Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 46–51
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Binding of helicid to human serum albumin: A hybrid spectroscopic approach and conformational study Yuanyuan Yue a,b, Jianming Liu b, Ren Liu b, Qiao Dong b, Jing Fan a,⇑ a b
School of Environment, Henan Normal University, Xinxiang, Henan 453007, PR China School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, Henan 453007, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The interaction of helicid with HSA
The Scatchard plots for the fluorescence quenching of HSA in the presence of helicid and molecular docking analysis of HSA with helicid.
was investigated. Helicid was located in the subdomain IIA of HSA. Binding parameters are important for understanding toxicity of helicid.
a r t i c l e
i n f o
Article history: Received 29 November 2013 Received in revised form 23 December 2013 Accepted 30 December 2013 Available online 8 January 2014 Keywords: Helicid Human serum albumin Fluorescence Three-dimensional fluorescence Molecular modeling
a b s t r a c t The interaction between human serum albumin and helicid was studied by steady-state fluorescence, ultraviolet–visible, circular dichroism, Fourier transform infrared techniques and molecular modeling. The binding site numbers, association constants, and corresponding thermodynamic parameters were used to investigate the quenching mechanism. The alternations of protein secondary structure in the presence of helicid were demonstrated using synchronous fluorescence, Fourier transform infrared, circular dichroism and three-dimensional fluorescence spectra. The molecular modeling results revealed that helicid could bind to hydrophobic pocket of HSA with hydrophobic and hydrogen bond force. The binding site of helicid in HSA was ascertained. Moreover, an apparent distance of 3.33 nm between the Trp214 and helicid was obtained via fluorescence resonance energy transfer method. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Helicia nilagirica Bedd, as a traditional Chinese herb, has been used for thousands of years to cure headache and insomnia in China [1]. Helicid, extracted from Helicia nilagirica Bedd, is one of the main constituents to treat neurasthenia, neurasthenia syndrome, ⇑ Corresponding author. Tel.: +86 373 3325805; fax: +86 373 3326445. E-mail address: [email protected] (J. Fan). 1386-1425/$ - see front matter Ó 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.12.108
and vascular headache. The clinical research indicated that helicid plays a neuroprotective role by affecting the excitotoxicity, nitric monoxide (NO) system, neuroglia, biomembrane, oxidative neurotoxicity, apoptosis [2–6]. Because of high efficacy and low toxicity, helicid is widely used in many synthetic drugs and have been successfully marketed. Human serum albumin (HSA) is the most abundant protein in serum and plasma. It is capable of binding to a wide variety of endogenous and exogenous compounds, such as fatty acids,
Y. Yue et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 46–51
hormones, and drugs, performing as carrier and transporter [7]. Owing to advantageous biochemical and pharmacological properties of HSA, HSA is playing an increasing role as a drug carrier in the clinical setting [8]. Therefore, the knowledge of interaction mechanisms between drug and HSA is very important for drug development. HSA is the best-studied model to understand the helicid delivery process. The present work focused on the molecular mechanism of helicid–HSA interactions by multi-spectroscopic methods including fluorescence, UV–vis absorption, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. The binding mechanism of helicid–HSA was investigated according to the fluorescence data. The CD, FT-IR, 3D and synchronous fluorescence were employed to investigate the secondary structure changes of HSA caused by helicid. The distance estimates from fluorescence resonance energy transfer (FRET) results suggested complex formed. These results are expected to provide some useful information for further discussing the toxicology of helicid.
47
at 220–500 nm, the initial excitation wavelength was set to 220 nm with increments of 5 nm, scanning number 27 with other parameters just the same as that of the steady-state fluorescence spectra. Fourier transform infrared spectroscopy The instrument was a Tensor 27 FT-IR spectrometer (Bruker, German) equipped with a Germanium attenuated total reflection (ATR) accessory, a DTGS KBr detector and a KBr beam splitter. All spectra were taken via the ATR method with a resolution of 4 cm1 using 64 scans. Samples measured in buffer. Spectra processing procedures: spectra of buffer were collected under the same conditions. Then, the absorbance of the buffer solution was subtracted from the spectra of the sample solution to obtain the FT-IR spectra of the protein. The subtraction criterion was that the original spectrum of protein solution between 2200 and 1800 cm1 was featureless; that is, no characteristic peak between 2200 and 1800 cm1 appear, and the curve is flatness [9].
Materials and methods Circular dichroism spectroscopy Materials Fatty acid free HSA was purchased from Sigma. Helicid was purchased from the National Institute for Control of Pharmaceutical and Bioproducts (China). The stock solution of HSA (3.0 105 M) was prepared in Tris–HCl buffer of pH 7.4. The stock solution (1.0 103 M) of helicid was prepared in double-distilled water. All other chemicals were obtained from Sigma–Aldrich. Ultrapure water from a Milli-Q ultrapure water purification system was used throughout the experiments. All pH values were measured with a pH-3 digital pH meter (Shanghai Lei Ci Device Works, Shanghai, China) with a combined glass electrode, which was calibrated with standard pH buffer solutions. Thermo/HAAKE DC30-K20 refrigerated circulator bath (±0.01 °C accuracy) was used to control the temperature of the samples.
CD spectra were obtained on a Jasco-810 model spectropolarimeter using a quartz cell with 0.1 cm path length at 298 K. CD measurements were carried out in the range of 200–250 nm and CD spectra were collected with the scan speed of 20 nm/min. Each sample was scanned three times at a bandwidth of 1.0 nm. The buffer solution was used as a blank and was automatically subtracted from the samples during scanning. CD results are expressed as molar ellipticity h. UV–vis absorbance Absorption spectra were obtained with a UV-1700 PharmaSpec (Shimadzu, Japan) at 298 K across 200–500 nm using a 1 cm quartz cell.
Steady-state fluorescence spectroscopy
Docking
The fluorescence spectra at 298 K were recorded using an FP-6500 spectrofluorometer (JASCO, Japan). The excitation wavelength was 280 nm. Both the excitation and emission slit widths were set at 5 nm. The emission spectra were recorded between 300 and 450 nm. Triplicate samples were measured. Each fluorescence spectrum of the protein in presence of different ligand concentrations were corrected for any possible inner filter effect using the following equation:
Ligand docking calculations were performed using version 6.9 of the Sybyl program [10]. As receptor structure, the structures of HSA (entry codes 1H9Z) were extracted from the Brookhaven Protein Data Bank (http://www.rcsb.org/pdb) and used with deleted H2O molecules [11]. Hydrogens were added to all ligands and the receptor prior to performing the docking runs. Ligand docking of
Icorr F ðkE ; kF Þ ¼ IF ðkE ; kF Þ
AðkE Þ Atot ðkE Þ
ð1Þ
where A represents the absorbance of the free protein, and Atot is the total absorbance of the solution at the excitation wavelength (kE). The intensity of fluorescence used in this paper is the corrected fluorescence intensity. Synchronous fluorescence spectroscopy Synchronous fluorescence spectra of HSA in the absence and presence of increasing amount of helicid were measured under the same conditions with steady-state fluorescence spectra. Three-dimensional fluorescence spectroscopy Three-dimensional fluorescence spectra were recorded under the following conditions: the emission wavelength was recorded
Fig. 1. Fluorescence emission spectra of tested helicid–HSA systems. (pH 7.40, T = 298 K): (a) 3.0 lM HSA; (b)–(j) 3.0 lM HSA in the presence of 3.32, 6.62, 9.90, 13.16, 16.39, 19.61 lM helicid. The inset shows the structure of helicid.
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Y. Yue et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 46–51
helicid molecules was conducted using the Lamarckian Genetic Algorithm with standard settings. FlexX program was applied to calculate the interaction mode between helicid and HSA. During the docking process, a maximum of 10 conformers were considered for the molecule. The conformation with the lowest energy was used for final analysis. Result and discussion
DG0 ¼ DH0 T DS0
ð4Þ
K is the binding constant at temperature T and R is gas constant. The thermodynamic parameters determined from linear Van’t Hoff plot (Fig. 2) were presented in Table 1. It can be seen that DG0 < 0, DH0 < 0 and DS0 > 0 indicated that the binding process was spontaneous, both hydrophobic interactions and hydrogen bonds played a major role in the reaction between helicid and HSA [15].
Fluorescence quenching of HSA by helicid
Molecular docking analysis
The fluorescence emission spectra of HSA spiked with various amounts of helicid were shown in Fig. 1. When the solution was excited at 280 nm, HSA displayed a strong fluorescence emission peak at 333 nm. The addition of helicid led to a distinct decrease of the fluorescence signal. These data indicated that helicid interacted with HSA and quenched its intrinsic fluorescence [12]. There are mainly two types of quenching mechanisms in solution. Dynamic quenching and static quenching are caused by diffusion and ground-state complex formation, respectively, and can be distinguished by their differing dependences on temperature and viscosity [13]. For dynamic quenching, higher temperatures result in faster diffusion and larger amounts of collision quenching, hence, the quenching constant values increase with increasing temperature. On the opposite side, static quenching will reduce the complex stability and decrease the quenching rate constants with increasing temperature [14]. To elaborate the fluorescence quenching mechanism, the fluorescence quenching data at different temperatures was utilized with the Scatchard equation
Conformational changes that HSA loading of helicid have been identified. HSA has three structurally homologous domains (I–III), each domain has two subdomains (A and B) possessing common structural elements, with six a-helices in subdomain A and four a-helices in subdomain B [16]. It is important to note that Trp214 is in subdomain IIA. Docking results for HSA from software packages were listed in Fig. 3 and Table 2. As shown in Fig. 3, the helicid molecule was surrounded by the hydrophobic residues, such as Ala291, Ala213, Ala258, Leu219, Leu238, Leu260, Phe211, Val216, Val241, Trp214. Therefore, it suggested that hydrophobic force was the main interaction force in the binding of helicid to HSA, which was supported by the thermodynamic analysis. Hydrogen bonds were also observed near the probe molecule, the hydrogen bond distances between interacting atoms of the amino acid residues of HSA and helicid were listed in Table 2. The results indicated that the formation of hydrogen bond decreased the hydrophilicity to stability in the HSA–helicid system [17]. From the docking simulation the observed free energy change of binding (DG0) for the complex HSA–helicid was found to be 17.614 kJ/ mol, which was not very close to the experimental data (24.25 kJ/mol, 296 K) [18]. The difference between experimental and theoretical results may be due to X-ray structure of the protein from crystals differs from that of the aqueous system used in this study.
r=Df ¼ nK rK
ð2Þ
r represents the number of moles of bound small molecules per mole of protein, Df represents the molar concentration of free helicid, n is binding site multiplicity per class of binding site and K is the association binding constant. A plot of (r/Df)/r is shown in Fig. 2. The good linearity plot indicated a single class binding site for helicid– HSA. The binding constants K and the number of binding sites (n) at different temperatures were listed in Table 1. The binding constant K deceased with an increase in temperature, it can be concluded that here quenching was primarily of static type. In order to characterize the acting forces between HSA and helicid, a thermodynamic process was considered to be responsible, which can be obtained by the Van’t Hoff equation. The following equations were employed:
ln K ¼ DH0 =RT þ DS0 =R
ð3Þ
Fig. 2. The Scatchard plots for the fluorescence quenching of HSA in the presence of helicid. The inset shows the van’t Hoff plot for helicid–HSA interaction.
Analysis of HSA conformation after helicid binding Synchronous fluorescence spectroscopy (SFS) is a rapid, sensitive and nondestructive method suitable for the analysis of changes in microenvironment around the chromophore [19]. The intrinsic fluorescence of albumins results from Trp and Tyr residues, while the spectra of free amino acid (Trp and Tyr) residues are overlap [20]. For the SFS technique, the selection of wavelength interval is one of the most important experimental parameter. When the Dk was fixed at 60 nm, the SFS gives the characteristic information of Trp residues [21]. The SFS of HSA in the presence of helicid were shown in Fig. 4. The results implied that helicid quenched the HSA fluorescence spectrum mainly by quenching the Trp residue. The addition of helicid to the HSA leaded to slight red shift of Trp fluorescence peak, indicating that the helicid changed the hydrophobic property of the microenvironment where residues located, which changed the HSA conformation. FT-IR spectroscopy is employed to characterize the secondary structure of proteins. IR spectra of proteins exhibit a number of amide bands, which represent different vibrations of the peptide moiety. Among the amide bands of the protein, the amide I band (1700–1600 cm1, mainly C@O stretch) and amide II band (1600–1500 cm1, CAN stretch coupled with NAH bending mode) both have a relationship with the secondary structure of protein, and the amide I band is more sensitive to the change of protein secondary structure than the amide II band [22]. The effects of helicid on structural conformation of HSA were evaluated by FT-IR spectra (Fig. 5). When HSA was titrated with helicid, the amide I peak at 1643.08 cm1 shifted to 1648.86 cm1 and the amide II at 1546.66 cm1 shifted to 1544.73 cm1. The spectra indicated that
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Y. Yue et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 46–51 Table 1 Binding and thermodynamic characteristics of the helicid–HSA system.
a
T (K)
K (104 M1)
Ra
n
DG0 (kJ mol1)
DS0 (J mol1 K1)
DH0 (kJ mol1)
289 296 303
2.25 1.99 1.55
0.9928 0.9963 0.9989
1.08 1.14 1.34
24.13 24.25 24.36
16.54
19.35
The correlation coefficient.
Fig. 4. The effect of helicid on the synchronous fluorescence spectra of HSA. (a) 3.0 lM HSA and (b–j) 3.0 lM HSA in the presence of 3.32, 6.62, 9.90, 13.16, 16.39, 19.61 lM helicid, Dk = 60 nm, pH = 7.40.
a helix% ¼ ð½h208 4000Þ=ð33000 4000Þ 100 Fig. 3. Docking poses of helicid with HSA. The residues of HSA are represented using gray ball and stick model and the helicid structure is represented by a green one. The hydrogen bond between helicid and HSA is represented using yellow dashed line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 2 Hydrogen bond distance and free energy change of binding between interacting atoms of the amino acid residues of HSA and helicid. H-bond interaction
H-bond interaction (Å)
DG0 (kJ mol1)
ARG222 ARG222 ARG222 ARG291
1.954 2.244 1.964 1.757
17.614
helicid interacted with both the C@O and CAN groups in the protein polypeptides and caused the rearrangement of the polypeptide carbonyl hydrogen bonding network [23]. A quantitative analysis of the HSA secondary structure for the free HSA and helicid added have been carried out. Fig. 6 exhibited the curve-fitted spectra of HSA infrared amide I bands. According to the curve-fit results, the a-helix structure content decreased from 48.2% to 44.6%, the b-sheet structure content of the HSA increased from 21.2% to 26.5%, the b-turn structure content increased from 23.3% to 25.0%. FTIR data indicated that binding with helicid significantly affected the structural conformation of HSA. The CD spectrum of HSA in the absence and presence of helicid were shown in Fig. 7. As shown in Fig. 7, the CD spectra of the HSA exhibited two negative bands in the ultraviolet region at 208 and 220 nm, which is a characteristic of a-helical structure of protein [24], the band intensity of HSA at 208 and 220 nm reduced indicating the changes in the protein secondary structure with the addition of helicid.
ð5Þ
From the above Eq. (5), the calculated results exhibit a reduction of the a-helical structure from 53.2% to 48.3%. These observations supported the fact that binding caused the conformational changes in the protein [25]. It is well-known that three dimensional (3D) fluorescence spectroscopy is scientific and credible to investigate the conformational change in protein [26]. When there is a shift at the Ex or Em wavelength around the fluorescence peak, or the appearance of a new peak or disappearance of existing peak, it suggest conformational changes in the protein [27]. Fig. 8 showed the 3D fluorescence spectral changes in the absence and presence of helicid. The peak a shown in the figure is the Rayleigh scattering peak. Whereas the strong peak b mainly reveals the spectral behavior of tryptophan, and the maximum emission wavelength and the fluorescence intensity of the residue associated with microenvironment polarity. Besides peak a or b, there is another strong fluorescence peak c, the second-ordered scattering peak (2kex = kem). Analysis from Fig. 8, the intensity of peak a or b decreased obviously but to different degree after the addition of helicid. The observed was attributed to the interaction of helicid with HSA induced conformational changes in HSA [28]. Energy transfer between HSA and helicid In order to determine the spatial distance between two points (a donor and an acceptor) in proteins, it is possible to use Förster’s nonradiative energy transfer (FRET) technique [29]. FRET technique has been successfully used to probe biological systems to study the structure, conformation, spatial distribution and assembly of complex proteins [30]. The efficiency (E) of energy transfer between the donor and the acceptor could be calculated by the follow equations,
E ¼ 1 F=F 0 ¼ R60 =ðR60 þ r 6 Þ
ð6Þ
50
Y. Yue et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 46–51
Fig. 5. FT-IR spectra of HSA (A) and FT-IR difference spectrum of HSA obtained by subtracting the spectrum of the helicid-free form from that of the helicid–HSA form (B) at 298 K. Tris–HCl buffer (pH 7.40); CHSA = 3.0 lM; Chelicid = 6.0 lM.
Fig. 6. Curve-fitted amide I region (1700–1600 cm1) of free HSA (A) and its helicid complex (B).
Fig. 7. CD spectra of HSA (3 lM) in the presence of helicid: 0 (a) and 6 lM (b).
R60 ¼ 8:79 1025 K 2 N4 U J J¼
X
X FðkÞeðkÞk4 Dk = FðkÞDk
ð7Þ ð8Þ
where F0 and F are the fluorescence intensities of HSA before and after addition of quencher, respectively. r is the binding distance between donor and receptor and R0 is the critical distance when the efficiency of excitation energy transferred to the acceptor is 50%; K2 is the spatial orientation factor of the dipole; n is the refractive index of the medium; U is the fluorescence quantum yield of the donor; and J is the overlap integral of the fluorescence emission spectrum of the donor with the absorption spectrum of the acceptor; F(k) is the corrected fluorescence intensity of the donor in the
Fig. 8. The three-dimensional fluorescence spectra of HSA (A) and helicid–HSA (B). HSA–helicid (1:2); C(HSA) = 3 lM, pH = 7.40.
wavelength range from k to k + Dk; e(k) is the extinction coefficient of the acceptor at k. From the overlapping of the absorption spectra of helicid and the emission spectra of HSA (Fig. 9), J can be
Y. Yue et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 46–51
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Acknowledgements This research was supported by the National Nature Science Foundation of China (Nos. 21103044 and 21205029), Ph.D. Programs Foundation of Ministry of Education of China (Nos. 20114104120003 and 20124104120004), and Foundation of Henan Educational Committee (12B150013). References
Fig. 9. The overlap of UV absorption spectrum of helicid with the fluorescence emission spectrum of HSA. The fluorescence emission spectrum of HSA (a) and the UV absorption spectrum of helicid (b). C(HSA) = C(helicid) = 3.32 lM.
calculated by integrating the spectra and the calculated J values was 5.64 1015 cm3 L mol1 for HSA. In our present case, K2 = 2/3, n = 1.336, U = 0.118 [31]. Hence, from Eqs. (6)–(8), we could calculate that R0 = 2.23 nm; E = 0.81 and r = 3.33 nm. Obviously, the donor (tryptophan residues of the HSA) to acceptor (helicid) distance was less than 8 nm, indicating an interaction between helicid and Trp-214 [32]. These data suggested that the energy transfer from HSA to helicid could occur with high probability [33]. Conclusions In summary, we have explored the interaction of helicid with HSA by optical spectroscopy technique (steady-state fluorescence, UV–vis absorption, CD, FT-IR, 3D fluorescence) and molecular modeling. Experimental results suggested that helicid could bind with HSA and quench the fluorescence of HSA. In addition to thermodynamic parameters, the values of binding constant and the number of binding sites of the helicid–HSA system were determined. The SFS, 3D fluorescence, CD and FT-IR results showed that the binding of helicid to HSA induced conformational changes of HSA. It was found that the hydrophobic interactions and hydrogen bond forces played a major role in the binding of helicid to HSA. The average binding distance between donor and acceptor molecules was calculated from FRET theory and found to be 3.33 nm for HSA–helicid systems. Additionally, docking calculations found helicid to be located in the hydrophobic pocket of HSA within subdomain IIA. The present study provides significant information for understanding of the process of helicid transportation in vivo and expected to provide some useful information for research the toxicology of helicid.
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Binding of helicid to human serum albumin: A hybrid spectroscopic approach and conformational study Yuanyuan Yue a,b, Jianming Liu b, Ren Liu b, Qiao Dong b, Jing Fan a,⇑ a b
School of Environment, Henan Normal University, Xinxiang, Henan 453007, PR China School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, Henan 453007, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The interaction of helicid with HSA
The Scatchard plots for the fluorescence quenching of HSA in the presence of helicid and molecular docking analysis of HSA with helicid.
was investigated. Helicid was located in the subdomain IIA of HSA. Binding parameters are important for understanding toxicity of helicid.
a r t i c l e
i n f o
Article history: Received 29 November 2013 Received in revised form 23 December 2013 Accepted 30 December 2013 Available online 8 January 2014 Keywords: Helicid Human serum albumin Fluorescence Three-dimensional fluorescence Molecular modeling
a b s t r a c t The interaction between human serum albumin and helicid was studied by steady-state fluorescence, ultraviolet–visible, circular dichroism, Fourier transform infrared techniques and molecular modeling. The binding site numbers, association constants, and corresponding thermodynamic parameters were used to investigate the quenching mechanism. The alternations of protein secondary structure in the presence of helicid were demonstrated using synchronous fluorescence, Fourier transform infrared, circular dichroism and three-dimensional fluorescence spectra. The molecular modeling results revealed that helicid could bind to hydrophobic pocket of HSA with hydrophobic and hydrogen bond force. The binding site of helicid in HSA was ascertained. Moreover, an apparent distance of 3.33 nm between the Trp214 and helicid was obtained via fluorescence resonance energy transfer method. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Helicia nilagirica Bedd, as a traditional Chinese herb, has been used for thousands of years to cure headache and insomnia in China [1]. Helicid, extracted from Helicia nilagirica Bedd, is one of the main constituents to treat neurasthenia, neurasthenia syndrome, ⇑ Corresponding author. Tel.: +86 373 3325805; fax: +86 373 3326445. E-mail address: [email protected] (J. Fan). 1386-1425/$ - see front matter Ó 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.12.108
and vascular headache. The clinical research indicated that helicid plays a neuroprotective role by affecting the excitotoxicity, nitric monoxide (NO) system, neuroglia, biomembrane, oxidative neurotoxicity, apoptosis [2–6]. Because of high efficacy and low toxicity, helicid is widely used in many synthetic drugs and have been successfully marketed. Human serum albumin (HSA) is the most abundant protein in serum and plasma. It is capable of binding to a wide variety of endogenous and exogenous compounds, such as fatty acids,
Y. Yue et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 46–51
hormones, and drugs, performing as carrier and transporter [7]. Owing to advantageous biochemical and pharmacological properties of HSA, HSA is playing an increasing role as a drug carrier in the clinical setting [8]. Therefore, the knowledge of interaction mechanisms between drug and HSA is very important for drug development. HSA is the best-studied model to understand the helicid delivery process. The present work focused on the molecular mechanism of helicid–HSA interactions by multi-spectroscopic methods including fluorescence, UV–vis absorption, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. The binding mechanism of helicid–HSA was investigated according to the fluorescence data. The CD, FT-IR, 3D and synchronous fluorescence were employed to investigate the secondary structure changes of HSA caused by helicid. The distance estimates from fluorescence resonance energy transfer (FRET) results suggested complex formed. These results are expected to provide some useful information for further discussing the toxicology of helicid.
47
at 220–500 nm, the initial excitation wavelength was set to 220 nm with increments of 5 nm, scanning number 27 with other parameters just the same as that of the steady-state fluorescence spectra. Fourier transform infrared spectroscopy The instrument was a Tensor 27 FT-IR spectrometer (Bruker, German) equipped with a Germanium attenuated total reflection (ATR) accessory, a DTGS KBr detector and a KBr beam splitter. All spectra were taken via the ATR method with a resolution of 4 cm1 using 64 scans. Samples measured in buffer. Spectra processing procedures: spectra of buffer were collected under the same conditions. Then, the absorbance of the buffer solution was subtracted from the spectra of the sample solution to obtain the FT-IR spectra of the protein. The subtraction criterion was that the original spectrum of protein solution between 2200 and 1800 cm1 was featureless; that is, no characteristic peak between 2200 and 1800 cm1 appear, and the curve is flatness [9].
Materials and methods Circular dichroism spectroscopy Materials Fatty acid free HSA was purchased from Sigma. Helicid was purchased from the National Institute for Control of Pharmaceutical and Bioproducts (China). The stock solution of HSA (3.0 105 M) was prepared in Tris–HCl buffer of pH 7.4. The stock solution (1.0 103 M) of helicid was prepared in double-distilled water. All other chemicals were obtained from Sigma–Aldrich. Ultrapure water from a Milli-Q ultrapure water purification system was used throughout the experiments. All pH values were measured with a pH-3 digital pH meter (Shanghai Lei Ci Device Works, Shanghai, China) with a combined glass electrode, which was calibrated with standard pH buffer solutions. Thermo/HAAKE DC30-K20 refrigerated circulator bath (±0.01 °C accuracy) was used to control the temperature of the samples.
CD spectra were obtained on a Jasco-810 model spectropolarimeter using a quartz cell with 0.1 cm path length at 298 K. CD measurements were carried out in the range of 200–250 nm and CD spectra were collected with the scan speed of 20 nm/min. Each sample was scanned three times at a bandwidth of 1.0 nm. The buffer solution was used as a blank and was automatically subtracted from the samples during scanning. CD results are expressed as molar ellipticity h. UV–vis absorbance Absorption spectra were obtained with a UV-1700 PharmaSpec (Shimadzu, Japan) at 298 K across 200–500 nm using a 1 cm quartz cell.
Steady-state fluorescence spectroscopy
Docking
The fluorescence spectra at 298 K were recorded using an FP-6500 spectrofluorometer (JASCO, Japan). The excitation wavelength was 280 nm. Both the excitation and emission slit widths were set at 5 nm. The emission spectra were recorded between 300 and 450 nm. Triplicate samples were measured. Each fluorescence spectrum of the protein in presence of different ligand concentrations were corrected for any possible inner filter effect using the following equation:
Ligand docking calculations were performed using version 6.9 of the Sybyl program [10]. As receptor structure, the structures of HSA (entry codes 1H9Z) were extracted from the Brookhaven Protein Data Bank (http://www.rcsb.org/pdb) and used with deleted H2O molecules [11]. Hydrogens were added to all ligands and the receptor prior to performing the docking runs. Ligand docking of
Icorr F ðkE ; kF Þ ¼ IF ðkE ; kF Þ
AðkE Þ Atot ðkE Þ
ð1Þ
where A represents the absorbance of the free protein, and Atot is the total absorbance of the solution at the excitation wavelength (kE). The intensity of fluorescence used in this paper is the corrected fluorescence intensity. Synchronous fluorescence spectroscopy Synchronous fluorescence spectra of HSA in the absence and presence of increasing amount of helicid were measured under the same conditions with steady-state fluorescence spectra. Three-dimensional fluorescence spectroscopy Three-dimensional fluorescence spectra were recorded under the following conditions: the emission wavelength was recorded
Fig. 1. Fluorescence emission spectra of tested helicid–HSA systems. (pH 7.40, T = 298 K): (a) 3.0 lM HSA; (b)–(j) 3.0 lM HSA in the presence of 3.32, 6.62, 9.90, 13.16, 16.39, 19.61 lM helicid. The inset shows the structure of helicid.
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Y. Yue et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 46–51
helicid molecules was conducted using the Lamarckian Genetic Algorithm with standard settings. FlexX program was applied to calculate the interaction mode between helicid and HSA. During the docking process, a maximum of 10 conformers were considered for the molecule. The conformation with the lowest energy was used for final analysis. Result and discussion
DG0 ¼ DH0 T DS0
ð4Þ
K is the binding constant at temperature T and R is gas constant. The thermodynamic parameters determined from linear Van’t Hoff plot (Fig. 2) were presented in Table 1. It can be seen that DG0 < 0, DH0 < 0 and DS0 > 0 indicated that the binding process was spontaneous, both hydrophobic interactions and hydrogen bonds played a major role in the reaction between helicid and HSA [15].
Fluorescence quenching of HSA by helicid
Molecular docking analysis
The fluorescence emission spectra of HSA spiked with various amounts of helicid were shown in Fig. 1. When the solution was excited at 280 nm, HSA displayed a strong fluorescence emission peak at 333 nm. The addition of helicid led to a distinct decrease of the fluorescence signal. These data indicated that helicid interacted with HSA and quenched its intrinsic fluorescence [12]. There are mainly two types of quenching mechanisms in solution. Dynamic quenching and static quenching are caused by diffusion and ground-state complex formation, respectively, and can be distinguished by their differing dependences on temperature and viscosity [13]. For dynamic quenching, higher temperatures result in faster diffusion and larger amounts of collision quenching, hence, the quenching constant values increase with increasing temperature. On the opposite side, static quenching will reduce the complex stability and decrease the quenching rate constants with increasing temperature [14]. To elaborate the fluorescence quenching mechanism, the fluorescence quenching data at different temperatures was utilized with the Scatchard equation
Conformational changes that HSA loading of helicid have been identified. HSA has three structurally homologous domains (I–III), each domain has two subdomains (A and B) possessing common structural elements, with six a-helices in subdomain A and four a-helices in subdomain B [16]. It is important to note that Trp214 is in subdomain IIA. Docking results for HSA from software packages were listed in Fig. 3 and Table 2. As shown in Fig. 3, the helicid molecule was surrounded by the hydrophobic residues, such as Ala291, Ala213, Ala258, Leu219, Leu238, Leu260, Phe211, Val216, Val241, Trp214. Therefore, it suggested that hydrophobic force was the main interaction force in the binding of helicid to HSA, which was supported by the thermodynamic analysis. Hydrogen bonds were also observed near the probe molecule, the hydrogen bond distances between interacting atoms of the amino acid residues of HSA and helicid were listed in Table 2. The results indicated that the formation of hydrogen bond decreased the hydrophilicity to stability in the HSA–helicid system [17]. From the docking simulation the observed free energy change of binding (DG0) for the complex HSA–helicid was found to be 17.614 kJ/ mol, which was not very close to the experimental data (24.25 kJ/mol, 296 K) [18]. The difference between experimental and theoretical results may be due to X-ray structure of the protein from crystals differs from that of the aqueous system used in this study.
r=Df ¼ nK rK
ð2Þ
r represents the number of moles of bound small molecules per mole of protein, Df represents the molar concentration of free helicid, n is binding site multiplicity per class of binding site and K is the association binding constant. A plot of (r/Df)/r is shown in Fig. 2. The good linearity plot indicated a single class binding site for helicid– HSA. The binding constants K and the number of binding sites (n) at different temperatures were listed in Table 1. The binding constant K deceased with an increase in temperature, it can be concluded that here quenching was primarily of static type. In order to characterize the acting forces between HSA and helicid, a thermodynamic process was considered to be responsible, which can be obtained by the Van’t Hoff equation. The following equations were employed:
ln K ¼ DH0 =RT þ DS0 =R
ð3Þ
Fig. 2. The Scatchard plots for the fluorescence quenching of HSA in the presence of helicid. The inset shows the van’t Hoff plot for helicid–HSA interaction.
Analysis of HSA conformation after helicid binding Synchronous fluorescence spectroscopy (SFS) is a rapid, sensitive and nondestructive method suitable for the analysis of changes in microenvironment around the chromophore [19]. The intrinsic fluorescence of albumins results from Trp and Tyr residues, while the spectra of free amino acid (Trp and Tyr) residues are overlap [20]. For the SFS technique, the selection of wavelength interval is one of the most important experimental parameter. When the Dk was fixed at 60 nm, the SFS gives the characteristic information of Trp residues [21]. The SFS of HSA in the presence of helicid were shown in Fig. 4. The results implied that helicid quenched the HSA fluorescence spectrum mainly by quenching the Trp residue. The addition of helicid to the HSA leaded to slight red shift of Trp fluorescence peak, indicating that the helicid changed the hydrophobic property of the microenvironment where residues located, which changed the HSA conformation. FT-IR spectroscopy is employed to characterize the secondary structure of proteins. IR spectra of proteins exhibit a number of amide bands, which represent different vibrations of the peptide moiety. Among the amide bands of the protein, the amide I band (1700–1600 cm1, mainly C@O stretch) and amide II band (1600–1500 cm1, CAN stretch coupled with NAH bending mode) both have a relationship with the secondary structure of protein, and the amide I band is more sensitive to the change of protein secondary structure than the amide II band [22]. The effects of helicid on structural conformation of HSA were evaluated by FT-IR spectra (Fig. 5). When HSA was titrated with helicid, the amide I peak at 1643.08 cm1 shifted to 1648.86 cm1 and the amide II at 1546.66 cm1 shifted to 1544.73 cm1. The spectra indicated that
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a
T (K)
K (104 M1)
Ra
n
DG0 (kJ mol1)
DS0 (J mol1 K1)
DH0 (kJ mol1)
289 296 303
2.25 1.99 1.55
0.9928 0.9963 0.9989
1.08 1.14 1.34
24.13 24.25 24.36
16.54
19.35
The correlation coefficient.
Fig. 4. The effect of helicid on the synchronous fluorescence spectra of HSA. (a) 3.0 lM HSA and (b–j) 3.0 lM HSA in the presence of 3.32, 6.62, 9.90, 13.16, 16.39, 19.61 lM helicid, Dk = 60 nm, pH = 7.40.
a helix% ¼ ð½h208 4000Þ=ð33000 4000Þ 100 Fig. 3. Docking poses of helicid with HSA. The residues of HSA are represented using gray ball and stick model and the helicid structure is represented by a green one. The hydrogen bond between helicid and HSA is represented using yellow dashed line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 2 Hydrogen bond distance and free energy change of binding between interacting atoms of the amino acid residues of HSA and helicid. H-bond interaction
H-bond interaction (Å)
DG0 (kJ mol1)
ARG222 ARG222 ARG222 ARG291
1.954 2.244 1.964 1.757
17.614
helicid interacted with both the C@O and CAN groups in the protein polypeptides and caused the rearrangement of the polypeptide carbonyl hydrogen bonding network [23]. A quantitative analysis of the HSA secondary structure for the free HSA and helicid added have been carried out. Fig. 6 exhibited the curve-fitted spectra of HSA infrared amide I bands. According to the curve-fit results, the a-helix structure content decreased from 48.2% to 44.6%, the b-sheet structure content of the HSA increased from 21.2% to 26.5%, the b-turn structure content increased from 23.3% to 25.0%. FTIR data indicated that binding with helicid significantly affected the structural conformation of HSA. The CD spectrum of HSA in the absence and presence of helicid were shown in Fig. 7. As shown in Fig. 7, the CD spectra of the HSA exhibited two negative bands in the ultraviolet region at 208 and 220 nm, which is a characteristic of a-helical structure of protein [24], the band intensity of HSA at 208 and 220 nm reduced indicating the changes in the protein secondary structure with the addition of helicid.
ð5Þ
From the above Eq. (5), the calculated results exhibit a reduction of the a-helical structure from 53.2% to 48.3%. These observations supported the fact that binding caused the conformational changes in the protein [25]. It is well-known that three dimensional (3D) fluorescence spectroscopy is scientific and credible to investigate the conformational change in protein [26]. When there is a shift at the Ex or Em wavelength around the fluorescence peak, or the appearance of a new peak or disappearance of existing peak, it suggest conformational changes in the protein [27]. Fig. 8 showed the 3D fluorescence spectral changes in the absence and presence of helicid. The peak a shown in the figure is the Rayleigh scattering peak. Whereas the strong peak b mainly reveals the spectral behavior of tryptophan, and the maximum emission wavelength and the fluorescence intensity of the residue associated with microenvironment polarity. Besides peak a or b, there is another strong fluorescence peak c, the second-ordered scattering peak (2kex = kem). Analysis from Fig. 8, the intensity of peak a or b decreased obviously but to different degree after the addition of helicid. The observed was attributed to the interaction of helicid with HSA induced conformational changes in HSA [28]. Energy transfer between HSA and helicid In order to determine the spatial distance between two points (a donor and an acceptor) in proteins, it is possible to use Förster’s nonradiative energy transfer (FRET) technique [29]. FRET technique has been successfully used to probe biological systems to study the structure, conformation, spatial distribution and assembly of complex proteins [30]. The efficiency (E) of energy transfer between the donor and the acceptor could be calculated by the follow equations,
E ¼ 1 F=F 0 ¼ R60 =ðR60 þ r 6 Þ
ð6Þ
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Fig. 5. FT-IR spectra of HSA (A) and FT-IR difference spectrum of HSA obtained by subtracting the spectrum of the helicid-free form from that of the helicid–HSA form (B) at 298 K. Tris–HCl buffer (pH 7.40); CHSA = 3.0 lM; Chelicid = 6.0 lM.
Fig. 6. Curve-fitted amide I region (1700–1600 cm1) of free HSA (A) and its helicid complex (B).
Fig. 7. CD spectra of HSA (3 lM) in the presence of helicid: 0 (a) and 6 lM (b).
R60 ¼ 8:79 1025 K 2 N4 U J J¼
X
X FðkÞeðkÞk4 Dk = FðkÞDk
ð7Þ ð8Þ
where F0 and F are the fluorescence intensities of HSA before and after addition of quencher, respectively. r is the binding distance between donor and receptor and R0 is the critical distance when the efficiency of excitation energy transferred to the acceptor is 50%; K2 is the spatial orientation factor of the dipole; n is the refractive index of the medium; U is the fluorescence quantum yield of the donor; and J is the overlap integral of the fluorescence emission spectrum of the donor with the absorption spectrum of the acceptor; F(k) is the corrected fluorescence intensity of the donor in the
Fig. 8. The three-dimensional fluorescence spectra of HSA (A) and helicid–HSA (B). HSA–helicid (1:2); C(HSA) = 3 lM, pH = 7.40.
wavelength range from k to k + Dk; e(k) is the extinction coefficient of the acceptor at k. From the overlapping of the absorption spectra of helicid and the emission spectra of HSA (Fig. 9), J can be
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Acknowledgements This research was supported by the National Nature Science Foundation of China (Nos. 21103044 and 21205029), Ph.D. Programs Foundation of Ministry of Education of China (Nos. 20114104120003 and 20124104120004), and Foundation of Henan Educational Committee (12B150013). References
Fig. 9. The overlap of UV absorption spectrum of helicid with the fluorescence emission spectrum of HSA. The fluorescence emission spectrum of HSA (a) and the UV absorption spectrum of helicid (b). C(HSA) = C(helicid) = 3.32 lM.
calculated by integrating the spectra and the calculated J values was 5.64 1015 cm3 L mol1 for HSA. In our present case, K2 = 2/3, n = 1.336, U = 0.118 [31]. Hence, from Eqs. (6)–(8), we could calculate that R0 = 2.23 nm; E = 0.81 and r = 3.33 nm. Obviously, the donor (tryptophan residues of the HSA) to acceptor (helicid) distance was less than 8 nm, indicating an interaction between helicid and Trp-214 [32]. These data suggested that the energy transfer from HSA to helicid could occur with high probability [33]. Conclusions In summary, we have explored the interaction of helicid with HSA by optical spectroscopy technique (steady-state fluorescence, UV–vis absorption, CD, FT-IR, 3D fluorescence) and molecular modeling. Experimental results suggested that helicid could bind with HSA and quench the fluorescence of HSA. In addition to thermodynamic parameters, the values of binding constant and the number of binding sites of the helicid–HSA system were determined. The SFS, 3D fluorescence, CD and FT-IR results showed that the binding of helicid to HSA induced conformational changes of HSA. It was found that the hydrophobic interactions and hydrogen bond forces played a major role in the binding of helicid to HSA. The average binding distance between donor and acceptor molecules was calculated from FRET theory and found to be 3.33 nm for HSA–helicid systems. Additionally, docking calculations found helicid to be located in the hydrophobic pocket of HSA within subdomain IIA. The present study provides significant information for understanding of the process of helicid transportation in vivo and expected to provide some useful information for research the toxicology of helicid.
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