Probing the binding of lomefloxacin to a calf thymus DNA-histone H1 complex by multi-spectroscopic and molecular modeling techniques

Journal of Molecular Liquids 256 (2018) 127–138

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Probing the binding of lomefloxacin to a calf thymus DNA-histone H1 complex by multi-spectroscopic and molecular modeling techniques Tahmineh Sohrabi a, Maral Hosseinzadeh a, Sima Beigoli b, Mohammad Reza Saberi c, Jamshidkhan Chamani a,⁎ a b c

Department of Biology, Faculty of Sciences, Mashhad Branch, Islamic Azad University, Mashhad, Iran Endoscopic and Minimally Invasive Surgery Research Center, Mashhad University of Medical Sciences, Mashhad, Iran Medical Chemistry Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

a r t i c l e

i n f o

Article history: Received 29 October 2017 Received in revised form 23 January 2018 Accepted 6 February 2018 Available online 07 February 2018 Keywords: LMF Histone H1 Acridine orange Ethidium bromide RLS Viscosity

a b s t r a c t The interaction between lomefloxacin (LMF) and a ctDNA-H1 complex was investigated by various spectroscopies, as well as viscometry and molecular modeling techniques. The RLS results pointed at different intensities of the ctDNA, H1 and ctDNA-H1 complex systems in the presence of LMF. The fluorescence intensity measurements of the ctDNA, H1 and ctDNA-H1 complex in the presence of LMF as binary and ternary systems at three temperatures exhibited regular quenching of the fluorescence emission curves signifying a static behavior of the binary and ternary systems. Stern-Volmer constant values of ctDNA-LMF complex formation were 7.94 × 107, 4.94 × 107 and 3.54 × 107 M−1 at 298, 303 and 308 K respectively. In the presence of H1, the Stern-Volmer constant values had been changed to 1.41 × 108, 0.84 × 108 and 0.21 × 108 M−1 at 298, 303 and 308 K respectively. The Stern-Volmer constant values of binary and ternary systems clearly revealed static quenching behavior of ctDNA upon interaction with LMF in absence and presence of H1. Thermodynamic parameters obtained from Van't Hoff plots revealed the different essence of interaction between LMF with ctDNA, H1 and ctDNA complex systems. The interaction between LMF with ctDNA and the ctDNA-H1 complex in the presence of ethidium bromide (EB) and acridine orange (AO) as intercalator probes showed that a competitive ness occurred between EB and AO with LMF, indicating that the LMF bound to ctDNA as an intercalator. Circular dichroism (CD) curves in far UV-CD were used to determine the enhancement of ellipticity in ctDNA upon interaction with LMF in the absence and presence of H1. The specific viscosity of ctDNA in the absence and presence of H1 increased with an enhancement of the LMF concentration which was another reason for LMF binding to ctDNA as an intercalator. Molecular modeling data confirmed the experimental results pertaining to the interaction behavior in the ctDNA-LMF complex in the absence and presence of H1. © 2018 Elsevier B.V. All rights reserved.

1. Introduction The interaction of small molecules with DNA is a subject at the interface between chemistry and biology. Small molecules can bind to DNA by covalent or non-covalent interactions [1]. Nowadays, the methods used to evaluate the binding interaction of DNA with small molecules involve docking and experimental techniques [2–5]. Nucleic acids play a main role in biological systems since they carry significant genetic information. DNA is a good target for antiviral and antibiotic drugs. There are several types of sites in the DNA molecules where binding of ligand complexes can occur: between two base pairs (intercalation), in the minor or major grooves, and on the outside of the helix. The interaction between small molecules and DNA can cause DNA damage in cancer

⁎ Corresponding author. E-mail address: [email protected] (J. Chamani).

https://doi.org/10.1016/j.molliq.2018.02.031 0167-7322/© 2018 Elsevier B.V. All rights reserved.

cells, thereby blocking the division of cancer cells and resulting in cell death [6–8]. Histones are alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. These are the main protein components of chromatin, acting as spools around which DNA winds and they also play a role in gene regulation [9–13] .The core of the nucleosomes is formed of two H2A-H2B dimers and a H3–H4 tetramer. The nucleosome core particle is composed of 147 bp of DNA wrapped around an octamer of two molecules each of the core histones H2A, H2B, H3 and H4. Five major families of histones exist: H1, H5, H2A, H2B, H3 and H4. Histones H2A, H2B, H3 and H4 are known as the core histones, while histones H1 and H5 are known as the linker histones [14–17]. The H1 histone family is the most divergent and heterogeneous group of histones and is generally a transcriptional repressor. The linker histone H1 is an important structural component of chromatin and provides its functional flexibility. Histone H1 binds the DNA entering and exiting the nucleosome, finishing two

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T. Sohrabi et al. / Journal of Molecular Liquids 256 (2018) 127–138

Scheme 1. The chemical structure of Lomefloxacin.

turns of DNA around the histone octamers. A typical H1 structure includes a central globular domain flanked by unstructured N and C-terminal tails. The C-terminal tails are required for H1-linker DNA binding and chromatin stabilization. Lomefloxacin (LMF, Scheme 1) is an antibiotic in a class of drugs called fluoroquinolones with an extensive spectrum of activity against different bacterial infections, such as bronchitis and urinary tract infections [18,19], involving gram-positive and -negative microorganisms. The medical functions have been extensively studied [20], and proven to prevent bacterial DNA biosynthesis by inhibiting the bacterial enzyme DNA gyrase [21,22]. LMF is almost completely absorbed when taken orally and is slowly eliminated, with its half-life of seven to 8 h. As a third-generation quinolone, it also has the advantage of being effective against some anaerobic bacteria. Here in, we studied the interaction between LMF and calf thymus DNA (ct DNA) in the absence and presence of H1and found a different affinity of LMF to ctDNA with different sides in ctDNA. Moreover, H1 plays an important role in different binding types of LMF to ctDNA. This subject is of great interest for gene transcription and translation.

performed as follows: fixed amounts of EB, AO and ctDNA were titrated with increasing amounts of LMF solution. The EB and AO were excited at 440 nm and 490 nm and emission spectra were recorded between 450 nm and 800 nm for EB, and between 500 nm and 700 nm for AO. Resonance light scattering (RLS) spectra were recorded by scanning both the excitation and emission monochromators of the spectrofluorometer with Δλ = 0. A resonance light scattering can be developed, and has proven to determine the aggregation of small molecules and the long-range assembly of drugs on biological templates. Suitable amounts of ctDNA and LMF were added to a cuvette. The spectrum was obtained by scanning from 220 nm to 600 nm. UV–visible absorption spectra were obtained with a Jasco V-630 spectrophotometer. The UV spectra of ctDNA and ct DNA-histone H1 with LMF were recorded in the wavelength range from 200 nm to 800 nm. The experiment was performed in the presence of a fixed concentration of LMF. The base-line concentration was obtained using a blank solution containing 6.8 × 10−5 mM of ctDNA and H1 in 10-mM Tris buffer. ctDNA-LMF and (ctDNA-H1) LMF complexes were titrated with KI and NaCl with increasing concentration of KI and NaCl. LMF was added to a 0.5 ml reaction mixture containing 10-mM Tris buffer (pH = 6.8) and the emission spectra were recorded after increasing the concentration of KI and NaCl. ctDNA melting experiments were performed by monitoring the absorption of ctDNA at 260 nm in the presence and absence of LMF at different temperatures using a spectrophotometer coupled to a thermocouple. The Tm values for ctDNA, ctDNA-LMF and (ctDNA-H1) LMF complexes were obtained from the transition midpoint of melting curves based on absorbance against temperature. CD spectra of ctDNA, H1, and the ctDNA-H1 complex and the enhancement of the LMF concentrations were recorded using a CD spectrophotometer. All the CD spectra were recorded in a range from 220 nm to 330 nm. The background spectrum of the buffer solution (10 mM Tris, pH = 6.8) deduced from the spectra of ctDNA-LMF and (ctDNA-H1) LMF and H1 with LMF. The inner filter effect was carried out for all experiments [29,30].

2. Materials and methods 2.1. Chemicals and reagents Histone H1, lomefloxacin, ctDNA, ethidium bromide (EB) and acridine orange (AO) were obtained from Sigma Chemical Co. (St. Louis, MO. USA), Ltd. and were used without purification. Tris-HCl was obtained from Merck chemical Co. The protein and ctDNA (1.37 × 10−4 M) were dissolved in 10 mM Tris buffer solutions at pH = 6.8. The LMF solution (0.01 mM) was provided by dissolution in Tris buffer. EB (3.2 × 10−4 M) and AO (2 × 10−7 M) were dissolved in 10-mM Tris buffer solutions at pH = 6.8. The ct-DNA solution gave an absorbance ratio (A260/A280) of ~1.8, demonstrating that ctDNA was free from protein [23–26]. The ctDNA solution concentration per nucleotide was assigned from the absorption at 260 nm. The molar extinction coefficient and concentration of ct DNA were 6600 M−1 cm−1 and 1.37 × 10−4 M, respectively [27,28]. 2.2. Methods 2.2.1. Spectrophotometric measurements Fluorescence measurements were performed at room temperature using a spectrofluorometer model F-2500 (Hitachi-Japan). The excitation wavelengths were set at 280 nm and 295 nm, and the emission wavelength was recorded between 300 nm and 600 nm. The changes in fluorescence intensity were observed by titrating the fixed amount of LMF with varying concentration of ct DNA and histone H1 at different temperatures (298 K, 303 K, 308 K). The competitive interaction between LMF, EB and AO as intercalator probes with ctDNA was

2.2.2. Viscosity measurements Viscosity measurements were performed using an Ostwald viscometer which was thermostated at 298 K in a constant temperature bath. The concentrations of ct DNA and ct DNA-H1 complexin buffer solution (pH = 6.8) in the absence and presence of LMF were fixed and the flow time were measured using a digital stopwatch. The mean values of three replicated measurements were used to evaluate the relative specific viscosity (η/η0)1/3, where η0 and η are the specific viscosity contributions of ct DNA and ct DNA-H1 in the absence and presence of LMF, respectively. 2.2.3. Molecular modeling The molecular docking studies were performed with the MOE software. The structure of LMF was drawn in ChemBioOffice-ChemDraw followed by MM energy minimization in ChemBioOffice-Chem3D while the ct DNA and histone H1 files were taken from a protein data bank (PDB), codes: 1bna and 5NLO, respectively. The base pair sequence of ct DNA was: DC-DG-DC-DG-DA-DA-DT-DT-DC-DG-DC-DG/DC-DGDC-DG-DA-DA-DT-DT-DC-DG-DC DG. To create the complex of ct DNA-H1 the HEX8 software was taken into account while the correlation type was “shape only” and the receptor and ligand range was set to 180° whereas the twist range was set to 360°. The result complex was saved as a PDB file and became the receptor source for the final docking with LMF in the MOE software. LMF was docked in MOE with ct DNA, histone H1, ct DNA-H1 complex after protonation of all atoms and requesting 30 final docking results in MOE. Other parameters were set according to the following: the docking placement methodology was a triangle matcher; the initial scoring methodology was London

T. Sohrabi et al. / Journal of Molecular Liquids 256 (2018) 127–138

dG; the retained specified number was 30; the post-placement refinement was force field; the final scoring methodology was London dG. The best conformation was selected based on energetics. 3. Result and discussion 3.1. Resonance light scattering (RLS) measurements The RLS technique can provide information of the process responsible for the formation of a complex. The RLS spectra of ct DNA, H1 and ctDNA-H1 were obtained in the presence of LMF (see Fig. 1). It was concluded that an interaction had occurred between histone H1, ctDNA, and the ct DNA-H1complex with LMF, as shown in Fig. 1(A–C) at pH = 6.8. As can be shown from Fig. 1, the RLS values of the LMF (ctDNA-H1) complex differed from those of the LMF-H1 and LMF-ctDNA complexes thus indicating that the interaction behavior of LMF (ctDNA-H1) was different from that of the LMF-H1 and LMF-ctDNA complexes and that increasing the RLS intensity caused a binding of LMF to H1 and ctDNA. According to Fig. 1(D), the RLS intensity for H1, ctDNA and ctDNA-H1 complex was investigated in three modes suggesting that the structural changes of ctDNA-H1 were different as compared with H1 and ctDNA. So, the higher ΔIRLS values for the LMF (ctDNA-H1) complex at different concentrations of LMF indicated that the kind of binding and interaction affinity of LMF to the (ctDNA-H1) complex depended on the ctDNA-H1 interaction.

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3.2. Circular dichroism (CD) spectroscopy CD spectroscopy is a practical method for analyzing the structure of materials such as DNA and protein, so the CD technique was used for determining the conformational changes of DNA and proteins [31–35]. Fig. 2(A, C) shows the changes in H1 and ct DNA-H1 complex upon LMF addition and there was only one peak in the negative zone. On the other hand, Fig. 2(B), ct-DNA exhibited a positive band (278 nm) and a negative band (249 nm). The fact that the positive band decreased in the area meant a more compact structure and the increasing negative band of ctDNA suggested the opening of the double helix structure of ctDNA and an increase in molar ellipticity without any considerable shift in the band maxima when the LMF concentration was raised. The consequent changes in ellipticity of ctDNA suggested an intercalative binding mode of LMF to the ctDNA molecule. Table 1 lists the secondary structural elements that were calculated. Obtained data indicated that the secondary structure of H1 and the ct DNA-H1 complex consisted of ~21.5% α-helix, ~24.1% β-sheet, ~31.7% turn and ~22.7% unordered coil for H1 and ~22.7% α-helix, ~25.8% βsheet, ~32.3% turn and ~19.2%unordered coil for the ct DNA-H1 complex. The results in Table 1 show that the percentage of α-helix, β-sheet and turn decreased gradually and that the percentage of unordered coil increased. From the results, we could conclude that the binding of LMF to H1 and the ctDNA-H1 complex caused conformational changes of H1 and ctDNA.

B

C

D

IRLS

A

Wavelength/ nm Fig. 1. (A) RLS spectra of the H1-LMF system, (B) ctDNA-LMF, (C) (H1-ct DNA) LMF at pH = 6.8, (D) Comparison of an ΔIRLS curve against the concentration of LMF, the hollow diamonds are H1-LMF, the solid circles are ctDNA-LMF, and the hollow circles are (ctDNA-H1) LMF.

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T. Sohrabi et al. / Journal of Molecular Liquids 256 (2018) 127–138 Table 1 Fractions of secondary structure of H1 and the ctDNA-H1 complex in the presence of LMF at pH = 6.8. Systems

α-Helix %

β-Sheet %

Turn %

Unordered coil%

H1-LMF (H1-ctDNA) LMF

21.5 ± 0.18 22.7 ± 0.18

24.1 ± 0.18 25.8 ± 0.18

31.7 ± 0.18 32.3 ± 0.18

22.7 ± 0.18 19.2 ± 0.18

concentration of EB and AO became enhanced, as shown in Figs. 3(A) and 5(A). Moreover, there occurred an intercalation binding between ctDNA and LMF. The quenching of the fluorescence intensity of

Fig. 2. (A) Circular dichroism spectra of H1 in the presence of increasing amounts of LMF, (B) ctDNA-LMF system, (C) (H1-ct DNA) LMF complex.

3.3. The competitive interaction of acridine orange and ethidium bromide binding to ctDNA and the ctDNA- H1 complex in the presence of LMF Acridine orange (AO) and ethidium bromide (EB) are the intercalator probes that were used to study the binding type between the molecule and ctDNA. They could intercalate between two adjacent base pairs in the ctDNA helix. The fluorescence intensity of AO (excited at 490 nm) and EB (excited at 440 nm) was weak, however, enhanced fluorescence intensity was observed upon the addition of ctDNA. So, with an increasing LMF concentration, the fluorescence of the ctDNAAO and ctDNA-EB complexes decreased demonstrating that the free

Fig. 3. (A) Fluorescence spectra of the competition between LMF and AO in the (ctDNAAO)LMF system. (B) Stern-Volmer plots of the fluorescence quenching of ctDNA-AO by LMF at different temperatures, (○) 298 K, (●) 303 K, (◇) 308 K at pH = 6.8. (C) Van't Hoff plot for the interaction of ctDNA-AO and LMF.

T. Sohrabi et al. / Journal of Molecular Liquids 256 (2018) 127–138 Table 2 The thermodynamic parameters of the (ctDNA-AO) LMF system at different temperatures at pH = 6.8.

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ctDNA-AO by the LMF was analyzed using the Stern-Volmer equation as below [36]:

ΔS0 (J·mol−1·K−1)

ΔH0 (kJ·mol−1)

ΔG0 (kJ·mol−1)

T

F0 =F ¼ 1 þ KSV ½Q 

−191.18

−79.41

−22.44 −21.49 −20.53

298 303 308

where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively, [Q] is the concentration of the quencher

Fig. 4. (A) Fluorescence spectra of the competition between LMF and AO in the (ctDNAH1-AO) LMF system. (B) Stern-Volmer plots for the fluorescence quenching of ctDNAH1-AO by LMF at different temperatures, (○) 298 K, (●) 303 K, (◇) 308 K at pH = 6.8. (C) Van't Hoff plot for the interaction of ctDNA-H1-AO and LMF.

ð1  3Þ

Fig. 5. (A) Fluorescence spectra of the competition between LMF and EB in the (ctDNA-EB) LMF system. (B) Stern-Volmer plots for the fluorescence quenching of ctDNA-EB by LMF at different temperatures, (○) 298 K, (●) 303 K, (◇) 308 K at pH = 6.8. (C) Van't Hoff plot for the interaction of ctDNA-EB and LMF.

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T. Sohrabi et al. / Journal of Molecular Liquids 256 (2018) 127–138 Table 4 The thermodynamic parameters of the (ctDNA-EB) LMF system at different temperatures at pH = 6.8. ΔS0 (J·mol−1·K−1)

ΔH0 (kJ·mol−1)

ΔG0 (kJ·mol−1)

T

−169.62

−70.19

−19.46 −18.79 −17.95

298 303 308

in temperature, the KSV values decreased thus pointing at a static behavior of the quenching fluorescence emission. Thermodynamic parameters such as changes in enthalpy (ΔH0), entropy (ΔS0) and Gibbs free energy (ΔG0) are very important in determining these binding forces. Log K ¼ −ΔH0 =ð2:303RTÞ þ ΔS0 =ð2:303RÞ

ð2  3Þ

ΔG0 ¼ ΔH0 −TΔS0

ð3  3Þ

and KSV is the Stern-Volmer quenching constant. Figs. 3(B) and 5(B) show the Stern-Volmer plots of the ctDNA-LMF complex in the presence of AO and EB at 298 K, 303 K and 308 K. It can be seen that with the raise

Here, K and R are respectively the binding constant and the gas constant. The values of ΔH0 and ΔS0 were measured from the slope and intercept of Van't Hoff plots in Figs. 3(C) and 5(C) for (AO-ctDNA) LMF and (EB-ctDNA) LMF. ΔG0 was calculated from Eq. (3-3) and the results can be seen in Tables 2 and 4 along with ΔH0 and ΔS0. The negative value of ΔG0 meant that the binding process was spontaneous, whereas the negative values of ΔH0 and ΔS0 indicated that hydrogen bonds and vander Waals forces played important roles in the binding of LMF to the (AO-ctDNA) and (EB-ctDNA) complexes [37–40]. The ΔH0 and ΔS0 values were found to be competitive of AO and EB with LMF upon interaction with ctDNA, so LMF bound to ctDNA as an intercalator. The fluorescence of the (ct DNA-H1-AO) and (ctDNA-H1-EB) complexes at 525 nm and 600 nm decreased consistently with increasing LMF as shown in Figs. 4(A) and 6(A). The competitive interaction between LMF and (ctDNA-H1-AO) and (ctDNA-H1-EB) was clear and the intercalation binding between DNA-H1 and LMF was affirmed. Figs. 4 (B) and 6(B) show the Stern-Volmer plots of interaction between LMF and (H1-ct DNA) in the presence of AO and EB at three different temperatures. According to the KSV values, the interaction of LMF to the AOctDNA and EB-ctDNA complexes in the presence of H1was different implying that H1 caused changes in binding affinity of LMF to ctDNA and that the interaction between ctDNA and H1 caused the structural changes to ctDNA. Therefore, H1 played an important role in the LMFctDNA complex formation. The values of ΔH0 and ΔS0 were measured from the slope and intercept of the Van't Hoff plots in Figs. 4(C) and 6 (C). ΔG0 was calculated and is listed in Tables 3 and 5 along with ΔH0 and ΔS0. The negative value of ΔG0 at the three temperatures signified that the binding process was spontaneous, and the negative values of ΔH0 and ΔS0 suggested that the vander Waals forces and hydrogen bonds played a main role. The interaction between ct DNA and LMF in absence and presence of H1 has been done using fluorescence spectroscopy. The Ksv values of ct DNA-LMF complex formation are 7.94 × 107, 4.94 × 107 and 3.54 × 107 M−1 at 298, 303 and 308 K respectively. In the presence of H1 in ternary system, the Ksv values of (ct DNA-H1) LMF complex formation are 1.41 × 108, 0.84 × 108 and 0.21 × 108 M−1 at 298, 303 and 308 K respectively (figures not shown). In both binary

Table 3 The thermodynamic parameters of the (ctDNA-H1-AO) LMF system at different temperatures at pH = 6.8.

Table 5 The thermodynamic parameters of the (ctDNA-H1-EB) LMF system at different temperatures at pH = 6.8.

Fig. 6. (A) Fluorescence spectra of the competition between LMF and EB in the (ctDNA-H1EB) LMF system. (B) Stern-Volmer plots for the fluorescence quenching of ctDNA-H1-EB by LMF at different temperatures, (○) 298 K, (●) 303 K, (◇) 308 K at pH = 6.8. (C) Van't Hoff plot for the interaction of ctDNA-H1-EB and LMF.

ΔS0 (J·mol−1·K−1)

ΔH0 (kJ·mol−1)

ΔG0 (kJ·mol−1)

T

ΔS0 (J·mol−1·K−1)

ΔH0 (kJ·mol−1)

ΔG0 (kJ·mol−1)

T

−183.84

−77.11

−22.32 −21.41 −20.49

298 303 308

−301.04

−106.73

−17.02 −15.51 −14

298 303 308

T. Sohrabi et al. / Journal of Molecular Liquids 256 (2018) 127–138

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Fig. 7. Effect of increasing amounts of LMF on the relative viscosity of ctDNA and ctDNA-H1 complex, (○) r = [LMF] / [ctDNA], (●) r = [LMF] / [ctDNA-H1] at pH = 6.8.

Fig. 9. Optimal thermal melting profiles of (○) ctDNA, (●) (ctDNA-H1) LMF, (◇) ctDNALMF.

and ternary systems, Ksv values have been decreased with enhancement temperature that showed the static quenching behavior of interaction between ct DNA and LMF in absence and presence of H1. Static quenching clearly revealed that the binding site of LMF on ct DNA in absence and presence of H1 was as intercalator form.

3.4. Viscometric studies

Fig. 8. (A) Absorption spectra of LMF by the NaCl in the presence of ctDNA and ctDNA-H1 complex, (○) (ctDNA-LMF) NaCl, (●) (ctDNA-H1-LMF) NaCl. (B) Absorption spectra of LMF by the KI in the presence of ctDNA and the ctDNA-H1 complex, (◇) (ctDNA-LMF) KI, (◆) (ctDNA-H1-LMF) KI.

The viscosity measurements were performed as a tool to clarify the binding mode of LMF to ctDNA [41–43]. The relative specific viscosity of the ctDNA sample clearly increased with the addition of LMF. As can be seen in Fig. 7, the relative specific viscosity of ctDNA increased with the LMF concentration in the absence and presence of H1. Therefore, the LMF bound to ctDNA by intercalative binding in the absence and presence of H1. The slope of the binary curve was higher than for

Fig. 10. (A) Stern-Volmer plots of LMF by (◆) ss ctDNA and (◇) ds ctDNA. (B) SternVolmer plots of LMF by (●) (ss ctDNA-H1) and (○) (ds ctDNA-H1) at pH = 6.8.

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the ternary system. Consequently, the binding of LMF to ctDNA in the absence of H1 was looser than in the presence of H1. In the ct DNA-H1 complex, LMF could bind not only to ct DNA but also to H1, which could explain the looser binding of LMF to ctDNA in the ternary system as opposed to in the binary one.

DNA). This was proof that the groove binding existed in the interaction of ctDNA and LMF aside from electrostatic binding. The higher slope of the ctDNA-LMF complex when increasing both the NaCl and KI concentrations showed a higher dependency of the ctDNA-LMF complex to ionic strength in the absence of histone H1.

3.5. Effect of ionic strength on the spectrum of ctDNA and ctDNA-H1 complex

3.6. DNA melting analysis

By increasing the concentration of the cation, the possibility for complexation between the cation and ctDNA phosphate backbone was enhanced. The phosphate backbone transports negative charges, thus, the ionic strength in the solution plays an important role in the interaction between small molecules and ctDNA. In this study, NaCl and KI were used to control the ionic intensity in order to determine whether electrostatic binding occurred between LMF with the ct DNA and the (H1-ct DNA) complex. In Fig. 8(A and B), the absorbance of a fixed concentration of the ctDNA-LMF and (H1-ct DNA) LMF complexes showed a considerable increase. The results demonstrated the competition between Na+ and K+ with LMF when binding with ct DNA and (H1-ct

The melting temperature (Tm) of ctDNA is described as the temperature at which half of the ctDNA strands are in the double-helical state whereas the others are in the random coil state [44–46]. The intercalative mode of binding can increase Tm by about 5–8 °C, but the non-intercalative binding did not cause a clear increase in Tm [47–49]. The relative absorbance of ctDNA and ctDNA-LMF were observed in the temperature range of 20–80 °C. Thermal denaturation profiles of ctDNA and ctDNA-H1 solutions, in the presence of LMF, were obtained from plotting the absorbance at 260 nm as a function of temperature. As can be seen from Fig. 9, the Tm values of ctDNA, ctDNA-LMF and (ctDNA-H1) LMF were 58 °C, 69 °C and 66 °C, respectively. These results strongly supported the intercalation of LMF in the ctDNA helix. On the

Fig. 11. (A) Molecular modeling of LMF with ctDNA. (B) LMF with H1. (C) LMF with the (ctDNA-H1) complex.

T. Sohrabi et al. / Journal of Molecular Liquids 256 (2018) 127–138

135

Fig. 11 (continued).

other hand, in the presence of H1, the Tm value of ctDNA decreased which was assigned to the lower stability of (ctDNA-H1) LMF as opposed to the ctDNA-LMF complex. So, the interaction between ctDNA and H1 caused a strong binding of LMF to ctDNA as an intercalator.

and 7.7 × 103 M−1, respectively, which means that the binding affinity of LMF to ss ctDNA in the presence of H1 decreased. This showed the key role of H1 in the ctDNA-LMF complex formation.

3.7. Comparison of dsctDNA and ssctDNA upon interaction with LMF in the absence and presence of H1

3.8. Molecular modeling

The behavior of native and denaturated ctDNA was compared. The results of the comparison experiments of ss ctDNA and ds ctDNA with LMF are presented in Fig. 10(A). The fluorescence quenching of LMF by ss ctDNA was less than that by ds ctDNA, which suggested that LMF intercalated into the helix of ctDNA. As can be seen in Fig. 10(B), in the presence of H1, the KSV value of the (dsctDNA-H1) LMF complex was higher than for (ss ctDNA-H1) LMF, which suggested an intercalator binding mode. The KSV values of the ds ctDNA-LMF and (ds ctDNA-H1) LMF complexes were 2.1 × 104 M−1 and 4.3 × 104 M−1, respectively. These KSV values proved that H1 caused the different behavior of the ct DNA-LMF complex formation. On the other hand, the KSV values of ss ctDNA-LMF and the (ss ctDNA-H1) LMF complex were 1 × 104 M−1

Docking studies provide insight into the interaction between the macromolecule and ligand. Molecular modeling has played an important role in designing new drugs [50,51]. From the docking calculation, the conformer with minimum binding energy was picked from the 30 minimum energy conformers out of 100 runs. It was seen from the results of 30 sets that almost all the binding sites of LMF were located in the groove of the double-helix ctDNA. From these results, we found that the ctDNA residues with A17, A18, C11, C9, G10, G16 played a main role at the binding site with the optimal energy. From the docking simulation the observed change in free energy (ΔG0) of the binding force ctDNA-LMF was calculated to be −23.73 kJ mol−1. According to this result it was concluded that LMF interacted with ctDNA through the minor groove binding (Fig. 11(A)).

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Fig. 11 (continued).

Molecular modeling of the interaction of LMF with histone H1 is shown in Fig. 11(B). The LMF molecule was just adjacent to a hydrophobic residue including: Ala145, Ala165, Ala176 of the histone H1 protein. The molecular modeling of the interaction of LMF with the ctDNA-H1 complex is shown in Fig. 11(C). The LMF was just adjacent to a hydrophobic residue including: Thr153 of the ctDNA-H1 complex. The free binding energy data calculated from experimental results of the fluorescent emission are accorded to the estimated ones from docking analyses (Table 6). The binding energy values obtained from molecular modeling and fluorescence emission are not identical because experiment conditions in two techniques are different. The fluorescence emission experiments have been done in solution environment that is vital condition but the molecular modeling results have been obtained in cyberspace. On the other hand, the ctDNA, H1 and ctDNA-H1 structures in solution are different to cyberspace. These subjects can be caused different results obtained from theoretical and experimental techniques. These

results clearly show the importance of histone for improving the affinity of the binding interaction between LMF and ctDNA. 4. Conclusion In the present work, the interaction of LMF with ctDNA, H1 and a ctDNA-H1 complex was studied by fluorescence spectroscopy, RLS, viscometry, CD spectroscopy and molecular modeling techniques. KI and NaCl quenching studies and competitive displacement with EB and AO revealed that LMF interacted with ctDNA through an intercalation mode. The binding constants and the number of binding sites were calculated from the fluorescence quenching data at different temperatures. The obtained thermodynamic parameters ΔG0 and ΔS0 suggested that vander Waals forces and hydrogen bonding played an important role in the binding of LMF to ctDNA and the ctDNA-H1 complex. The intercalative binding was much more reasonable when taking into

T. Sohrabi et al. / Journal of Molecular Liquids 256 (2018) 127–138 Table 6 Docking data of ctDNA, H1 and ctDNA-H1 with LMF by the moe program. The free binding energy data of models bind to ctDNA, H1 and ctDNA-H1 moieties of the complex are separated in the table as ctDNA-LMF, H1-LMF and (ctDNA-H1) LMF respectively. Parameters

ctDNA-LMF H1-LMF (ctDNA-H1) LMF

Number of models Lowest amount free binding energy (kJ mol−1) Maximum amount of free binding energy (kJ mol−1) Mean (kJ mol−1) Standard deviation

104 −23.73

101 −22.19

107 −21.27

−17.72

−18.23

−17.19

−27.31 2.39

−28.13 2.17

−26.61 2.61

account the ionic strength effects, through experiments on the ability of LMF to bind to ds ctDNA and ss ctDNA, KI and NaCl quenching studies and viscosity measurements. CD spectroscopy revealed that LMF interacted with ctDNA and ctDNA-H1 through an outside binding mode which could lead to conformational changes in ctDNA. The molecular modeling further confirmed that LMF bound to the minor groove of ctDNA with a binding energy of −11.16 kJ mol−1. The results of the molecular modeling were consistent with those from the other methods.

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