Stability of trans-Resveratrol Associated with Transport Proteins

Article pubs.acs.org/JAFC

Stability of trans-Resveratrol Associated with Transport Proteins Manuela Pantusa,† Rosa Bartucci,†,‡ and Bruno Rizzuti*,§ †

Department of Physics, University of Calabria, Ponte P. Bucci, Cubo 31C, 87036 Rende (CS), Italy CNISM Unit, University of Calabria, Ponte P. Bucci, Cubo 31C, 87036 Rende (CS), Italy § CNR-IPCF UOS of Cosenza, LiCryL and CEMIF.Cal, University of Calabria, Ponte P. Bucci, Cubo 33B, 87036 Rende (CS), Italy ‡

ABSTRACT: Spectrophotometry and fluorescence combined with docking and molecular dynamics simulations are used to study the effect of the carrier proteins β-lactoglobulin and human serum albumin on the degradative trans-to-cis conversion of resveratrol. The spectroscopic measurements quantify the concentration of resveratrol isoforms after 2 h of irradiation with light at 340 nm, showing that their ratio depends linearly on temperature between 20 and 50 °C and obeys an Arrhenius law with activation energies of photoisomerization of 7.8 and 11.2 kcal/mol for β-lactoglobulin and albumin, respectively, compared to 5.1 kcal/mol in solution. Thus, both proteins protect trans-resveratrol from degradation, with albumin being more effective than βlactoglobulin. The computational techniques clarify details of the binding of trans-resveratrol to the proteins and show that the stabilizing effect correlates with an increase of the dihedral order parameter of the ligand. These findings suggest that transport proteins are viable carriers to stabilize and deliver resveratrol in vivo in the biologically effective trans form. KEYWORDS: resveratrol, β-lactoglobulin, human serum albumin, absorbance, fluorescence, docking, molecular dynamics

■

INTRODUCTION Resveratrol is a phenolic compound of the stilbene family that is naturally present in some plants, such as grapes, peanuts, and mulberries, to protect against fungal infections or environmental stresses.1,2 Resveratrol is also found in food and beverages, particularly red wine.3,4 From a structural point of view, resveratrol consists of two aromatic rings, one single- and another double-hydroxylated joined by a methylene bridge, and can occur in two isomers, trans- and cis-resveratrol (Figure 1).

These limitations are overcome via loading the polyphenol into water-soluble carriers, which also offer protection from degradative processes. Among soluble carriers, proteins are optimal for the loading and cellular delivery of drugs. Resveratrol has been shown to have an affinity for various proteins, such as breast cancer resistance protein and human cyclin-dependent kinase,12 insulin,13 buttermilk proteins,14,15 plasmatic lipoproteins,16 and other transport proteins such as hemoglobin,17 β-lactoglobulin,18−20 and albumins.17,21−26 β-Lactoglobulin (βLG) and human serum albumin (HSA) are the most abundant proteins in bovine milk and in human blood plasma, respectively, and they are considered model carriers for a number of ligands. βLG (162 residues, 18.3 kDa) is a member of the lipocalin protein family, involved in the binding and transport of hydrophobic and amphiphilic molecules such as fatty acids and retinoids.27 It is characterized by nine β-strands and a three-turn α-helix. Eight out of the nine β-strands are antiparallel and form a β-barrel sandwich with a central cavity, usually referred to as the protein calyx, which constitutes the main binding site.28 Other external sites have been characterized or proposed for several ligands.27 HSA (585 residues, 66 kDa) has an α-helical structure and can bind, store, and transport a wide variety of exogenous and endogenous molecules that include drugs, metals, and, with strong affinity, fatty acids.29 Within the protein, two drug sites for binding aromatic and heterocyclic compounds30 and 11 sites for medium- and long-chain fatty acids have been identified.31 In this paper we study the stability of trans-resveratrol bound to βLG and HSA as compared to when it is free in solution, either in aqueous buffer or in ethanol. UV light at 340 nm has

Figure 1. Chemical structures of (left) trans- and (right) cisresveratrol.

The trans isomer is the natural form and converts to the cis isomer as a consequence of exposure to sunlight or UV radiation.5 To the biologically active trans isomer are ascribed health benefits that make resveratrol particularly interesting for applications in the pharmaceutical and nutraceutical fields.6 The beneficial health properties include antioxidant and antiatherosclerotic effects,7 inhibition of blood platelet aggregation,8 cardioprotective activity,9 and chemopreventive action against cancer proliferation.10,11 In spite of its biological positive effects, resveratrol shows limitations such as low solubility in aqueous media, and consequently in biological fluids,1,11 weak absorption after oral administration, and rapid metabolization in vivo, resulting in poor bioavailability. In addition, the conversion of the trans form into the less active cis isomer reduces resveratrol action. © 2014 American Chemical Society

Received: Revised: Accepted: Published: 4384

December 13, 2013 April 28, 2014 April 29, 2014 April 29, 2014 dx.doi.org/10.1021/jf405584a | J. Agric. Food Chem. 2014, 62, 4384−4391

Journal of Agricultural and Food Chemistry

Article

The apo form of βLG (3NPO entry28 in the Protein Data Bank (PDB) repository) was used. Docking results were further tested with the βLG structure in complex with retinol and retinoic acid (1GX8 and 1GX9 entries,27 respectively). For HSA, the crystallographic structures in association with palmitic acid (1E7H entry31 of the PDB) and with both myristic acid and warfarin (1H9Z entry38) were considered. In particular, the latter was used to compare directly our docking results with those previously reported by Lu and co-workers.17 Although extracted from complexes, only the coordinates of protein atoms were used in all cases. Docking calculations to estimate the binding affinity of resveratrol and to obtain the starting structure for the protein−ligand complexes were performed by using AutoDock Vina,39 allowing full flexibility for the ligand. Computations were repeated to ensure the reproducibility of the docking location, and the affinity energy values are calculated over multiple runs. Molecular Dynamics Simulations. After docking, protein− resveratrol complexes were solvated in a rhombic dodecahedron with a minimum distance of 1 nm from any box edge, resulting in 6571 water molecules for βLG and 30415 for HSA. The systems were neutralized by adding 8 and 15 Na+ counterions for βLG and HSA, respectively. Energy minimization was performed to relax the system and avoid bad atomic contacts. Simulations were carried out by using the GROMACS 4.5 software package40 with the AMBER ff99SB force field.41 The GAFF force field42 was used for resveratrol, and the TIP3P model43 for water. Long-range electrostatic interactions were calculated through the particle-mesh Ewald (PME) method.44 Periodic boundary conditions were applied to avoid edge effects, and sampling was performed in the isothermal−isobaric ensemble. A Bussi−Donadio−Parrinello thermostat45 and Berendsen barostat46 were used, with reference values of 300 K and 105 Pa, respectively. Production runs were carried out for 50 ns, saving the simulation trajectories with a sampling time of 1 ps. The software package VMD47 was used for trajectory visualization.

been used as the external perturbation to probe the photostability of trans-resveratrol. At this wavelength neither the two proteins nor cis-resveratrol absorb, and only transresveratrol is excited. Samples are investigated prior and after 2 h of continuous illumination at different temperatures, between 20 and 50 °C. The study is carried out experimentally by spectrophotometry and intrinsic resveratrol fluorescence, which allow quantifying the fraction of the two resveratrol isoforms present after irradiation and to assess the stabilizing effect of the proteins. Jointly, docking calculations and molecular dynamics (MD) simulations have been performed to gain insights into the binding modes and the dynamical behavior of resveratrol in interaction with both βLG and HSA, providing details on the mechanism of stabilization of the molecule.

■

MATERIALS AND METHODS

Materials and Sample Preparation. trans-Resveratrol (3,4′,5trihydroxy-trans-stilbene), βLG (A and B variant, purity ≥90%), and HSA (fatty acid-free and globulin-free, purity approximately 99%) were from Sigma-Aldrich (St. Louis, MO, USA). The reagent-grade salts for the 10 mM phosphate buffer solution (PBS) at pH 7.4 were from Merck (Darmstadt, Germany), and ethanol was from Carlo Erba (Milan, Italy). All materials were used as purchased with no further purification. Bidistilled water was used throughout. Solutions of trans-resveratrol either in PBS or in ethanol were prepared daily at a concentration of 5 μM, which is below the resveratrol solubility in water (0.023 mg/mL). A gentle stirring was used to dissolve it in PBS. Resveratrol concentration in both PBS and ethanol was determined by spectrophotometry, by using a molar extinction coefficient ε304 = 30 135 M−1 cm−1 and ε308 = 30 000 M−1 cm−1, respectively.5,32 Protein−resveratrol complexes were prepared by mixing an amount of protein stock solution (at a concentration of 50 μM) and transresveratrol solution in PBS at the molar ratio of 10:1. HSA and βLG concentration were determined spectrophotometrically by using ε280 = 35 219 M−1 cm−1 and ε278 = 17 600 M−1 cm−1, respectively.33,34 To protect resveratrol from degradation due to radiation effects, sample handling and storage were carried out under light-protected conditions. Absorbance and Fluorescence Measurements. Absorbance data were acquired with a JASCO 7850 spectrophotometer equipped with a TPU-436 Peltier thermostated cell holder (accuracy ±0.5 °C) and an EHC-441 temperature programmer, using 1 cm optical path quartz cuvettes. Temperature was measured directly by a YSI thermistor dipped into the cuvette. Intrinsic trans-resveratrol fluorescence data were collected on an LS 50B spectrofluorometer (PerkinElmer, Beaconsfield, UK) equipped with a Peltier temperature programmer PTP-1 (accuracy ±0.5 °C) using 1 cm path length cuvettes. The excitation wavelength was 340 nm, whereas the slit widths of excitation and emission were 6 and 4 nm, respectively. Buffer background was subtracted from the raw emission spectra. Absorbance and fluorescence measurements were carried out at temperatures of 20, 25, 37, 45, and 50 °C on either freshly prepared nonilluminated resveratrol-containing samples (t = 0 h) or samples illuminated for 2 h with light at 340 nm. Irradiation was performed within the spectrofluorometer after loading the sample into the cuvette. A 2 h time interval of irradiation was suitable for obtaining maximum conversion of the trans- to the cis-resveratrol isomer. The reproducibility of the results has been assessed by performing three independent experiments. Docking Calculations. The binding of trans-resveratrol to both βLG and HSA was first predicted by molecular docking and subsequently simulated in the presence of explicit solvent. The structure of resveratrol was built by using the UCSF Chimera software.35 The starting structures of the two proteins were modeled on the basis of their crystallographic configurations, as previously described.36,37

■

RESULTS AND DISCUSSION Photostability of trans-Resveratrol in Solution and in the Carrier Proteins βLG and HSA. The absorbance and fluorescence spectra at 37 °C of resveratrol in PBS before illumination (black lines) and after 2 h of continuous irradiation at 340 nm (red lines) are reported in Figure 2A and B, respectively. The absorbance spectrum of nonirradiated resveratrol shows a broad maximum between 300 and 320 nm, as expected for trans-resveratrol in aqueous solution at

Figure 2. (A) Absorbance and (B) fluorescence spectra at 37 °C of trans-resveratrol in PBS before (black line) and after (red line) exposure for 2 h to radiation at 340 nm. 4385

dx.doi.org/10.1021/jf405584a | J. Agric. Food Chem. 2014, 62, 4384−4391


Article

neutral pH.5,32,48 By comparing the absorbance curves of the irradiated and nonirradiated sample in Figure 2A it is evident that UV light induces in the absorbance a marked attenuation of about 55%, a shift to lower wavelength of λmax, and an enhancement at 260 nm. These spectral features are typical of cis-resveratrol.32,48 They indicate that resveratrol is not stable and tends to isomerize from trans to cis form when perturbed with radiation at 340 nm. The emission spectrum of the illuminated sample in Figure 2B is characterized by a pronounced quenching of the fluorescence quantum yield compared to the nonilluminated one. Similarly, this is due to the formation of the cis isoform upon irradiation at 340 nm, at which only trans-resveratrol is excited. The attenuation of fluorescence intensity cannot be attributed to resveratrol aggregation, because of the low concentration used, 5 μM. In fact, aggregation is reported to occur for resveratrol concentrations higher than 12.5 μM at pH 5.5 and 37 μM at pH 10.5.49 Thus, the results in Figure 2 clearly indicate that trans-resveratrol in PBS solution, exposed for 2 h to radiation at 340 nm, partially converts to cis-resveratrol, resulting in a coexistence of trans- and cis-resveratrol populations in the samples. Absorbance and fluorescence measurements have been carried out on resveratrol−PBS samples at temperatures both lower and higher than 37 °C (i.e., 20, 25, 45, and 50 °C) (curves not shown). The spectra of irradiated samples are markedly and progressively affected by the temperature increase, in a manner consistent with the progressive conversion of trans- to cis-resveratrol. In contrast, no significant spectral variations are recorded in nonilluminated samples at each temperature, indicating that the sole temperature does not affect the stability of trans-resveratrol, whereas it affects the ratio of the two isoforms that coexist in solution upon UV irradiation. The stability of trans-resveratrol against UV irradiation has been also investigated in ethanol solution. Absorbance and fluorescence curves (not shown) indicate, also in this solvent, a temperature-dependent degradation of trans-resveratrol due to light irradiation. Resveratrol binds to βLG with a binding constant18,20 between 104 and 106 M−1 and to HSA with a binding constant25 of about 105 M−1. The number of resveratrol molecules bound per protein is 1 for HSA25 and 118 or 1.120 for βLG. The excess of protein in solution (protein to resveratrol molar ratio is 10:1) gives a saturation in the fluorescence signal, ensuring that all trans-resveratrol is bound to the proteins. The absorbance and fluorescence spectra for resveratrol in βLG and the fluorescence spectra for resveratrol in HSA recorded at 37 °C before (black lines) and after 2 h (red lines) of continuous irradiation at 340 nm are shown in Figure 3A, B, and C, respectively. For the HSA−resveratrol complex the absorbance measurements are not given because of the high superposition of the spectrum of the protein with those of both trans- and cisresveratrol. The absorbance spectra in Figure 3A are obtained by subtracting the βLG spectrum from the spectrum of the βLG− resveratrol complex. By comparing the curves in Figure 3 (relative to resveratrol−protein complexes) with those reported in Figure 2 (relative to resveratrol−PBS samples), it emerges that the formation of cis-resveratrol consequent to UV illumination also occurs when the ligand is bound to the protein, although it is clearly reduced.

Figure 3. (A) Absorbance and (B) fluorescence spectra at 37 °C of trans-resveratrol bound to βLG before (black line) and after (red line) exposure for 2 h to radiation at 340 nm. (C) Fluorescence spectra of trans-resveratrol bound to HSA as in (B).

The absorbance and fluorescence data have been analyzed to estimate the concentration of trans-, Ctrans, and cis-resveratrol, Ccis, present in solution after 2 h of exposure to light at 340 nm. The concentrations of the two resveratrol isoforms are determined from the absorbance measurements by the following formulas, which are valid when starting from a 100% trans-resveratrol solution of known initial concentration, C0 = A0/εtrans:5 Ctrans =

Ccis =

⎞ 1 ⎛ RA − C 0⎟ ⎜ R − 1 ⎝ lεtrans ⎠

⎞ 1 ⎛ A − RC0⎟ ⎜ 1 − R ⎝ lεcis ⎠

(1)

(2)

where R = εtrans,304 nm/εcis,304 nm = 30 135/9749 ≈ 3.09 in 10 mM phosphate buffer solution5 and R = εtrans,308 nm/εcis,308 nm = 30 000/12 500 = 2.4 in ethanol,32 εtrans and εcis are the molar extinction coefficients of trans- and cis-resveratrol, respectively, A is the measured absorbance at 304 nm in buffer and at 308 nm in ethanol, and l is the optical path length. The fractions of the two isoforms are simply given by f trans = Ctrans/C0 and fcis = Ccis/C0. From the fluorescence data, the fraction of trans isomers is f trans =Ft/F0 and the corresponding concentration is Ctrans = f transC0, where Ft is the fluorescence maximum after irradiation (t = 2 h) and F0 the fluorescence maximum before irradiation (t = 0 h). The fraction and the concentration of cis isomers are given by fcis = 1 − f trans and Ccis = C0 − Ctrans, respectively. Table 1 reports the data deduced by using the equations given above. The values indicate that comparable fractions are obtained from the two techniques at each temperature. The values of f trans decrease ( fcis increase) with a temperature increase. For resveratrol in PBS, 2 h of UV illumination promotes the formation of a marked cis population already at 20 °C, about 73%, which increases to 86% at 50 °C. In addition, 4386



Article

Table 1. Percentage Fractions, f trans, of trans-Resveratrol Free in PBS and in Ethanol and Bound to βLG and HSA PBS T ± 0.5 (°C) 20.0 25.0 37.0 45.0 50.0

absorbance 27.4 26.1 16.9 16.5 13.8

± ± ± ± ±

0.9 0.9 0.8 0.8 0.7

βLG

ethanol fluorescence 26.7 27.2 18.1 17.2 14.5

± ± ± ± ±

2.1 2.4 1.4 1.2 1.0

fluorescence

absorbance 18.7 17.9 11.7 11.0 10.2

± ± ± ± ±

0.6 0.6 0.5 0.4 0.4

21.1 18.8 14.0 13.5 12.1

the fractions of cis isomer, fcis, are slightly higher in ethanol, ranging from 79%−81% to 88−90% in the whole temperature range. This finding should be taken into account in the context of the possible use of ethanol in dissolving resveratrol for pharmaceutical applications. A number of studies have dealt with the isomerization of resveratrol in solution. It has been reported that, except in buffers at high pH, trans-resveratrol is stable in the dark from hours to several days,32 whereas different treatments, including exposure to sunlight,5 direct illumination with tubular lamps that emit at all visible wavelengths,18 and UV irradiation,25,32,48,50 induce the formation of cis isomers starting from a pure trans-resveratrol solution. Specifically, for a UVilluminated resveratrol solution, the formation of the cis isoform is influenced by physicochemical parameters such as resveratrol concentration, pH of the dispersion media, and wavelength and duration of the excitation.25,32,48,50 In addition, it has been demonstrated that no other products are present except transand cis-resveratrol when UV light interacts with a resveratrol solution.48 Within this framework, it is of interest to compare our results for resveratrol free in solution with those obtained in the above-mentioned studies. At 20 °C, upon illumination at 260 nm (i.e., a wavelength suitable to excite cis-resveratrol) of a 5.7 μM solution, 50.9% of trans-resveratrol is converted into cisresveratrol after 150 min, and 59.6% is converted after 330 min.50 By using HPLC/UV, Camont et al. report that 8 h of sunlight exposure reduces to 30% the concentration of transresveratrol starting from a pure 100 μM solution.5 Trela and Waterhouse report that a 418 μM solution of trans-resveratrol in ethanol was converted to 90.6% cis-resveratrol after 100 min of illumination at 366 nm and to 63% after 10 h at 254 nm.32 The data in Table 1 relative to the two proteins clearly indicate that they decrease resveratrol isomerization induced by radiation at 340 nm. Indeed, compared to that obtained in PBS or ethanol, at any temperature the fraction of trans-resveratrol is increased (cis-resveratrol is reduced) in the presence of βLG and even more in the presence of HSA. This occurs more efficiently at low than at high temperature. For comparison, by using spectrophotometry, Liang et al. reported that complexing resveratrol with βLG provides a slight increase in photostability relative to resveratrol−buffer solution.18 In fact, samples exposed to white light are almost stable in trans-configuration for up to 21 h, when absorbance at 305 nm is decreased to about 80% of the initial value in the absence of βLG and to about 90% in its presence. For the HSA−resveratrol complex, after repeated fluorescence measurements at λex = 315 nm, the fraction of resveratrol in trans form is 50.5% compared to 34% for resveratrol in PBS.25 Arrhenius plots of the temperature dependences of Ctrans/Ccis deduced from fluorescence data for resveratrol in PBS (squares), ethanol (circles), βLG (triangles), and HSA (diamonds) are given in Figure 4. In all cases, the temperature dependence is linear, conforming well to an Arrhenius law with

± ± ± ± ±

0.7 0.9 1.0 0.7 0.6

absorbance 38.9 31.0 27.9 17.7 15.2

± ± ± ± ±

0.9 0.8 0.8 0.6 0.6

HSA fluorescence 38.6 32.6 24.3 18.1 14.9

± ± ± ± ±

2.3 2.6 1.5 0.9 0.7

fluorescence 61.2 53.1 37.8 25.9 20.4

± ± ± ± ±

2.4 2.1 1.5 1.3 1.0

Figure 4. Temperature dependence of Ctrans/Ccis ratio deduced from fluorescence data for resveratrol in PBS (squares), ethanol (circles), βLG (triangles), and HSA (diamonds). The lines are least-squares linear fits of the data according to Arrhenius law.

an activation energy of photoisomerization of 5.1 ± 0.7 and 4.1 ± 0.4 kcal/mol for resveratrol in PBS and ethanol, respectively. These values translate to 7.8 ± 0.3 and 11.2 ± 0.4 kcal/mol for resveratrol in βLG and HSA, respectively. The activation energy of photoisomerization for transresveratrol in PBS at pH 4 was previously reported to be 3.7 ± 0.3 kcal/mol upon excitation at 260 nm for different time periods and at temperatures between 20 and 50 °C.50 On the whole, the spectroscopic data indicate that the two proteins, although not being able to prevent the formation of the cis isomer, are able to protect trans-resveratrol from degradation, and in this respect HSA is more effective than βLG. Docking and MD Simulations of Protein−Resveratrol Complexes. Docking of trans-resveratrol to βLG was carried out by using the unliganded protein form and resulted in two poses of the ligand on the protein surface, as shown in Figure 5A. Pose 1 corresponds to several slightly different binding modes with resveratrol placed at the entrance of the protein calyx and an estimated energy of −6.2 ± 0.3 kcal/mol. Pose 2 occurs in an external region on the opposite site with respect to the protein calyx, with one −OH group of resveratrol forming a hydrogen bond with the exposed carboxyl oxygen of Trp19, and a binding energy of −5.8 ± 0.2 kcal/mol. In a single case, a binding mode of pose 1 was found with resveratrol more deeply inserted into the protein calyx. However, stable association within this site can be excluded on the basis of experimental data19 indicating that binding of resveratrol takes place in another site because it occurs even at 4387



Article

Figure 5. trans-Resveratrol bound to (A) βLG and (B) HSA, with the ligand in the docking poses (blue and purple) and after MD simulations (orange). For HSA, superposition with crystallographic binding sites for fatty acids (FA1, FA3, FA4, and FA9) is evidenced. Details of protein residues interacting with the ligand in (C) βLG and (D) HSA are also shown.

low pH, when the inner region of the hydrophobic cavity is inaccessible,27 and at high temperature when the calyx is disrupted.51 Therefore, this particular binding mode of pose 1 can be considered an artifact, possibly due to the docking scoring function not taking into account the specificity of the binding site of βLG, which is an extreme case of a large and yet completely dehydrated cavity.52 Additional docking calculations were carried out by using the crystallographic βLG structure containing within the protein calyx either retinol or retinoic acid, both possessing a β-ionone ring whose steric hindrance resembles that of either of the two resveratrol rings. The structure of βLG alone was extracted from the complexes with both retinol and retinoic acid and used to accommodate trans-resveratrol. Nevertheless, estimates for binding modes and affinity of resveratrol to these protein structures were found to be similar to the unliganded protein form. Classical MD simulations were performed with the ligand initially placed according to the binding modes obtained by the docking procedure, to determine the main features of βLG− resveratrol interaction when all the degrees of freedom of the protein are free to move and in the presence of solvent. Figure 6 shows the simulated number of water molecules within a distance of 0.3 nm with respect to resveratrol. When the ligand is initially placed in pose 1, at the opening of the protein calyx, it is in contact on average with 40 water molecules compared with about 75 contacts when it is fully solvated in water. Thus, the curve indicates that resveratrol remains within the upper region of the funnel, without penetrating the main βLG binding site. A similar behavior was observed in five other simulation runs, with the molecule variously interacting with several protein residues, such as forming transient hydrogen bonds with either O-Met107, O-Ser116, or Oγ-Ser116. Migration of resveratrol deep into the binding site was never observed in simulation. Simulations starting with the ligand in pose 2 (Figure 5A) show that resveratrol initially explores the surroundings of the docking placement while remaining in contact with the protein main chain in correspondence with the Trp19 residue. In this position, resveratrol is in contact on average with 60 water molecules (see Figure 6). Quick reduction of the number of

Figure 6. Number of water molecules in contact with trans-resveratrol (blue line) free in the solvent, (black line) at the entrance of the βLG calyx (pose 1), and (red line) close to Trp19 (pose 2), as a function of the simulation time. Data are averaged over 50 ps for clarity of presentation.

contacts, which is visible in the curve at 16−18 ns, indicates that resveratrol partially penetrates the protein surface. As shown in Figure 5C, the ligand accommodates in between the side chain of Arg124, which is exposed to the solvent, and the side chain of Trp19, which is more distant from the protein surface. In this position resveratrol is more specifically attached to the protein and protected from the solvent compared with any location at the calyx entrance. Both hydrophobic and hydrophilic contacts contribute to secure the ligand to the protein, the former playing a major role as previously suggested on the basis of experimental data.19,20 The docking procedure was repeated by using protein structures obtained in simulation after 20 ns. A binding energy of −7.1 ± 0.5 kcal/mol was found for resveratrol in the binding site between Trp19 and Arg124. In contrast, no appreciable variation was observed for the binding affinity of resveratrol at the entrance of the protein calyx, compared with the starting protein structure. Therefore, the overall simulation results suggest that the region close to the Trp19 residue is the most 4388



Article

Ile142, Phe134, and Lys162, as shown in Figure 5D. The first two residues are close to resveratrol from the beginning of the simulation; thus they move away from their starting position. Similarly, when placed in pose 2, resveratrol moves about 0.4 nm from the starting position. To assess the strain exerted by the protein matrix, the distribution of values of the dihedral angle involved in the transto-cis conversion was calculated and compared to the equilibrium value found for resveratrol free in water, θeq = 180 ± 22°. The results reported in Figure 7 show that the

likely binding site for resveratrol to βLG, in agreement with fluorescence quenching19 and other techniques.20 Association of trans-resveratrol in the protein region in between Trp19 and Arg124 residues provides a straightforward explanation of the binding mechanism and of the stabilizing effect due to βLG on the trans-to-cis transition. In fact, in this binding site the ligand molecule is restrained in its dynamics and mostly protected from the solvent. Nevertheless, the protein does not provide a complete protection from the solvent, and it does not totally inhibit the possibility of rotation around the double bond in the methylene bridge of resveratrol. Furthermore, a fraction of resveratrol molecules may bind in nonspecific or low-affinity locations on the protein surface, including the entrance of the protein calyx, thus being unrestrained in the dynamics and markedly more in contact with the solvent compared to the binding location in correspondence with Trp19 and prone to convert to the cis form. This hypothesis would also explain why for βLG the number of resveratrol molecules bound per protein could vary from 1 to 1.1.18,20 For the HSA−resveratrol complex, the docking results indicate that resveratrol can bind HSA in three different poses (see Figure 5B), corresponding to sites that can be occupied by fatty acids or other small ligands. These sites are hereafter indicated with their conventional nomenclature.31 Pose 1 was found in correspondence with the binding site FA1 of HSA and shows the highest affinity, with an interaction energy of −8.9 ± 0.2 kcal/mol. In this site resveratrol can associate in either of two binding modes. One of the two rings of resveratrol occupies the position corresponding to a fatty acid tail, whereas the other ring is placed either instead of the lipid headgroup or pointing toward the center of the protein (see Figure 5B). Similarly, pose 2 shows an interaction energy of −8.5 ± 0.2 kcal/mol and overlaps totally or in part with the binding site FA4, with two different binding modes. In fact, one of the two resveratrol rings can alternatively be in the place of the tail of a fatty acid in the binding site FA3. Such a combination is not unusual for HSA, because the two binding sites FA3 and FA4 can accommodate ligands bulkier than fatty acids by rearranging into the single cavity DS2, or drug site 2.30 Finally, pose 3 has a binding affinity of −7.5 ± 0.3 kcal/mol and coincides with the binding site FA9, which is a cleft in the upper region of the central protein crevice and can be occupied by capric acid, but not by other fatty acids with a longer chain.31 It is interesting to note that our docking results do not detect any binding mode in the cavity between subdomain IB/IIA of the protein, which was previously predicted17 as the only binding site for resveratrol by using the AutoDock 3.5 program.53 In contrast, the drug site DS2 was already reported as the only binding site for resveratrol in bovine serum albumin26 by using AutoDock 4.2. Such differences in the results can probably be attributed to the improvement of the scoring function of AutoDock, as well as to the higher accuracy of the new-generation function later adopted by AutoDock Vina,39 which was used in our calculations. MD simulations were performed on the HSA−resveratrol complex, with the ligand placed in the different poses selected through the docking. The results show that, except when placed in pose 3 (FA9) at the central protein crevice, resveratrol partially diffuses away from the starting position due to dynamical rearrangements of the protein matrix. In particular, when initially placed in pose 1, it relocates about 0.5 nm from the starting position and ends up being surrounded by Tyr161,

Figure 7. Distribution of dihedral angle for trans-resveratrol calculated in the time interval 25−50 ns in the binding sites of HSA: (black line) site FA1, (red line) site FA4, (blue line) site FA9.

highest strain is found in correspondence with the binding site FA9, as indicated by both the higher difference found for the average value and the lower variance in the distribution around this value. In contrast, less strain is found in correspondence with the other two positions, indicating that resveratrol is more easily accommodated within the protein in these sites. Table 2 reports the dihedral order parameter S2 and water contacts of trans-resveratrol in the different binding sites of Table 2. Dihedral Order Parameter and Solvent Contacts, Calculated in Simulation in the Time Interval 25−50 ns, for trans-Resveratrol Free in Water or Starting from Different Poses in Either βLG or HSA environment

pose

binding site

water βLG HSA HSA HSA

2 1 2 3

Trp19/Arg124 FA1 FA4 FA9

order parameter 0.86 0.89 0.91 0.94 0.95

± ± ± ± ±

0.01 0.01 0.01 0.01 0.01

solvent contacts 75 25 23 27 28

± ± ± ± ±

3 6 7 6 8

HSA. Data for resveratrol free in water and associated with βLG are also reported for comparison. The parameter S2 ranges from 0, for a dihedral angle free to sample any conformation, to 1 for a perfectly rigid system.54 Thus, it gives a quantitative indication of the degree of inhibition for a rotation around the central methylene bridge of resveratrol. The values found in the simulation show that the dihedral order parameter correlates with the stability of resveratrol against the trans-to-cis conversion found in our absorbance and fluorescence measurements: it is lower in the aqueous solvent, intermediate in βLG, and higher in HSA. In contrast, no clear correlation appears by comparing the contacts between transresveratrol and water in βLG and HSA. Although resveratrol 4389



■

complexed with HSA is more deeply buried and in contact with more amino acid compared to βLG, the difference of solvent contacts for the two proteins is not striking because the binding sites of HSA are hydrophobic cavities accessible to the solvent.55 Finally, docking of trans-resveratrol was performed again on HSA by using the simulation structures closer to the average configuration in the interval 25−50 ns. Only the protein structures were considered, and the search space for the docking was restricted to the region occupied by each ligand. The resulting affinity energies obtained placing the ligand in the three binding sites are, respectively, 8.9 ± 0.4, 8.1 ± 0.5, and 7.1 ± 0.3 kcal/mol. These values are consistent with the ones obtained for the three poses in the initial docking procedure. However, the energetic differences among the three binding sites in this case are more evident and in better agreement with previous experiments.25 In fact, the stoichiometry of binding between resveratrol and HSA is not modified by the presence of 3 mol of stearic acid per mole of protein, whereas the amount of associated resveratrol is reduced when 5 mol of stearic acid is present. This suggests that the binding site FA1, which is not one of the three high-affinity binding sites for longchain fatty acids,56 is the one preferred by resveratrol. In contrast, the binding site FA4 has a high affinity for long-chain fatty acids;31,56 thus it is reasonable to consider it at most a secondary binding site for resveratrol. Furthermore, the binding site FA9 cannot accommodate a long-chain fatty acid;31 thus it cannot be a primary binding site for resveratrol, as in this case the stoichiometry of the HSA−resveratrol complex would have no or little change with varying amounts of stearic acid. The overall results provide a possible explanation for the different degree of protection against the trans-to-cis transition of resveratrol in βLG and HSA. Limitations in the contact with the solvent, and especially restrictions in the freedom of fluctuation due to the hindrance of the protein matrix, likely stabilize the trans-resveratrol molecule. In this respect, the simulation data show that HSA protects resveratrol more efficiently than βLG. In particular, van der Waals interactions with a relatively high number of hydrophobic amino acid residues contribute to fasten resveratrol in HSA. Furthermore, the binding energy of trans-resveratrol to HSA is considerably lower than for βLG, thus providing a more stable complex. Other factors may also play an additional role, such as the fact that the binding site of HSA is more internal, so that the ligand may not easily escape back into the solvent. In conclusion, the findings of the present work indicate that transport proteins can be conveniently used to store and deliver resveratrol in the trans form and suggest that other protein carriers may be further investigated with the aim of dispensing in vivo resveratrol or other similar ligands.

■

Article

REFERENCES

(1) King, R. E.; Bomser, J. A.; Min, D. B. Bioactivity of resveratrol. Compr. Rev. Food Sci. Food Saf. 2006, 5, 65−70. (2) Gulcin, I. Antioxidant properties of resveratrol: A structureactivity insight. Innov. Food Sci. Emerg. Technol. 2010, 11, 210−218. (3) Langcake, P.; Pryce, R. The production of resveratrol by Vitis vinifera and other members of the vitaceae as a response to infection or injury. J. Physiol. Plant Pathol. 1976, 9, 77−86. (4) Celotti, E.; Ferrarini, R.; Zironi, R.; Conte, L. S. Resveratrol content of some wines obtained from dried Valpolicella grapes: Recioto and Amarone. J. Chromatogr. A 1996, 730, 47−52. (5) Camont, L.; Cottart, C. H.; Rhayem, Y.; Nivet-Antoine, V.; Djelidi, R.; Collin, F.; Beaudeux, J. L.; Bonnefont-Rousselot, D. Simple spectrophotometric assessment of the trans-/cis-resveratrol ratio in aqueous solutions. Anal. Chim. Acta 2009, 634, 121−128. (6) Fremont, L. Minireview - Biological effects of resveratrol. Life Sci. 2000, 66, 663−673. (7) Orallo, F. Comparative studies of the antioxidant effects of cisand trans-resveratrol. Curr. Med. Chem. 2006, 13, 87−98. (8) Olas, B.; Wachowicz, B. Resveratrol, a phenolic antioxidant with effects on blood platelet functions. Platelets 2005, 16, 251−260. (9) Bradamante, S.; Barenghi, L.; Villa, A. Cardiovascular protective effects of resveratrol. Cardiovasc. Drug Rev. 2004, 22, 169−188. (10) Jang, M. S.; Cai, E. N.; Udeani, G. O.; Slowing, K. V.; Thomas, C. F.; Beecher, C. W. W.; Fong, H. H. S.; Farnsworth, N. R.; Kinghorn, A. D.; Mehta, R. G.; Moon, R. C.; Pezzuto, J. M. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 1997, 275, 218−220. (11) Asensi, M.; Medina, I.; Ortega, A.; Carretero, J.; Bano, M. C.; Obrador, E.; Estrela, J. M. Inhibition of cancer growth by resveratrol is related to its low bioavailability. Free Radical Biol. Med. 2002, 33, 387− 398. (12) Feng, L.; Jin, J.; Zhang, L. F.; Yan, T.; Tao, W. Y. Analysis of the resveratrol-binding protein using phage-displayed random peptide library. Acta Biochem. Biophys. Sin. 2006, 38, 342−348. (13) Chen, Y. Y.; Xiao, L.; Cui, J. H.; Chen, G. F.; Zhang, J.; Wang, P. Biological relevance of the interaction between resveratrol and insulin. Food Biophys. 2013, 8, 282−289. (14) Ye, J. H.; Thomas, E.; Sanguansri, L.; Liang, Y. R.; Augustin, M. A. Interaction between whole buttermilk and resveratrol. J. Agric. Food Chem. 2013, 61, 7096−7101. (15) Bourassa, P.; Bariyanga, J.; Tajmir-Riahi, H. A. Binding sites of resveratrol, genistein, and curcumin with milk α- and β-caseins. J. Phys. Chem. B 2013, 117, 1287−1295. (16) Belguendouz, L.; Fremont, L.; Gozzelino, M. T. Interaction of transresveratrol with plasma lipoproteins. Biochem. Pharmacol. 1998, 55, 811−816. (17) Lu, Z.; Zhang, Y. Y.; Liu, H.; Yuan, J. L.; Zheng, Z. L.; Zou, G. L. Transport of a cancer chemopreventive polyphenol, resveratrol: Interaction with serum albumin and hemoglobin. J. Fluoresc. 2007, 17, 580−587. (18) Liang, L.; Tajmir-Riahi, H. A.; Subirade, M. Interaction of βlactoglobulin with resveratrol and its biological implications. Biomacromolecules 2008, 9, 50−56. (19) Liang, L.; Subirade, M. Study of the acid and thermal stability of β-lactoglobulin-ligand complexes using fluorescence quenching. Food Chem. 2012, 132, 2023−2029. (20) Kanakis, C. D.; Tarantilis, P. A.; Polissiou, M. G.; Tajmir-Riahi, H. A. Probing the binding sites of resveratrol, genistein, and curcumin with milk β-lactoglobulin. J. Biomol. Struct. Dyn. 2013, 31, 1455−1466. (21) Jannin, B.; Menzel, M.; Berlot, J. P.; Delmas, D.; Lancon, A.; Latruffe, N. Transport of resveratrol, a cancer chemopreventive agent, to cellular targets: Plasmatic protein binding and cell uptake. Biochem. Pharmacol. 2004, 68, 1113−1118. (22) N’soukpoe-Kossi, C. N.; St-Louis, C.; Beauregard, M.; Subirade, M.; Carpentier, R.; Hotchandani, S.; Tajmir-Riahi, H. A. Resveratrol binding to human serum albumin. J. Biomol. Struct. Dyn. 2006, 24, 277−283.

AUTHOR INFORMATION

Corresponding Author

*Phone: +39 0984 496078. Fax: +39 0984 494401. E-mail: [email protected]. Funding

M.P. acknowledges financial support from EU and Regione Calabria by Regional Operative Program Calabria ESF 2007/ 2013, IV Axis Human Capital, Operative Objective M2, Action D5. Notes

The authors declare no competing financial interest. 4390



Article

(23) Xiao, J. B.; Chen, X. Q.; Jiang, X. Y.; Hilczer, M.; Tachiya, M. Probing the interaction of trans-resveratrol with bovine serum albumin: a fluorescence quenching study with Tachiya model. J. Fluoresc. 2008, 18, 671−678. (24) Bourassa, P.; Kanakis, C. D.; Tarantilis, P.; Pollissiou, M. G.; Tajmir-Riahi, H. A. Resveratrol, genistein, and curcumin bind bovine serum albumin. J. Phys. Chem. B 2010, 114, 3348−3354. (25) Pantusa, M.; Sportelli, L.; Bartucci, R. influence of stearic acids on resveratrol-HSA interaction. Eur. Biophys. J. Biophy. 2012, 41, 969− 977. (26) Liu, X. L.; Shang, Y. H.; Ren, X. D.; Li, H. Molecular modeling and spectroscopic studies on the interaction of transresveratrol with bovine serum albumin. J. Chem. 2013, 2013, 494706−1−7. (27) Kontopidis, G.; Holt, C.; Sawyer, L. The ligand-binding site of bovine β-lactoglobulin: evidence for a function? J. Mol. Biol. 2002, 318, 1043−1055. (28) Loch, J.; Polit, A.; Gorecki, A.; Bonarek, P.; Kurpiewska, K.; Dziedzicka-Wasylewska, M.; Lewinski, K. Two modes of fatty acid binding to bovine β-lactoglobulin − Crystallographic and spectroscopic studies. J. Mol. Recognit. 2011, 24, 341−349. (29) Peters, T. All about Albumin: Biochemistry, Genetics and Medical Applications; Academic Press: San Diego, 1997. (30) Sudlow, G.; Birkett, D. J.; Wade, D. N. Further characterization of specific drug binding sites on human serum albumin. Mol. Pharmacol. 1976, 12, 1052−1061. (31) Bhattacharya, A. A.; Grune, T.; Curry, S. Crystallographic analysis reveals common modes of binding of medium and long-chain fatty acids to human serum albumin. J. Mol. Biol. 2000, 303, 721−732. (32) Trela, B. C.; Waterhouse, A. L. Resveratrol: Isomeric molar absorptivities and stability. J. Agric. Food Chem. 1996, 44, 1253−1257. (33) Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. How to measure and predict the molar absorption-coefficient of a protein. Protein Sci. 1995, 4, 2411−2423. (34) Dufour, E.; Roger, P.; Haertle, T. Binding of benzo(α)pyrene, ellipticine, and cis-parinaric acid to β-lactoglobulin − Influence of protein modifications. J. Protein Chem. 1992, 11, 645−652. (35) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF chimera - A visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605−1612. (36) Guzzi, R.; Rizzuti, B.; Bartucci, R. Dynamics and binding affinity of spin-labeled stearic acids in β-lactoglobulin: Evidences from EPR spectroscopy and molecular dynamics simulation. J. Phys. Chem. B 2012, 116, 11608−11615. (37) Rizzuti, B.; Pantusa, M.; Guzzi, R. The role of lys525 on the head-group anchoring of fatty acids in the highest affinity binding site of albumin. Spectrosc.-Int. J. 2010, 24, 159−163. (38) Petitpas, I.; Bhattacharya, A. A.; Twine, S.; East, M.; Curry, S. Crystal structure analysis of warfarin binding to human serum albumin − Anatomy of drug site I. J. Biol. Chem. 2001, 276, 22804−22809. (39) Trott, O.; Olson, A. J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010, 31, 455−61. (40) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 2008, 4, 435−447. (41) Hornak, V.; Abel, R.; Okur, A.; Strockbine, B.; Roitberg, A.; Simmerling, C. Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins 2006, 65, 712−725. (42) Wang, J. M.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. Development and testing of a general Amber force field. J. Comput. Chem. 2004, 25, 1157−1174. (43) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983, 79, 926−935. (44) Darden, T.; York, D.; Pedersen, L. Particle mesh Ewald: An N•log(N) method for Ewald sums in large systems. J. Chem. Phys. 1993, 98, 10089−10092.

(45) Bussi, G.; Donadio, D.; Parrinello, M. Canonical sampling through velocity rescaling. J. Chem. Phys. 2007, 126, 014101−014108. (46) Berendsen, H. J. C.; Postma, J. P. M.; Di Nola, A.; Haak, J. R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 1984, 81, 3684−3690. (47) Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual Molecular Dynamics. J. Mol. Graph. Model. 1996, 14, 33−38. (48) Goldberg, D. M.; Ng, E.; Karumanchiri, A.; Yan, J.; Diamandis, E. P.; Soleas, G. J. Assay of Resveratrol glucosides and isomers in wine by direct-injection high-performance liquid-chromatography. J. Chromatogr. A 1995, 708, 89−98. (49) Lopez-Nicolas, J. M.; Garcia-Carmona, F. Aggregation state and pKa values of (E)-resveratrol as determined by fluorescence spectroscopy and UV-visible absorption. J. Agric. Food Chem. 2008, 56, 7600− 7605. (50) Figueiras, T. S.; Neves-Petersen, M. T.; Petersen, S. B. Activation energy of light induced isomerization of resveratrol. J. Fluoresc. 2011, 21, 1897−1906. (51) Mousavi, S. H. A.; Bordbar, A. K.; Haertle, T. Changes in structure and in interactions of heat-treated bovine beta-lactoglobulin. Protein Pept. Lett. 2008, 15, 818−825. (52) Qvist, J.; Davidovic, M.; Hamelberg, D.; Halle, B. A dry ligandbinding cavity in a solvated protein. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 6296−6301. (53) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.; Olson, A. J. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem. 1998, 19, 1639−1662. (54) van der Spoel, D.; Berendsen, H. J. C. Molecular dynamics simulations of Leu-enkephalin in water and DMSO. Biophys. J. 1997, 72, 2032−2041. (55) De Simone, F.; Guzzi, R.; Sportelli, L.; Marsh, D.; Bartucci, R. Electron spin-echo studies of spin-labelled lipid membranes and free fatty acids interacting with human serum albumin. BBA-Biomembranes 2007, 1768, 1541−1549. (56) Simard, J. R.; Zunszain, P. A.; Ha, C. E.; Yang, J. S.; Bhagavan, N. V.; Petitpas, I.; Curry, S.; Hamilton, J. A. Locating high-affinity fatty acid-binding sites on albumin by X-ray crystallography and NMR spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17958−17963.

4391


Stability of trans-Resveratrol Associated with Transport Proteins

Recommend Documents