuticals and pesticides, J. Agric. Food Chem. 66 (2018) 3315e3323. [64] M.T. Colvin, R. Silvers, Q.Z. Ni, T.V. Can, I. Sergeyev, M. Rosay, K.J. Donovan, B. Michael, J. Wall, S. Linse, R.G. Griffin, Atomic resolution structure of monomorphic Ab42 amyloid fibrils, J. Am. Chem. Soc. 138 (2016) 9663e9674. [65] A. Jadhav, R. Dash, R. Hirwani, M. Abdin, Sequence and structure insights of kazal type thrombin inhibitor protein: studied with phylogeny, homology modeling and dynamic MM/GBSA studies, Int. J. Biol. Macromol. 108 (2018) 1045e1052.

903

[66] L. Zhang, Y. Li, Y. Yuan, Y. Jiang, Y. Guo, M. Li, X. Pu, Molecular mechanism of carbon nanotube to activate Subtilisin Carlsberg in polar and non-polar organic media, Sci. Rep. 6 (2016) 36838. [67] T. Sohrabi, M. Hosseinzadeh, S. Beigoli, M.R. Saberi, J. Chamani, Probing the binding of lomefloxacin to a calf thymus dna-histone h1 complex by multispectroscopic and molecular modeling techniques, J. Mol. Liq. 256 (2018) 127e138. [68] N. Shakibapour, F. Dehghani Sani, S. Beigoli, H. Sadeghian, J. Chamani, Multispectroscopic and molecular modeling studies to reveal the interaction between propyl acridone and calf thymus dna in the presence of histone h1: binary and ternary approaches, J. Biomol. Struct. Dyn. (2018) 1e43.