Mol Biol Rep (2014) 41:1693–1702 DOI 10.1007/s11033-013-3018-0

Detection of interaction between lysionotin and bovine serum albumin using spectroscopic techniques combined with molecular modeling Yuting Hu • Guowen Zhang • Jiakai Yan

Received: 25 September 2013 / Accepted: 30 December 2013 / Published online: 8 January 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract A combination of fluorescence, UV–Vis absorption, circular dichroism (CD), Fourier transform infrared (FT-IR) and molecular modeling approaches were employed to determine the interaction between lysionotin and bovine serum albumin (BSA) at physiological pH. The fluorescence titration suggested that the fluorescence quenching of BSA by lysionotin was a static procedure. The binding constant at 298 K was in the order of 105 L mol-1, indicating that a high affinity existed between lysionotin and BSA. The thermodynamic parameters obtained at different temperatures (292, 298, 304 and 310 K) showed that the binding process was primarily driven by hydrogen bond and van der Waals forces, as the values of the enthalpy change (DH°) and entropy change (DS°) were found to be -40.81 ± 0.08 kJ mol-1 and -35.93 ± 0.27 J mol-1 K-1, respectively. The surface hydrophobicity of BSA increased upon interaction with lysionotin. The site markers competitive experiments revealed that the binding site of lysionotin was in the subdomain IIA (site I) of BSA. Furthermore, the molecular docking results corroborated the binding site and clarified the specific binding mode. The results of UV–Vis absorption, CD and FT-IR spectra demonstrated that the secondary structure of BSA was altered in the presence of lysionotin. Keywords Lysionotin  Bovine serum albumin  Interaction  Spectroscopy  Molecular modeling

Y. Hu  G. Zhang (&)  J. Yan State Key Laboratory of Food Science and Technology, Nanchang University, No. 235, Nanjing East Road, Nanchang 330047, Jiangxi, China e-mail: [email protected]

Introduction Serum albumin, the most abundant protein in the circulatory system, is capable of binding and transporting varieties of endogenous and exogenous substances such as fatty acids, dyes and drugs [1, 2]. The distribution, free concentration and the metabolism of various drugs can be significantly influenced by drug-protein interactions in the bloodstream. Consequently, it is of imperative importance to investigate the interaction of different endogenous and exogenous substances with the protein. Bovine serum albumin (BSA), one of the major components in plasma protein, is composed of three structurally homologous domains (I–III), and each domain contains two subdomains (A, B). It has two tryptophan residues which possess intrinsic fluorescence: Trp-134, lying on the surface of sub-domain IB, and Trp-213, locating in the hydrophobic binding pocket of sub-domain IIA [3]. The binding sites of BSA for endogenous and exogenous ligands may be in these domains, and the principal regions of drugs binding sites of albumin are often located in hydrophobic cavities of sub-domains IIA and IIIA which are called sites I and II, respectively [4]. Studies have reported that BSA and human serum albumin (HSA) display approximately 76 % sequence homology, and the 3-D structure of BSA is believed to be similar to that of HSA [5]. Therefore, BSA has often been selected as model protein in the related research by virtue of its medical importance, stability, low cost and availability. Lysionotin (5,7-dyhydroxy-40 ,6,8-methoxyflavone, structure shown in Fig. 1) widely exists in fewflower lysionotus herb, which is a flavonoid compound with multiple pharmacological effects such as antibacterial, anti-inflammatory, antihypertensive and free radical-scavenging activities [6, 7]. In recent

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years, exploring the binding of flavonoids to proteins has attracted lots of interests among researchers because flavonoids widely exist in natural plants and exhibit significant pharmacological activities. Bi et al. have revealed that three flavonoids including naringenin, hesperetin and apigenin can interact with BSA and quench BSA fluorescence by a static procedure. The binding constants at 291 K were obtained to be 4.08 9 104 L mol-1 for naringenin, 5.40 9 104 L mol-1 for hesperetin, and 5.32 9 104 L mol-1 apigenin, respectively [8]. Feroz et al. [9] have reported the interaction of flavokawain B (FB), a multitherapeutic flavonoid from Alpinia mutica with HSA. Their results showed that FB binding to HSA mainly takes place in subdomain IIA and leads to both secondary and tertiary structural alterations in the protein. To the best of our knowledge, the study on the interaction of lysionotin with proteins has not been previously reported. Many spectroscopic techniques, such as fluorescence, UV–Vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy have become popular methods for studying ligands-protein interactions. Fluorescence and UV–Vis absorption spectroscopy can reveal the binding affinity of small molecules with proteins and be helpful to understand their binding mechanisms based on their sensitivity, reproducibility and convenience. CD and FT-IR spectroscopy are reliable methods for detecting the contents of secondary conformation forms of proteins, which can explain the conformational changes of proteins induced by ligands [10]. In addition, molecular modeling is becoming more and more popular to identify the interaction as well as the feasible binding site and binding mode between ligands and proteins [11]. The purpose of this work was to explore the binding mechanism of lysionotin with BSA and the effect of the flavonoid on the protein structure at physiological pH (pH 7.4) by fluorescence, UV–Vis absorption, CD and FT-IR spectroscopy, coupled with molecular docking. We hope that the results obtained from this study can provide worthy information for understanding the pharmacological actions of the flavonoid at molecular level.

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Experimental Chemicals and materials BSA, essentially fatty acid free, was purchased from SigmaAldrich Co. (St. Louis, MO, USA) and used without further purification. The stock solution of BSA (5.00 9 10-4 mol L-1) was prepared with a pH 7.4 Tris–HCl buffer (0.10 mol L-1 Tris base, 0.10 mol L-1 HCl and 0.10 mol L-1 NaCl) and then diluted to the required concentrations with the buffer. Lysionotin (analytical grade) was obtained from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The stock solution of lysionotin (5.00 9 10-4 mol L-1) was prepared in absolute methanol. 8-Anilino-1-naphthalene-sulfonic acid (ANS) was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). The stock solution of ANS (9.00 9 10-5 mol L-1) was prepared in ultrapure water. All chemicals were of analytical reagent grade, and ultrapure water was used throughout the experiment. All stock solutions were stored at 0–4 °C. Apparatus All fluorescence spectra were measured with the use of a 1.0 cm quartz cell on a Hitachi spectrofluorimeter model F-7000 (Hitachi, Japan) equipped with a 150 W xenon lamp and a thermostat bath. UV–Vis absorption spectra were performed on a Shimadzu UV-2450 spectrophotometer (Shimadzu, Japan) using a 1.0 cm quartz cell. FT-IR spectra were measured on a Thermo Nicolet-5700 FT-IR spectrometer (Thermo Nicolet Co., USA) equipped with a germanium attenuated total reflection (ATR) accessory, a DTGS KBr detector, and a KBr beam splitter. CD spectra were recorded on a Bio-Logic MOS 450 CD spectrometer (Bio-Logic, France) using a quartz cuvette of 1.0 mm path length. Procedures Fluorescence measurements

Fig. 1 Molecular structure of lysionotin

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The fluorescence measurements were performed at different temperatures (292, 298, 304 and 310 K) at wavelengths between 290 and 500 nm with an excitation wavelength at 280 nm. The excitation and emission bandwidths were both set at 2.5 nm. BSA concentration was kept at 5.50 9 10-7 mol L-1 and lysionotin concentrations were varied from 0 to 9.80 9 10-6 mol L-1. Appropriate blanks corresponding to the buffer are subtracted to correct background fluorescence. To eliminate the possibility of re-absorption and inner filter effect arising from UV absorption, the fluorescence

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data were corrected for absorption of excitation light and emitted light according to the relationship [12]: Fc ¼ Fm eðA1 þA2 Þ=2

ð1Þ

where Fc and Fm are the corrected and measured fluorescence, respectively. A1 and A2 represented the absorbance of the drug at excitation and emission wavelengths, respectively.

The displacement experiments were performed using different site markers viz., warfarin, ibuprofen and digitoxin for sites I, II and III, respectively, by keeping the concentrations of BSA and the markers constant at 5.50 9 10-7 mol L-1, and then gradually adding lysionotin solution (to give a final concentration of 9.80 9 10-6 mol L-1). Fluorescence quenching spectra were measured at 298 K over a range of 290–500 nm. The binding constants of lysionotin–BSA system in the presence of above site markers were calculated by the fluorescence data. Determination of protein surface hydrophobicity (PSH) Surface hydrophobicity properties of BSA were evaluated by fluorimetric titration experiments with the use of ANS as a fluorescence probe. The fluorescence measurements were employed by keeping the BSA concentration constant (5.50 9 10-7 mol L-1) while gradually increased ANS concentration (from 0 to 5.36 9 10-6 mol L-1). The excitation and emission wavelengths were 380 and 487 nm, respectively. There was a linear relationship between the fluorescence intensity (F) and the ANS concentration (c) only for the dilute solutions of ANS (F = Bc, B was the proportionality coefficient between fluorescence intensity and ANS concentration). It was assumed that all ANS molecules (0–3.00 9 10-7 mol L-1) were bound to the BSA, the value of B could be calculated from the slope of linear portion of the plot of F versus [ANS]. The concentration of the bound ANS molecules was obtained using the following equation: [ANS]bound = F/B, and the concentration of the free ANS molecules was calculated from the difference between total and bound ANS concentrations ([ANS]free = [ANS]total - [ANS]bound) [13]. From the Scatchard plot, the protein surface hydrophobicity (PSH) index in the absence and presence of the drug could be determined by the following relationship [14]: Fmax ½BSAKdapp

free

d

d

UV–Vis absorption spectra

Displacement experiments

PSH ¼

dissociation constant of the fluorescent ANS–BSA complex, is the binding affinity of ANS to BSA in the and 1/Kapp d absence and presence of lysionotin [14]. The values of 1/Kapp d and Fmax were obtained from the slope and the x-intercept of Scatchard plot, respectively (in this study, Scatchard plot was the plot of fluorescence intensity/[ANS]free versus F fluorescence intensity, that is ½ANS ¼  KFapp þ FKmax app ). 

The UV–Vis absorption spectra of BSA in the absence and presence of lysionotin, and the absorption spectra of corresponding concentration of lysionotin solution were recorded over a wavelength range of 200–300 nm in pH 7.4 Tris–HCl buffer solution at room temperature. All observed absorption spectra were corrected for the buffer absorbance. Circular dichroism (CD) measurements The CD spectra of BSA incubated with lysionotin at molar ratios ([lysionotin]/[BSA]) of 0:1, 1:1 and 2:1 were recorded in pH 7.4 Tris–HCl buffer solution at room temperature. The CD measurements were carried out at wavelengths between 200 and 250 nm and a scan speed of 60 nm min-1 under constant nitrogen flush. All observed CD spectra were corrected for buffer signal, and results were expressed as CD ellipticity in mdeg. The contents of different secondary conformation forms of BSA, e.g., a-helix, b-sheet, b-turn, and random coil, were analyzed from CD spectroscopic data by the online SELCON3 program (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml). FT-IR measurements FT-IR measurements were carried out on a Thermo Nicolet-5700 FT-IR spectrometer. All spectra were taken via the ATR method with a resolution of 4 cm-1 and 64 scans. The FT-IR spectra of BSA in the absence and presence of lysionotin were recorded in the range of 1,800–1,400 cm-1 at pH 7.4 Tris–HCl buffer and room temperature. The molar ratio of lysionotin to BSA was maintained at 2:1. The corresponding spectra of buffer solution were measured under the same conditions and taken as blank, which were subtracted to obtain the FT-IR spectra of the sample solution. The secondary structure compositions of BSA and its lysionotin complex were estimated by the FT-IR spectra and curve-fitted results of amide I band.

ð2Þ

where Fmax is the maximum fluorescence intensity at the saturated ANS concentration which indicated the number of surface hydrophobic sites of BSA, Kapp is the apparent d

Molecular docking studies Molecular modeling studies were carried out to explore the probable binding of lysionotin with BSA by using the

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docking program AutoDock (vers. 4.2) along with the AutoDock Tools (ADT). The X-ray crystal structure of BSA (PDB ID: 3V03) was downloaded from the RCSB Protein Data Bank (http://www.rcsb.org/pdb) and used by deleted water molecules. The 3D structure of lysionotin was generated in Sybyl 9 1.1 (Tripos Inc., St. Louis, USA). Docking calculations were performed using the Lamarckian genetic algorithm (LGA) method. From the docking results, the best scoring (i.e., with the lowest docking energy) docked model of a compound was chosen to represent its most favorable binding mode predicted by AutoDock. To bring docking simulations into force, a grid box was defined to enclose the active site with dimensions ˚ and a grid spacing of 0.981 A ˚ . The of 126 9 54 9 80 A grid maps for energy scoring were calculated using AutoGrid and other miscellaneous parameters were assigned the default values given by AutoDock. The output from AutoDock was visualized with PyMol.

Results and discussion Fluorescence quenching studies The fluorescence emission spectra of BSA in the absence and presence of lysionotin are displayed in Fig. 2a, where BSA exhibited a strong fluorescence emission peak at 348 nm. With increasing amounts of lysionotin to BSA solution, the fluorescence intensity of BSA decreased significantly and a blue shift of the peak (from 348 to 346 nm) was observed, suggesting that lysionotin interacted with BSA and quenched its intrinsic fluorescence, and the microenvironment around the fluorophores in BSA was changed, resulting in an increase of hydrophobicity in the vicinity of the fluorophores [15]. Furthermore, the occurrence of an isosbestic point at 406 nm in the lysionotin–BSA system might be rationalized in terms of the existence of bound and free lysionotin at equilibrium [16]. The fluorescence quenching mechanism is usually classified as dynamic quenching and static quenching, which can be distinguished by their different dependence on temperature and viscosity, or preferably by lifetime measurements. For dynamic quenching, higher temperatures result in faster diffusion and larger amounts of collisional quenching so that the quenching constant increases with increasing temperature, and the reverse effect would be observed for static quenching [17]. The Stern–Volmer equation is often applied to characterize the mechanism of the fluorescence quenching: F0 ¼ 1 þ KSV ½Q ¼ 1 þ Kq s0 ½Q F

ð3Þ

where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. KSV is the

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Fig. 2 a Emission spectra of BSA in the presence of lysionotin at different concentrations (pH 7.4, T = 298 K, kex = 280 nm, kem = 348 nm). c(BSA) = 5.50 9 10-7 mol L-1, and c(lysionotin) = 0, 1.00, 1.99, 2.98, 3.97, 4.95, 5.93, 6.90, 7.87, 8.84, and 9.80 9 10-6 mol L-1 for curves a ? k, respectively. Curve x the fluorescence spectra of lysionotin only, c(lysionotin) = 1.00 9 10-6 mol L-1. b The Stern–Volmer plots for the BSA–lysionotin system at four different temperatures

Stern–Volmer quenching constant. Kq is the quenching rate constant of the biomolecule, which can be obtained by KSV (Kq = KSV/s0). s0 is the average lifetime of fluorescence without quencher, the value of s0 is about 10-8 s [17]. [Q] is the concentration of the quencher. The Stern–Volmer curves of F0/F versus [Q] at four different temperatures (292, 298, 304 and 310 K) are shown in Fig. 2b. The KSV values for the lysionotin–BSA system were calculated from the slope of these plots and are listed in Table 1. The results showed that the values of KSV decreased with increasing temperature, and the Kq values obtained were (1.58 ± 0.01) 9 1013, 13 (1.40 ± 0.01) 9 10 , (1.32 ± 0.01) 9 1013, and 13 (1.01 ± 0.01) 9 10 L mol-1s-1 at 292, 298, 304, and 310 K, respectively, which were much greater than the maximum scatter collision quenching constant of various quenchers with biopolymers, 2.0 9 1010 L mol-1 s-1 [17]. The evidence indicated that the fluorescence

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quenching mechanism of BSA by lysionotin was not initiated by dynamic collision but by static quenching. Binding constant and location of binding site Assuming small molecules bind independently to a set of equivalent sites on a macromolecule, the binding constant (K) and the number of binding sites (n) can be determined by the double logarithm regression curve of log [(F0 - F)/F] versus log [Q] according to the following equation: log

F0  F ¼ log K þ n log½Q F

ð4Þ

From the intercept and slope value of the regression curve based on Eq. (4), the values of K and n at four different temperatures (292, 298, 304 and 310 K) were obtained (Table 1). The calculated K values suggested that a high affinity existed between lysionotin and BSA, and the values of K decreased with the increase temperatures, indicating that the capacity of lysionotin binding to BSA was reduced [18]. The increasing temperature led to the increasing diffusion coefficient and the reduction of stability of the lysionotin–BSA complex. The values of n were approximately equal to 1, suggesting the presence of a single class of lysionotin binding sites on the BSA molecule. Sudlow et al. [19] have reported two major specific ligands binding sites in serum albumin and the principal regions usually located in hydrophobic cavities subdomains IIA and IIIA corresponding to site I and site II, which specifically bind to warfarin and ibuprofen, respectively. The binding location of digitoxin has been found to be independent of sites I and II and defined as site III [20]. To identify the binding site of lysionotin in BSA, displacement reactions were performed with warfarin, ibuprofen and digitoxin as site markers. The corresponding values of binding constant were obtained to be (6.50 ± 0.05) 9 104 L mol-1 for warfarin–lysionotin–BSA system, (1.91 ± 0.13) 9 105 L mol-1 for ibuprofen–lysionotin–BSA system, and (1.50 ± 0.05) 9 105 L mol-1 for digitoxin–lysionotin–BSA system at 298 K, based on Eq. (4). Apparently, the binding constant of lysionotin–BSA system decreased markedly (from 1.86 ± 0.04 9 105 to

6.50 ± 0.05 9 104 L mol-1) with the addition of warfarin, while the addition of ibuprofen or digitoxin influenced the binding constant to a lesser extent. The results revealed that lysionotin shared the same binding site with warfarin. Therefore, it could be concluded that the lysionotin was mainly located in the region of subdomain IIA (Sudlow’s site I) in BSA [21]. Thermodynamic parameters and acting forces Generally, hydrogen bonds, van der Waals forces, electrostatic forces, and hydrophobic interactions are four representative types of interaction forces exiting in small ligands binding to biological molecules. Ross and Subramanian [22] have characterised the signs and magnitudes of the thermodynamic parameters associated with various kinds of interaction force that may take place during the process of ligand binding to protein. To characterize the forces involved in the lysionotin–BSA interaction, thermodynamic parameters for the binding reaction were determined from the van’t Hoff equation: DH  DS log K ¼  þ ð5Þ 2:303RT 2:303R where R is the gas constant (8.314 J mol-1 K-1). The temperatures used were 292, 298, 304 and 310 K. The values of the enthalpy change (DH°) and the entropy change (DS°) were obtained from the slope and intercept, respectively, of the van’t Hoff plot based on log K versus 1/T and listed in Table 1. The free energy change (DG°) could be estimated from the equation: DG ¼ DH   TDS :

ð6Þ

As seen from Table 1, the negative values of DG° indicated the spontaneous process of the interaction between lysionotin and BSA. The DH° and DS° values were -40.81 ± 0.08 kJ mol-1 and -35.93 ± 0.27 J mol-1 K-1, respectively, which implied the binding was an exothermic process, and the hydrogen bond and van der Waals forces were the predominant driving forces for the reaction [23].

Table 1 The quenching constants (KSV), binding constants (K), number of binding sites (n) and relative thermodynamic parameters for the interaction of lysionotin with BSA at different temperatures T (K)

KSV (9105 L mol-1)

Ra

K (9105 L mol-1)

n

Rb

DH° (kJ mol-1) -40.81 ± 0.08

292

1.58 ± 0.01

0.9991

2.64 ± 0.09

1.05 ± 0.02

0.9989

298

1.40 ± 0.01

0.9996

1.86 ± 0.04

1.03 ± 0.01

0.9989

DG° (kJ mol-1)

DS° (J mol-1 K-1)

-30.32 ± 0.16

-35.93 ± 0.27

-30.10 ± 0.16

304

1.32 ± 0.01

0.9995

1.41 ± 0.05

1.01 ± 0.01

0.9995

-29.89 ± 0.16

310

1.01 ± 0.01

0.9993

0.98 ± 0.04

1.00 ± 0.01

0.9992

-29.67 ± 0.16

a

R is the correlation coefficient for the KSV values

b

R is the correlation coefficient for the K values

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Energy transfer between BSA and lysionotin The non-radiative energy transfer occurs from the donor (Trps in BSA) to acceptor (lysionotin) when the emission spectrum of donor overlaps with the absorption spectrum of the acceptor (Fig. 3). Then the distance between donor and acceptor could be determined based on the Fo¨ster’s nonradiative energy transfer theory [24]. According to Fo¨ster’s theory, the energy transfer efficiency E, is not only related to the distance between the donor and acceptor but also influenced by the critical energy transfer distance R0, which is as follows: R6 F E ¼ 6 0 6 ¼1 F0 R0 þ r

ð7Þ

where F0 and F are the fluorescence intensities of BSA in the absence and presence of lysionotin and r is the distance between lysionotin and the tryptophan residue of BSA. R0 is the critical distance when their transfer efficiency is 50 %, which can be calculated based on the following equation: R60 ¼ 8:79  1025 j2 N4 UJ

ð8Þ

In Eq. (8), j2 is the spatial orientation factor of the dipole for random orientations as in a fluid solution; N is the refractive index of the medium; U is the fluorescence quantum yield of the donor in the absence of the acceptor, and J is the overlap integral between the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor. J can be described by the equation: J¼

RF ðkÞeðkÞk4 Dk RF ðkÞDk

ð9Þ

where F (k) is the fluorescence intensity of the donor at wavelength k, e (k) is the molar absorption coefficient of the acceptor at wavelength k. For ligand–BSA interaction, j2 = 2/3, N = 1.336 and U = 0.118. According to Eqs. (7)–(9), the values of the parameters were J = 1.54 9 10-14 cm3 L mol-1, R0 = 2.74 nm, E = 0.087, and r = 4.06 nm. Obviously, the distance between lysionotin and BSA was less than 8 nm, and 0.5 R0 \ r \ 1.5 R0, this suggested that the energy transfer from Trps in BSA to lysionotin occurred with high probability. Furthermore, the larger r value in comparison to R0 further supported the presence of static quenching mechanism in the binding of lysionotin to BSA as demonstrated earlier for other ligand–protein interactions [25].

Fig. 3 The spectral overlaps of the fluorescence spectra of BSA (a) with the absorption spectra of lysionotin (b). c(BSA) = c(lysionotin) = 1.00 9 10-6 mol L-1

known to bind to the hydrophobic areas accessible to the aqueous solvent. Upon binding, its fluorescence is dramatically enhanced so that exposed hydrophobic surface areas may be quantitatively determined and ANS may be used for monitoring changes in protein surface hydrophobicity induced by drug binding. The fluorescence intensity of ANS was measured at a fixed concentration of BSA (5.50 9 10-7 mol L-1) and increasing concentrations of ANS (Fig. 4a). A typical hyperbolic response was obtained. It was noted that in the presence of lysionotin, BSA presented different curvilinear response than in the absence of the drug. From the Scatchard plot (Fig. 4b), the Kapp d and Fmax values could be obtained from the slope and the x-intercept. In comparison with BSA, the values of Kapp Fmax, and PSH were altered from d , (0.75 ± 0.02) 9 10-6 to (0.84 ± 0.02) 9 10-6 mol L-1, from 207.53 ± 1.78 to 241.88 ± 1.97, and from 7,509.00 ± 48.30 to 7,814.18 ± 53.46, respectively, at the molar ratio of lysionotin to BSA of 1:1. The increasing values of KKapp d in the presence of lysionotin showed looser binding of ANS to the lysionotin–BSA complex. Additionally, compared with BSA, the value of PSH was increased 4.06 % in the lysionotin–BSA system. Thus, it might be concluded that the binding of lysionotin to BSA was accompanying with increase in protein surface hydrophobicity as decrement of ANS binding to BSA in the presence of lysionotin [26]. Conformational changes of BSA induced by lysionotin UV–Vis absorption studies

Determination of the protein surface hydrophobicity (PSH) The hydrophobicity of proteins can be determined by several methods. ANS (an extrinsic fluorescence probe) is

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UV–Vis absorption spectroscopy is an effective method in detecting the protein structure changes and complex formation of ligands and proteins. According to the literature [27], the strong absorption of BSA at 208 nm resulted from

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negative bands in the UV region at around 210 and 221 nm, which were characteristic of a-helix of protein and both contributed to n ? p* transfer for the peptide bond of a-helical structure [29]. As shown in Fig. 6, the CD intensity of BSA increased without any significant shift of the peaks with increasing molar ratio of lysionotin to BSA (from 0:1 to 2:1), which indicated that a-helical content of BSA increased upon interaction with lysionotin [30]. The contents of different secondary structures of BSA were calculated by the online Dichroweb software. Compared to free BSA, the contents of ahelix and b-turn increased from 58.2 to 58.9, 59.7 %, from 11.4 to 11.7, 11.8 %, the contents of b-sheet and random coil structures decreased from 7.2 to 6.8, 6.6 % and from 23.2 to 22.6, 21.9 %, respectively, at the molar ratios of lysionotin to BSA of 1:1 and 2:1. These results indicated that the binding of lysionotin to BSA caused secondary structure changes of the protein with the increase of helical stability, which was similar to that reported previously about the interaction of quinoline yellow with BSA [31]. FT-IR analysis

Fig. 4 a Binding of ANS to BSA in the absence (filled triangle) and presence (filled circle) of lysionotin. b Scatchard plots for the titration with increasing concentrations of ANS to BSA in the absence (filled triangle) and presence (filled circle) of lysionotin. Both BSA and lysionotin concentrations were 5.50 9 10-7 mol L-1

the p ? p* transition of BSA’s characteristic polypeptide backbone structure C=O and was related to the changes in the conformation of peptide backbone associated with helix-coil transformation in the difference spectra of proteins. Figure 5 shows the difference absorption spectra of BSA with various amounts of lysionotin obtained by subtracting the corresponding spectra of free lysionotin from those of lysionotin– BSA complex. Obviously, with increasing amounts of lysionotin added to BSA solution, the intensity of the absorption peak of BSA at 208 nm decreased. The results indicated that the interaction of lysionotin with BSA resulted in the changes in the BSA conformation [28]. CD studies Further evidence for conformational changes in BSA was obtained by CD studies, which is a sensitive technique to monitor the conformational changes in the protein. The CD spectra of BSA in the absence and presence of lysionotin are presented in Fig. 6. The CD spectra of BSA exhibited two

Additional evidence regarding the conformational changes of BSA after binding with lysionotin was obtained from FT-IR results. Infrared spectra of proteins show a number of amide bands, which represent different vibrations of the peptide moiety. Among these amide bands of protein, the amide I band (1,700–1,600 cm-1, mainly C=O stretch) and amide II band (1,600–1,500 cm-1, C–N stretch coupled with N–H bending mode) both have a relationship with the secondary structure of protein, and amide I band is more sensitive to the change of protein secondary than amide II band [32]. Figure 7a displays the FT-IR difference spectra of BSA before and after binding with lysionotin. The peak position of amide I band shifted from 1,651 to 1,656 cm-1 and the amide II band moved from 1,540 to 1,546 cm-1 with the decrease in the intensity upon the addition of lysionotin to BSA. The changes of these peak positions indicated that lysionotin interacted with the C=O and C–N groups in the protein structural subunits, which led to the rearrangement of the polypeptide carbonyl hydrogen bonding pattern and ultimately changed the secondary structure of BSA [33]. According to the literature [34], the band range 1,615–1,637 cm-1 was generally assigned to b-sheet, 1,638–1,648 cm-1 to random coil, 1,649–1,660 cm-1 to ahelix, 1,660–1,680 cm-1 to b-turn, and 1,680–1,692 cm-1 to b-antiparallel, respectively. The content of each secondary structure of BSA can be calculated from the integrated areas of the component bands in amide I band. According to Fig. 7b, free BSA contained major amounts of a-helix 58.7 %, random coil 24.1 %, b-turn 10.4 %, bsheet 3.2 %, and b-antiparallel 2.6 %. Upon the binding

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(A) 1540 a

Absorbance

1651 1546

1656

1800

b

1700

1600

1500

1400

-1

Wavenumbers (cm ) α-helix β-anti β-sheet β-turn random coil

(B) 1652 Absorbance

Fig. 5 The UV–Vis spectra of the lysionotin–BSA system in pH 7.4 at room temperature. c(BSA) = 5.50 9 10-7 mol L-1, and c(lysionotin) = 0, 1.99, 3.97, 5.93, 7.87, and 9.80 9 10-6 mol L-1for curves a ? f, respectively; Curve m shows the absorption spectrum of lysionotin only. c(lysionotin) = 1.99 9 10-6 mol L-1

58.7% 2.6% 3.2% 10.4% 24.1%

1638

1670 1686 1700

1624

1680

1660

1640

1620

1600

-1

Wavenumbers (cm )

(C)

with lysionotin (Fig. 7c), the contents of a-helix, b-turn and b-sheet increased from 58.7 % to 59.6 %, from 10.4 % to 13.5 % and from 3.2 % to 3.7 %, while the contents of random coil and b-antiparallel decreased from 24.1 % to 20.8 % and from 2.6 % to 2.4 %, respectively, at a molar ratio of lysionotin to BSA of 2:1. This result was in accordance with that obtained from CD analysis, demonstrating that the binding of lysionotin to BSA gave rise to the conformational changes in BSA. The conformational changes induced the protein to be exposed to a more hydrophobic environment [35]. Molecular docking analysis Through the docking calculation, the conformer with lowest binding energy was obtained from the 10 minimum

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Absorbance

Fig. 6 The CD spectra of BSA in the presence of increasing amounts of lysionotin. c(BSA) = 2.00 9 10-6 mol L-1, the molar ratios of lysionotin to BSA were 0:1 (a), 1:1 (b), 2:1 (c), respectively

α-helix β-anti β-sheet β-turn random coil

1656

59.6% 2.4% 3.7% 13.5% 20.8%

1639 1673 1624

1691 1700

1680

1660

1640

1620

1600

-1

Wavenumbers (cm ) Fig. 7 a The FT-IR spectra of free BSA (a) and difference spectra [(lysionotin–BSA)–lysionotin solution] (b) in pH 7.4 buffer solution in the region of 1800–1,400 cm-1. c(BSA) = 2.00 9 10-5 mol L-1; the molar ratio of lysionotin to BSA was 2:1. The curve-fitted amide I region (1,700–1,600 cm-1) of free BSA (b) and its lysionotin complex (c)

energy conformers from 100 runs and displayed in Fig. 8a. Lysionotin bound deeply into the pocket of sub-domain IIA in the minimum energy conformer. As shown in Fig. 8b, the probe molecule lysionotin was surrounded by the

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(DH°) and entropy change (DS°), were calculated to be -40.81 ± 0.08 kJ mol-1 and -35.93 ± 0.27 J mol-1 K-1, respectively, suggesting that the binding process was primarily driven by hydrogen bond and van der Waals forces. The binding of lysionotin to BSA increased the surface hydrophobicity of BSA. Sudlow’s site I of BSA located in domain IIA was assigned to be the most probable binding site for lysionotin on BSA, and the molecular docking results confirmed and illustrated the binding site and specific binding mode. Binding of lysionotin to BSA led to conformational changes in the protein as analyzed by UV–Vis absorption, CD and FT-IR spectra. In brief, the combination of the multispectroscopic methods with molecular modeling technique utilized in this work was decisive in the depiction of the lysionotin–BSA complex. Since the binding interaction of flavonoids with proteins is greatly important in biomedical science, it is our hope that this study provides new grounds for further investigations of the pharmaceutical potential of lysionotin, and its possible uses in monitoring biological functions.

Fig. 8 a Predicted binding mode of lysionotin docked into BSA on molecular surface using the Autodock 4.2. The active site of BSA (colored domain) interacting with lysionotin was shown as molecular surface structures. b Interacting amino acid residues around the binding sites of lysionotin at sub-domain IIA. The dashed lines (green) represent hydrogen-bonding interaction. (Color figure online)

hydrophobic side chains and also the positively charge residues, namely, Thr190, Ser191, Ala193, Arg194, Gln195, Arg198, Trp213, Arg217, Gln220, Lys221, Glu291, Lys294, etc. A hydrogen bond formed between the oxygen atom at B ring of lysionotin and the hydrogen atom ˚ . This hydrogen of Arg198 with the distance of 2.168 A bonding supports a decrease in hydrophilicity within the complex of BSA and lysionotin [11]. The molecular docking results indicated that lysionotin actually interacted with the primary amino acid residues located within the sub-domain IIA of BSA, that is to say, the lysionotin bound to BSA in site I.

Conclusions Fluorescence, UV–Vis absorption, CD and FT-IR spectroscopic methods were applied to investigate the interaction between BSA and lysionotin at physiological pH. The results indicated that the fluorescence of BSA was quenched by lysionotin through a static quenching mechanism. The binding constant at 298 K was of the order of 105 L mol-1, indicating that a high affinity existed between lysionotin and BSA. The thermodynamic parameters, enthalpy change

Acknowledgments We gratefully acknowledge the financial support of the National Natural Science Foundation of China (Nos. 21167013 and 31060210), the Natural Science Foundation of Jiangxi Province (20114BAB204019), and the Research Program of State Key Laboratory of Food Science and Technology of Nanchang University (Nos. SKLF-ZZB-201305, SKLF-ZZA-201302 and SKLFKF-201203).

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