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Glycerol induced stability enhancement and conformational changes of β-lactoglobulin
Food Chemistry 308 (2020) 125596
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Glycerol induced stability enhancement and conformational changes of βlactoglobulin Xiaoxia Chena,b, Haiyang Zhangc, Yacine Hemarb,d, Na Lie, Peng Zhoua,b,
T
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a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province 214122, China International Joint Research Laboratory for Functional Dairy Protein Ingredients, Jiangnan University, Wuxi, Jiangsu Province 214122, China c Department of Biological Science and Engineering, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 100083 Beijing, China d Riddet Institute, Palmerston North, New Zealand e National Center for Protein Sciences Shanghai, Chinese Academy of Sciences, Shanghai 201204, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: β-Lactoglobulin Glycerol Stability Molecular dynamics Small angle X-ray scattering
The protective mechanism of glycerol on β-lactoglobulin were studied in 0–60% glycerol solutions by experimental and molecular simulation approaches. Results showed that the stability of β-lactoglobulin increased with glycerol concentration, with little secondary structure changes induced by glycerol. The tertiary structure altered slightly with glycerol concentration, resulting in a stronger near UV circular dichroism signal and intrinsic tryptophan fluorescence quenching, indicating aromatic side chains closer to hydrophobic microenvironment. The Rg of β-lactoglobulin increased with glycerol concentration without dimer dissociation, due to expansion of the quaternary structures. Moreover, the flexibility (RMSF) of β-lactoglobulin decreased by glycerol. Distance between areas enclosing Asp33 and Arg40 from separate chains did not increase, suggesting possibly reinforced electrostatic attractions. In conclusion, the stabilization of β-lactoglobulin in glycerol solution is probably the comprehensive results of the decreased molecular flexibility, the strengthened hydrophobic interaction in individual chain, and the possibly reinforced electrostatic attractions between two chains of β-lactoglobulin.
1. Introduction Glycerol is an excellent cosolvent stabilizing protein, routinely used in pharmaceutical dosage formulation (Kamerzell, Esfandiary, Joshi, Middaugh, & Volkin, 2011), antibody preservation (Wakankar et al., 2010) and protein crystallization (Sedgwick, Cameron, Poon, & Egelhaaf, 2007). A good amount of research has been conducted to evaluate the protective effect of glycerol on protein stability. In the presence of glycerol, denaturation temperature of proteins is enhanced to various extents (Back, Oakenfull, & Smith, 1979). Preferential hydration, a non-contact protective effect as proposed by Gekko and Timasheff (1981), is one of the major interpretations for the glycerol stabilizing effect on protein. In addition, prevention from aggregation by glycerol also contributes to the protein stability (Vagenende, Yap, & Trout, 2009). Denaturation temperature (Td) has been known as an effective way to evaluate the stability of protein. According to the literature, the increase of Td of protein because of glycerol depends on the type of proteins (Damodaran, 2013), meaning that proteins with different structures respond differently to glycerol protection. Moreover, the native structure of the protein is a one of the key factors
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determining its stability (Pucci & Rooman, 2017). The β-lactoglobulin monomer is constituted of 162 amino acid residues (Braunitzer, Chen, Schrank, & Stangl, 1972), part of which fold into nine β-sheets and three α-helices and further form a β-barrel structure involving eight of the β-sheets (Brownlow et al., 1997) which is the structural characteristic of lipocalin. Hydrophobic effect has been considered as the driving force of the globular protein folding and a major contribution to their stability (Pace, Shirley, Mcnutt, & Gajiwala, 1996). A hydrophobic pocket is enclosed in the β-barrel structure, not only enabling β-lactoglobulin to carry apolar molecules but also stabilizing the steric structure of the protein itself. At neutral pH, β-lactoglobulin exists in dimer (Vijayalakshmi, Krishna, Sankaranarayanan, & Vijayan, 2010), with dimerization due to both hydrophobic interaction and electrostatic attraction (Mercadante et al., 2012). From an applied view point, β-lactoglobulin is responsible for the film making properties of whey protein (Cavot and Lorient, 1997). It is and ideal ingredient for edible films preparation because of its molecular mobility and ability to form intermolecular bonding (Bourtoom, 2008; Lacroix & Cooksey, 2005). Glycerol is routinely used as a plasticizer to increase the flexibility and decrease the brittleness of edible
Corresponding author at: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province 214122, China. E-mail address: [email protected] (P. Zhou).
https://doi.org/10.1016/j.foodchem.2019.125596 Received 12 May 2019; Received in revised form 23 September 2019; Accepted 24 September 2019 Available online 15 October 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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used to determine the secondary and tertiary structure of β-lactoglobulin in glycerol solutions. The β-lactoglobulin stocks solution was diluted 50 and 500 times by glycerol solutions at concentration of 1 mg/ mL and 0.1 mg/mL for near-UV (250–320 nm) and far-UV (190–260 nm) measurements, respectively. The samples were equilibrated at 4 °C for 12 h. Cuvettes with 10 mm and 2 mm path-lengths were used during near-UV and far-UV measurement. The scanning was performed in a continuous mode of 100 nm/min. Glycerol solutions were taken as background and subtracted. All measurements were carried out in duplicates. The quantification of secondary structure was analyzed by Dichroweb (Whitmore & Wallace, 2004, 2008) using SELCON3 and reference Set4 in the range from 200–260 nm.
films (Ramos et al., 2013). During the preparation of the films, whey protein solution (5–10%) is initially heated until the proteins are denatured then glycerol is added, since glycerol is been known as protein stabilizer, depressing the thermal denaturation of protein. (Mchugh & Krochta, 1994). In our previous work, we reported that the stability of the two major proteins in whey, α-lactalbumin and β-lactoglobulin, varied in extremely different way with the increase in glycerol concentration (Chen, Bhandari, & Zhou, 2019). The stability of β-lactoglobulin was improved by glycerol, following a linear correlation with glycerol concentration up to 80% (Chen et al., 2019). Noteworthy, the theory was proposed under the condition that glycerol concentration was up to 50%. A latest research pointed that the hydration shell of protein was well preserved at glycerol concentration under ~40% (Timasheff, 1993). However, the hydration shell was partially penetrated and water molecules were replaced by glycerol at glycerol concentration beyond 50% (Hirai et al., 2018), indicating that preferential hydration could no longer explain the mechanism of stabilization of protein under high glycerol concentration. To determine the possible mechanism involved in the stabilization of β-lactoglobulin by glycerol, heat stability and protein conformation were investigated in the presence of glycerol at concentration ranging from 0 to 60% (v/v). As previously demonstrated, effects of glycerol are entwined with salts and protons (Yancey, Clark, Hand, Bowlus, & Somero, 1982). To simplify the system, a binary model solvent system without salts was used. The pH of glycerol solution was around 7, a condition under which β-lactoglobulin exists as a dimer. Experimental detection by differential scanning calorimetry (DSC), circular dichroism (CD), and fluorescence spectrum were used to shed light on the stability changes at a molecular level. Besides, molecular dynamics (MD) simulation and synchrotron small angle x-ray scattering (SAXS) were used to monitor and consolidate the conformation of β-lactoglobulin in a glycerol solution.
2.5. Intrinsic fluorescence spectrometry A fluorescence Spectrometer (Fluoro Max4, HORIBA Jobin Yvon, Pairs, France) was used to evaluate the tryptophan micro-environment of β-lactoglobulin in glycerol solutions. The β-lactoglobulin stock solution was diluted 100 times by glycerol solutions to 0.5 mg/mL and equilibrate at 4 °C for 12 h. The excitation wavelength was 295 nm at a slit width of 10 nm. Spectrum from 300 to 450 nm was recorded at a rate of 200 nm/min. The fluorescence intensity was normalized by taking the maximal intensity in water as a reference (set as 1). All measurements were carried out in duplicates. 2.6. Synchrotron facility based small-angle X-ray scattering (SAXS) SAXS was performed at the B19U2 beamline (Shanghai Synchrotron Radiation Facility, Shanghai, China) of National Center for Protein Sciences Shanghai (NCPSS), equipped with PILATUS 1 M detector (DECTRIS Ltd., Baden-Daettwil, Switzerland). The wavelength (λ) of the beam was 1.03 Å with energy of 12 KeV, and the scattering vector (q = 4π sinθ/λ, with θ is the scattering angle) in the range from 0.0084 to 0.3199 Å−1. SAXS data were collected as 20 × 1 s exposures on a 60 μL sample at 25 °C. The scattering data of glycerol solution were collected and used as blanks. BioXTAS RAW (Nielsen et al., 2009) was used to convert the SAXS profiles. The SAXS profiles were subtracted by the corresponding blanks, averaged and normalized using BioXTAS RAW. An ATSAS2.7.2 package was used for the calculation of the invariants and distance distribution. The PRIMUS program (Konarev, Volkov, Sokolova, Koch, & Svergun, 2010) was used to estimate the radius of gyration (Rg) in the Guinier region where qRg < 1.3.
2. Materials and methods 2.1. Materials β-lactoglobulin (Bio Pure, JE-003-6-922) powders were kindly provided by Agropur Dairy Cooperative (Minnesota, MN, USA). Glycerol (analytical grade) was purchased from Alfa Aesar (Thermo Fisher Scientific Co., Shanghai, China). Ultra-pure water was used throughout the experiments. 2.2. Preparation of stock solution
2.7. Molecular dynamics simulation Glycerol solutions were prepared into concentration of 0–60% (v/ v). Glycerol was mixed with water by volume and the weights of both fractions were recorded in order to estimate their molar ratio. A stock solution of β-lactoglobulin was prepared at concentration of 50 mg/mL and degassed using a vacuum chamber (YZF-6050, Yao Instrument, Shanghai, China). The protein stock solution was stored at 4 °C and used within 2 days.
Molecular structure of glycerol molecule (PubChem CID:753) was optimized at HF/6-31G* in gas phase with the Gaussian 09 software (Gaussian, Inc., Wallingford, CT, USA) and modeled using the general Amber force field (GAFF) (Wang, Wolf, Caldwell, Kollman, & Case, 2004). Solvent box of glycerol solution at concentration of 10–60% (v/ v) was built with rigid TIP3P water model and equilibrated for 10 ns before use. A 100-ns MD simulation was performed at 298.15 K with 2 fs step using GROMACS 5.1.4 (Abraham et al., 2015). The crystal structure of β-lactoglobulin dimer (PDB ID: 1beb) and monomer (PDB ID: 3blg) were taken from Protein Data Bank (Brownlow et al., 1997; Qin et al., 1998) and the missing residues were mended by Discovery Studio (BIOVIA Scientific Innovation, San Diego, CA, USA). The Amber ff99SB force field (Lindorff-Larsen et al., 2010) was used to model β-lactoglobulin. The protein was placed in a cubic box under periodic boundary condition and then solvated using glycerol solution. The Na+ ion was added to the system for neutrality. Prior to the production simulation, an energy minimization was performed, followed by a 50-ps NVT simulation and a 100-ps NPT simulation. Throughout the simulation, the temperature was maintained by the v-rescale algorithm
2.3. Differential scanning calorimetry (DSC) β-lactoglobulin was dissolved in glycerol solutions at a concentration of 5 (w/v) %, which was subsequently degassed using vacuum chamber. Micro-DSC (DSCIII, SETARAM Instrumentation, Lyon, France) was used to monitor the thermal stability of protein in 0 to 60% glycerol solutions. The temperature and heat flow were calibrated with indium. Samples were heated from 25 to 110 °C at 1 °C/min. The heat flow of samples were normalized to the weight of protein. 2.4. Circular dichroism Circular dichroism (CD) (Jasco-710, Jasco Co., Tokyo, Japan) was 2
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3.2. Structure of monomeric β-lactoglobulin in glycerol solution The secondary and tertiary structure of β-lactoglobulin in glycerol solution was measured by far-UV and near-UV CD, as shown in Fig. 2A and B, respectively. Taking into account the strong UV absorbance of hydroxyl group of glycerol in the vacuum ultraviolet region (λ < 200 nm) into consideration, the far-UV CD spectra were presented in a wavelength range from 200 to 260 nm. A typical β-sheet dominated spectra was observed with a broad negative band centered at 215 nm (Barteri, Gaudiano, Mei, & Rosato, 1998). The far-UV CD spectra showed little difference at different glycerol concentration. As shown in Table S1, the proportions of helix and sheet structure decreased while that of random coils increased in the presence of glycerol. However, they did not further change with glycerol concentration, indicating that the secondary structure of β-lactoglobulin was hardly influenced by the increase in glycerol concentration (Barbiroli et al., 2017). The tertiary structure of β-lactoglobulin in glycerol solution was characterized by near-UV CD (Fig. 2B). The spectra presented two sharp negative bands at 285 nm and 293 nm due to the vibrational fine structure of tryptophan (Barteri et al., 1998). With increasing glycerol, the bands strengthened and reached a minimum for the β-lactoglobulin in 50% glycerol solution, indicating changes in the exposure of tryptophan (Kelly, Jess, & Price, 2005). Stemming from aromatic groups the near-UV CD results indicated that the asymmetry of buried aromatic side chains improved because they are closer to hydrophobic environment. It suggests that the interior hydrophobic interactions might be strengthened with increasing glycerol concentration. Hydrophobic interaction at the interior of β-barrel structure contributed remarkably to the stability of β-lactoglobulin. Hence, the promotion of hydrophobic interaction by glycerol could be one of the factors which enhanced the stability of β-lactoglobulin. To investigate the micro-environment of tryptophan in the presence of glycerol, intrinsic fluorescence spectrum was used. Fig. 2C and D showed that the intrinsic fluorescence intensity of tryptophan quenched with glycerol concentration and reached a minimum (80% of the initial intensity) at 50% glycerol, with a 3 nm red shift. The Trp19 and Trp61 contribute to approximately 80% and 20% of the intrinsic fluorescence, respectively (Cho, Batt, & Sawyer, 1994). In 60% glycerol solution, approximate 80% intrinsic fluorescence of tryptophan remained probably owing to the quenching of Trp61 fluorescence, Trp61 being on the surface of β-lactoglobulin. A possible cause of the quenching and red shift of tryptophan fluorescence is its exposure to water (Viseu, Melo, Carvalho, Correia, & Sílvia, 2007), which suggests that the solvent accessibility to the hydrophobic area might increase. Besides, endogenous quenchers, such as proximity of Cys66-Cys160 to Trp61, might also result in the fluorescence quenching (Bao, Wang, Shi, Suying Dong, & Ma, 2007; Harvey, Bell, & Brancaleon, 2007).
Fig. 1. DSC thermograms of 5% β-lactoglobulin in glycerol solutions. The G10 to G60 represent 10% to 60% of glycerol concentration.
(Deighan, Bonomi, & Pfaendtner, 2012) and pressure was controlled by the Parrinello-Rahman algorithm (Parrinello & Rahman, 1981). Nonbonded interactions were calculated with a cut-off of 10 Å, and the coulomb interaction was treated by Particle mesh Ewald (PME). The bond length was constrained using the LINCS algorithm. (Hess, Bekker, Berendsen, & Fraaije, 1997) The root-mean-square fluctuation (RMSF), solvent accessible surface area (SASA), and mean-smallest distance (MSD) of residues were achieved from the MD results by GROMACS. Theoretical SAXS of βlactoglobulin was calculated from dimer (PDBID: 1beb) after MD simulation using a CRYSOL program (Svergun, 2010) in the package of ATSAS.
3. Results and discussion 3.1. Thermal stability of β-lactoglobulin in glycerol solution The thermal stability of 5% β-lactoglobulin was monitored in glycerol solution with concentration varying from 0 to 60% by micro-DSC. As shown in Fig. 1, the denaturation temperature of β-lactoglobulin increased with the glycerol concentration, which is in good agreement with not only our previous study in a high-protein content model system (Chen et al., 2019) but also with the work of Chanasattru (Chanasattru, Decker, & Mcclements, 2007). In water, the β-lactoglobulin denatured at 75.9 °C, consistent with the previous report under neutral condition (Huang, Skar, Vegarud, Langsurd, & Draget, 2009). Damodaran (2013) reported that the denaturation temperature of βlactoglobulin increased by 1.5 °C in the presence of 1 M glycerol (approximately 10%) while it was 1.3 °C in this study. Additionally, to consolidate the protective effects of glycerol, stability of 5% β-lactoglobulin in 70% and 80% glycerol solution was also measured, in which conditions the denaturation temperature was 3.7 and 7.1 °C higher than in 60% glycerol solution, respectively (data shown in Fig. S1). Unfortunately, glycerol at above high concentration affects some of the experimental methods used in this study. Thus, the structural characterization experiments were performed at glycerol concentration of 60% or below.
3.3. Structure of dimeric β-lactoglobulin in glycerol solution Synchrotron SAXS was used to monitor the steric structure of βlactoglobulin in glycerol solution. The Rg at 5 mg/mL was estimated in the Guinier region (Table 1). In water, the Rg of β-lactoglobulin was 20.7 ± 0.3 Å. As reported previously, the Rg of β-lactoglobulin under physiological condition was 21 Å, which is in good agreement with our experimental result (Panick, Malessa, & Winter, 1999). The Rg increased with glycerol concentration, particularly when glycerol was higher than 40%. The increase of the Rg of myoglobin was previously observed in the presence of 60% glycerol as well. If the overall conformation is unchanged, a lower value of Rg is expected owing to the density effect of glycerol (Barteri, Gaudiano, & Santucci, 1996). This confirms that glycerol influenced the conformation of β-lactoglobulin in solution. A little increase of the Rg without dramatic deformation of the spatial structure, to certain extents, indicated protein being in a more favorable environment (Li, Tan, Zhang, & Feng, 2010). The increase of 3
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Fig. 2. Structural characteristics of β-lactoglobulin in glycerol solutions. Changes in secondary (A) and tertiary (B) structure at β-lactoglobulin concentration of 0.1 mg/mL (in A) and 1 mg/mL (in B), respectively. Tryptophan intrinsic fluorescence (C) of 5 mg/mL β-lactoglobulin in glycerol solution. The decrease of the intrinsic fluorescence intensity peak vs the glycerol concentration (D). Error bars correspond to standard deviations. The G10 to G60 represent 10% to 60% of glycerol concentration.
and chain B are more distant in the case of 60% glycerol, e.g. the distance between stands I and CD loops in separate chains, as illustrated in Fig. 4B and D (marked by square No. 2 and 3), which might be also responsible for the changes in Rg and the surface accessible to the solvent. No dissociation was observed by MD simulation although the particular regions in between two individual β-lactoglobulin monomer became further in 60% glycerol. Moreover, as shown in Fig. 4B and D, the region marked by square No. 1 showed little changes, where the Asp33-Arg40 respectively from chain A and chain B are. The electrostatic attraction between Asp33-Arg40 in individual chain was considered as one of the major factors maintaining the dimer. According to previous report, the β-lactoglobulin dimer dissociated at pH < 3 which was the pKa of aspartic acid side chain (Mercadante et al., 2012). The pH of samples studied here is around 7.5 (Figure S2). As a result, the dimer was not expected to dissociate in 60% glycerol. Additionally, electrostatic interaction was one of long-range interaction, and glycerol decreases the dielectric constant of the solution, which is in favor of the
Rg will also results in an increase of the surface area accessible to the solvent, as obtained by the MD simulation using GROMACS (Fig. 3). The hydrophobic surface, where the amount of charge was less than 0.2, increased slightly with glycerol concentration while the hydrophilic surface grew with the increasing glycerol in the environment, possibly stemming from either the outward orientation of hydrophilic side chain or the increase of distance between two monomers. As seen in Fig. 4 A and C, an obvious expansion of the N-terminal indicated the likelihood of the residues located at flexible regions becoming more outward-oriented in 60% glycerol, contributing to the increase of molecular size and solvent accessible surface. On the other hand, the β-lactoglobulin existed as a dimer under neutral condition, which was stabilized particularly by Asp33-Arg40 interaction in the AB loop along with the antiparallel stands I (Sakurai & Goto, 2002). However, at a low salt concentration and low β-lactoglobulin concentration, the dissociation of the dimer occurs (Renard, Lefebvre, Griffin, & Griffin, 1998), indicating that the interactions of two monomers become weak. In the present system, the stands I in chain A Table 1 Radius of gyration (Rg) of β-lactoglobulin at 5 mg/mL in glycerol solution. Glycerol (%)
0
10
20
30
40
50
60
Rg (Å)
20.6 ± 0.3a
20.8 ± 0.1a
21.4 ± 0.2b
21.5 ± 0.3b
21.6 ± 0.2b
22.2 ± 1.4bc
23.4 ± 0.5c
Different letters represent the significant differences (p < 0.05). 4
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3.4. Conformational fluctuation of β-lactoglobulin in glycerol aqueous MD simulation was conducted to investigate the changes in conformation which was difficult to be well-characterized by current experimental methodology with respect of time and space scale. The equilibrated structure of β-lactoglobulin dimer after 100-ns MD simulation was extracted and compared with the SAXS result. As shown in Fig. 5A, the calculated SAXS profiles of simulated β-lactoglobulin structures under different glycerol concentrations matched those obtained from Synchrotron SAXS. The consistency of experimental and MD SAXS data was superior in higher glycerol concentrations, particularly in the q range from 0.01 to 0.15 Å−1, the range providing the information of macromolecules’ shape. Nevertheless, protein was considered as a weak scattering source. To guarantee a sufficient scattering difference between protein and solution, SAXS data were not collected under a higher glycerol concentration than 60%. The flexibility of protein was generally described by the root-meansquare fluctuation (RMSF) of residues from the crystal structure as shown in Fig. 5C and D. In water, RMSF of residues was lower in regular secondary structures than those which are not, e.g. β-sheet stands A, C, F, G, H, and I fluctuated less than 1 Å, as well as the helices other that the one close to C terminal. Nevertheless, residues distributed in the loop or random coil regions tended to be more flexible, with RMSF more than 2 Å or even higher, e.g. N-terminal, CD loop and EF loop. With the increase of glycerol concentration, the fluctuation of less flexible regions changed little, remaining at their low fluctuation level. The flexibility of residues in the loop among β-sheets apparently decreased, including the CD loop, EF loop and C-terminal helix, which
Fig. 3. Solvent accessible surface area of β-lactoglobulin dimer with increasing glycerol concentration obtained by MD simulation.
electrostatic interaction, e.g. Asp33-Arg40, thus, in favor of the stabilization of the dimer. The interaction between Asp33-Arg40 was briefly studied by quantum chemical calculation, as results shown in Fig. S3 and Table S1.
Fig. 4. Conformation of β-lactoglobulin dimer after 100 ns MD simulation in water (A) and 60% glycerol (C) and the mean smallest distance between residues of βlactoglobulin in water (B) and 60% glycerol (D) with residues in chain A given residue index from 1 to 162 and chain B from 163 to 324. 5
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Fig. 5. Conformation of β-lactoglobulin in glycerol solutions revealed by MD. Comparison of MD simulated structures (red lines) of β-lactoglobulin to SAXS data (black symbols) (A) in glycerol solution at concentration from 0 to 60%. The repaired integrity of β-lactoglobulin crystal (B). And root means squared fluctuation of residues in chain A (C) and chain B (D) of β-lactoglobulin in glycerol aqueous solutions. The regions highlighted in green stands for nine strands of β-sheet A to I. The pink regions stands for helix structures. The white regions stands for the loops between neighboring β-sheet strands or coil. The G20 to G60 represent 20% to 60% of glycerol concentration. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
greatly contribute to the stability enhancement of β-lactoglobulin. Noteworthy, the N-terminal residues remained to be the most flexible residue regardless of the glycerol concentration.
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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors gratefully acknowledge the subsidization from the National Key Research and Development Program of China (2017YFD0400600), the National First-class Discipline Program of Food Science and Technology (JUFSTR20180201), the Natural Science Foundation of China (31471697, U1832144, 21977013), 111 Project (BP0719028), and the Youth Innovation Promotion Association of Chinese Academy of Science (grant no. 2017319). We thank the staff of NCPSS beamline BL19U2 beamline for assistance during data collection. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125596. References Abraham, M. J., Murtola, T., Schulz, R., Páll, S., Smith, J. C., Hess, B., & Lindahl, E. (2015). Gromacs: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX, 1–2(C), 19–25. Back, J. F., Oakenfull, D., & Smith, M. B. (1979). Increased thermal stability of proteins in the presence of sugars and polyols. Biochemistry, 18(23), 5191. Bao, Z., Wang, S., Shi, W., Suying Dong, A., & Ma, H. (2007). Selective modification of Trp19 in β-lactoglobulin by a new diazo fluorescence probe. Journal of Proteome Research, 6(9), 3835–3841.
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Ramos, Ó. L., Reinas, I., Silva, S. I., Fernandes, J. C., Cerqueira, M. A., Pereira, R. N., ... Malcata, F. X. (2013). Effect of whey protein purity and glycerol content upon physical;properties of edible films manufactured therefrom. Food Hydrocolloids, 30(1), 110–122. Renard, D., Lefebvre, J., Griffin, M. C., & Griffin, W. G. (1998). Effects of pH and salt environment on the association of beta-lactoglobulin revealed by intrinsic fluorescence studies. International Journal of Biological Macromolecules, 147(2–3), 41–49. Sakurai, K., & Goto, Y. (2002). Manipulating monomer-dimer equilibrium of bovine beta -lactoglobulin by amino acid substitution. Journal of Biological Chemistry, 277(28), 25735–25740. Sedgwick, H., Cameron, J. E., Poon, W. C. K., & Egelhaaf, S. U. (2007). Protein phase behavior and crystallization: Effect of glycerol. The Journal of Chemical Physics, 127(12), 125102. Svergun, D. (2010). Crysol – A program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates. Journal of Applied Crystallography, 28(6), 768–773. Timasheff, S. N. (1993). The control of protein stability and association by weak interactions with water: How do solvents affect these processes? Annual Review of Biophysics and Biomolecular Structure, 22(22), 67–97. Vagenende, V., Yap, M. G. S., & Trout, B. L. (2009). Mechanisms of protein stabilization and prevention of protein aggregation by glycerol. Biochemistry, 48(46), 11084–11096. Vijayalakshmi, L., Krishna, R., Sankaranarayanan, R., & Vijayan, M. (2010). An asymmetric dimer of beta-lactoglobulin in a low humidity crystal form-structural changes that accompany partial dehydration and protein action. Proteins-structure Function & Bioinformatics, 71(1), 241–249. Viseu, M. I., Melo, E. P., Carvalho, T. I., Correia, R. F., & Sílvia, M. B. C. (2007). Unfolding kinetics of -lactoglobulin induced by surfactant and denaturant: A stopped-flow/ fluorescence study. Biophysical Journal, 93(10), 3601–3612. Wakankar, A. A., Liu, J. U. N., Vandervelde, D., Wang, Y. J., Shire, S. J., & Borchardt, R. T. (2010). The effect of cosolutes on the isomerization of aspartic acid residues and conformational stability in a monoclonal antibody. Journal of Pharmaceutical Sciences, 96(7), 1708–1718. Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A., & Case, D. A. (2004). Development and testing of a general amber force field. Journal of Computational Chemistry, 25(9), 1157–1174. Whitmore, L., & Wallace, B. A. (2008). Protein secondary structure analyses from circular dichroism spectroscopy: Methods and reference databases. Biopolymers, 89(5), 392–400. Whitmore, L., & Wallace, B. A. (2004). Dichroweb, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Research, 32, W668–W673. Yancey, P., Clark, M., Hand, S., Bowlus, R., & Somero, G. (1982). Living with water stress: Evolution of osmolyte systems. Science, 217(4566), 1214–1222.
transition. Biophysical Journal, 115(2), 313–327. Huang, I. J., Skar, H. M., Vegarud, G. E., Langsurd, T., & Draget, K. I. (2009). Electrostatic effects on β-lactoglobulin transitions during heat denaturation as studied by differential scanning calorimetry. Food Hydrocolloids, 23, 2287–2293. Kamerzell, T. J., Esfandiary, R., Joshi, S. B., Middaugh, C. R., & Volkin, D. B. (2011). Protein–excipient interactions: Mechanisms and biophysical characterization applied to protein formulation development. Advanced Drug Delivery Reviews, 63(13), 1118–1159. Kelly, S. M., Jess, T. J., & Price, N. C. (2005). How to study proteins by circular dichroism. Biochimica et Biophysica Acta, 1751(2), 119–139. Konarev, P. V., Volkov, V. V., Sokolova, A. V., Koch, M. H. J., & Svergun, D. I. (2010). Primus: A windows pc-based system for small-angle scattering data analysis. Journal of Applied Crystallography, 36(5), 1277–1282. Lacroix, M., & Cooksey, K. (2005). Edible films and coatings from animal-origin proteins. In J. H. Han (Ed.). Innovations in food packaging (pp. 301–317). San Diego, California: Elsevier Academic Press. Lindorff-Larsen, K., Piana, S., Palmo, K., Maragakis, P., Klepeis, J. L., Dror, R. O., & Shaw, D. E. (2010). Improved side-chain torsion potentials for the amber ff99sb protein force field. Proteins-structure Function and Bioinformatics, 78(8), 1950–1958. Mchugh, T. H., & Krochta, J. M. (1994). Sorbitol- vs glycerol-plasticized whey protein edible films: Integrated oxygen permeability and tensile property evaluation. Journal of Agricultural and Food Chemistry, 42(4), 841–845. Mercadante, D., Melton, L. D., Norris, G. E., Loo, T. S., Williams, M. A. K., Dobson, R. C. J., & Jameson, G. B. (2012). Bovine β-lactoglobulin is dimeric under imitative physiological conditions: Dissociation equilibrium and rate constants over the ph range of 2.5–7.5. Biophysical Journal, 103(2), 303–312. Nielsen, S., Toft, K. N., Snakenborg, D., Jeppesen, M. G., Jacobsen, J., Vestergaard, B., ... Arleth, L. (2009). BioXTAS RAW, a software program for high-throughput automated small-angle X-ray scattering data reduction and preliminary analysis. Journal of Applied Crystallography, 42(5), 959–964. Pace, C. N., Shirley, B. A., Mcnutt, M., & Gajiwala, K. (1996). Forces contributing to the conformational stability of proteins. Federation of American Societies for Experimental Biology, 10(1), 75–83. Panick, G., Malessa, R., & Winter, R. (1999). Differences between the pressure- and temperature-induced denaturation and aggregation of beta-lactoglobulin a, b, and ab monitored by FTIR spectroscopy and small-angle x-ray scattering. Biochemistry, 38(20), 6512–6519. Parrinello, M., & Rahman, A. (1981). Polymorphic transitions in single crystals: A new molecular dynamics method. Journal of Applied Physics, 52(12), 7182–7190. Pucci, F., & Rooman, M. (2017). Physical and molecular bases of protein thermal stability and cold adaptation. Current Opinion in Structural Biology, 42, 117–128. Qin, B. Y., Bewley, M. C., Creamer, L. K., Baker, H. M., Baker, E. N., & Jameson, G. B. (1998). Structural basis of the Tanford transition of bovine β-lactoglobulin. Biochemistry, 37(40), 14014–14023.
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Glycerol induced stability enhancement and conformational changes of βlactoglobulin Xiaoxia Chena,b, Haiyang Zhangc, Yacine Hemarb,d, Na Lie, Peng Zhoua,b,
T
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a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province 214122, China International Joint Research Laboratory for Functional Dairy Protein Ingredients, Jiangnan University, Wuxi, Jiangsu Province 214122, China c Department of Biological Science and Engineering, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 100083 Beijing, China d Riddet Institute, Palmerston North, New Zealand e National Center for Protein Sciences Shanghai, Chinese Academy of Sciences, Shanghai 201204, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: β-Lactoglobulin Glycerol Stability Molecular dynamics Small angle X-ray scattering
The protective mechanism of glycerol on β-lactoglobulin were studied in 0–60% glycerol solutions by experimental and molecular simulation approaches. Results showed that the stability of β-lactoglobulin increased with glycerol concentration, with little secondary structure changes induced by glycerol. The tertiary structure altered slightly with glycerol concentration, resulting in a stronger near UV circular dichroism signal and intrinsic tryptophan fluorescence quenching, indicating aromatic side chains closer to hydrophobic microenvironment. The Rg of β-lactoglobulin increased with glycerol concentration without dimer dissociation, due to expansion of the quaternary structures. Moreover, the flexibility (RMSF) of β-lactoglobulin decreased by glycerol. Distance between areas enclosing Asp33 and Arg40 from separate chains did not increase, suggesting possibly reinforced electrostatic attractions. In conclusion, the stabilization of β-lactoglobulin in glycerol solution is probably the comprehensive results of the decreased molecular flexibility, the strengthened hydrophobic interaction in individual chain, and the possibly reinforced electrostatic attractions between two chains of β-lactoglobulin.
1. Introduction Glycerol is an excellent cosolvent stabilizing protein, routinely used in pharmaceutical dosage formulation (Kamerzell, Esfandiary, Joshi, Middaugh, & Volkin, 2011), antibody preservation (Wakankar et al., 2010) and protein crystallization (Sedgwick, Cameron, Poon, & Egelhaaf, 2007). A good amount of research has been conducted to evaluate the protective effect of glycerol on protein stability. In the presence of glycerol, denaturation temperature of proteins is enhanced to various extents (Back, Oakenfull, & Smith, 1979). Preferential hydration, a non-contact protective effect as proposed by Gekko and Timasheff (1981), is one of the major interpretations for the glycerol stabilizing effect on protein. In addition, prevention from aggregation by glycerol also contributes to the protein stability (Vagenende, Yap, & Trout, 2009). Denaturation temperature (Td) has been known as an effective way to evaluate the stability of protein. According to the literature, the increase of Td of protein because of glycerol depends on the type of proteins (Damodaran, 2013), meaning that proteins with different structures respond differently to glycerol protection. Moreover, the native structure of the protein is a one of the key factors
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determining its stability (Pucci & Rooman, 2017). The β-lactoglobulin monomer is constituted of 162 amino acid residues (Braunitzer, Chen, Schrank, & Stangl, 1972), part of which fold into nine β-sheets and three α-helices and further form a β-barrel structure involving eight of the β-sheets (Brownlow et al., 1997) which is the structural characteristic of lipocalin. Hydrophobic effect has been considered as the driving force of the globular protein folding and a major contribution to their stability (Pace, Shirley, Mcnutt, & Gajiwala, 1996). A hydrophobic pocket is enclosed in the β-barrel structure, not only enabling β-lactoglobulin to carry apolar molecules but also stabilizing the steric structure of the protein itself. At neutral pH, β-lactoglobulin exists in dimer (Vijayalakshmi, Krishna, Sankaranarayanan, & Vijayan, 2010), with dimerization due to both hydrophobic interaction and electrostatic attraction (Mercadante et al., 2012). From an applied view point, β-lactoglobulin is responsible for the film making properties of whey protein (Cavot and Lorient, 1997). It is and ideal ingredient for edible films preparation because of its molecular mobility and ability to form intermolecular bonding (Bourtoom, 2008; Lacroix & Cooksey, 2005). Glycerol is routinely used as a plasticizer to increase the flexibility and decrease the brittleness of edible
Corresponding author at: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province 214122, China. E-mail address: [email protected] (P. Zhou).
https://doi.org/10.1016/j.foodchem.2019.125596 Received 12 May 2019; Received in revised form 23 September 2019; Accepted 24 September 2019 Available online 15 October 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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used to determine the secondary and tertiary structure of β-lactoglobulin in glycerol solutions. The β-lactoglobulin stocks solution was diluted 50 and 500 times by glycerol solutions at concentration of 1 mg/ mL and 0.1 mg/mL for near-UV (250–320 nm) and far-UV (190–260 nm) measurements, respectively. The samples were equilibrated at 4 °C for 12 h. Cuvettes with 10 mm and 2 mm path-lengths were used during near-UV and far-UV measurement. The scanning was performed in a continuous mode of 100 nm/min. Glycerol solutions were taken as background and subtracted. All measurements were carried out in duplicates. The quantification of secondary structure was analyzed by Dichroweb (Whitmore & Wallace, 2004, 2008) using SELCON3 and reference Set4 in the range from 200–260 nm.
films (Ramos et al., 2013). During the preparation of the films, whey protein solution (5–10%) is initially heated until the proteins are denatured then glycerol is added, since glycerol is been known as protein stabilizer, depressing the thermal denaturation of protein. (Mchugh & Krochta, 1994). In our previous work, we reported that the stability of the two major proteins in whey, α-lactalbumin and β-lactoglobulin, varied in extremely different way with the increase in glycerol concentration (Chen, Bhandari, & Zhou, 2019). The stability of β-lactoglobulin was improved by glycerol, following a linear correlation with glycerol concentration up to 80% (Chen et al., 2019). Noteworthy, the theory was proposed under the condition that glycerol concentration was up to 50%. A latest research pointed that the hydration shell of protein was well preserved at glycerol concentration under ~40% (Timasheff, 1993). However, the hydration shell was partially penetrated and water molecules were replaced by glycerol at glycerol concentration beyond 50% (Hirai et al., 2018), indicating that preferential hydration could no longer explain the mechanism of stabilization of protein under high glycerol concentration. To determine the possible mechanism involved in the stabilization of β-lactoglobulin by glycerol, heat stability and protein conformation were investigated in the presence of glycerol at concentration ranging from 0 to 60% (v/v). As previously demonstrated, effects of glycerol are entwined with salts and protons (Yancey, Clark, Hand, Bowlus, & Somero, 1982). To simplify the system, a binary model solvent system without salts was used. The pH of glycerol solution was around 7, a condition under which β-lactoglobulin exists as a dimer. Experimental detection by differential scanning calorimetry (DSC), circular dichroism (CD), and fluorescence spectrum were used to shed light on the stability changes at a molecular level. Besides, molecular dynamics (MD) simulation and synchrotron small angle x-ray scattering (SAXS) were used to monitor and consolidate the conformation of β-lactoglobulin in a glycerol solution.
2.5. Intrinsic fluorescence spectrometry A fluorescence Spectrometer (Fluoro Max4, HORIBA Jobin Yvon, Pairs, France) was used to evaluate the tryptophan micro-environment of β-lactoglobulin in glycerol solutions. The β-lactoglobulin stock solution was diluted 100 times by glycerol solutions to 0.5 mg/mL and equilibrate at 4 °C for 12 h. The excitation wavelength was 295 nm at a slit width of 10 nm. Spectrum from 300 to 450 nm was recorded at a rate of 200 nm/min. The fluorescence intensity was normalized by taking the maximal intensity in water as a reference (set as 1). All measurements were carried out in duplicates. 2.6. Synchrotron facility based small-angle X-ray scattering (SAXS) SAXS was performed at the B19U2 beamline (Shanghai Synchrotron Radiation Facility, Shanghai, China) of National Center for Protein Sciences Shanghai (NCPSS), equipped with PILATUS 1 M detector (DECTRIS Ltd., Baden-Daettwil, Switzerland). The wavelength (λ) of the beam was 1.03 Å with energy of 12 KeV, and the scattering vector (q = 4π sinθ/λ, with θ is the scattering angle) in the range from 0.0084 to 0.3199 Å−1. SAXS data were collected as 20 × 1 s exposures on a 60 μL sample at 25 °C. The scattering data of glycerol solution were collected and used as blanks. BioXTAS RAW (Nielsen et al., 2009) was used to convert the SAXS profiles. The SAXS profiles were subtracted by the corresponding blanks, averaged and normalized using BioXTAS RAW. An ATSAS2.7.2 package was used for the calculation of the invariants and distance distribution. The PRIMUS program (Konarev, Volkov, Sokolova, Koch, & Svergun, 2010) was used to estimate the radius of gyration (Rg) in the Guinier region where qRg < 1.3.
2. Materials and methods 2.1. Materials β-lactoglobulin (Bio Pure, JE-003-6-922) powders were kindly provided by Agropur Dairy Cooperative (Minnesota, MN, USA). Glycerol (analytical grade) was purchased from Alfa Aesar (Thermo Fisher Scientific Co., Shanghai, China). Ultra-pure water was used throughout the experiments. 2.2. Preparation of stock solution
2.7. Molecular dynamics simulation Glycerol solutions were prepared into concentration of 0–60% (v/ v). Glycerol was mixed with water by volume and the weights of both fractions were recorded in order to estimate their molar ratio. A stock solution of β-lactoglobulin was prepared at concentration of 50 mg/mL and degassed using a vacuum chamber (YZF-6050, Yao Instrument, Shanghai, China). The protein stock solution was stored at 4 °C and used within 2 days.
Molecular structure of glycerol molecule (PubChem CID:753) was optimized at HF/6-31G* in gas phase with the Gaussian 09 software (Gaussian, Inc., Wallingford, CT, USA) and modeled using the general Amber force field (GAFF) (Wang, Wolf, Caldwell, Kollman, & Case, 2004). Solvent box of glycerol solution at concentration of 10–60% (v/ v) was built with rigid TIP3P water model and equilibrated for 10 ns before use. A 100-ns MD simulation was performed at 298.15 K with 2 fs step using GROMACS 5.1.4 (Abraham et al., 2015). The crystal structure of β-lactoglobulin dimer (PDB ID: 1beb) and monomer (PDB ID: 3blg) were taken from Protein Data Bank (Brownlow et al., 1997; Qin et al., 1998) and the missing residues were mended by Discovery Studio (BIOVIA Scientific Innovation, San Diego, CA, USA). The Amber ff99SB force field (Lindorff-Larsen et al., 2010) was used to model β-lactoglobulin. The protein was placed in a cubic box under periodic boundary condition and then solvated using glycerol solution. The Na+ ion was added to the system for neutrality. Prior to the production simulation, an energy minimization was performed, followed by a 50-ps NVT simulation and a 100-ps NPT simulation. Throughout the simulation, the temperature was maintained by the v-rescale algorithm
2.3. Differential scanning calorimetry (DSC) β-lactoglobulin was dissolved in glycerol solutions at a concentration of 5 (w/v) %, which was subsequently degassed using vacuum chamber. Micro-DSC (DSCIII, SETARAM Instrumentation, Lyon, France) was used to monitor the thermal stability of protein in 0 to 60% glycerol solutions. The temperature and heat flow were calibrated with indium. Samples were heated from 25 to 110 °C at 1 °C/min. The heat flow of samples were normalized to the weight of protein. 2.4. Circular dichroism Circular dichroism (CD) (Jasco-710, Jasco Co., Tokyo, Japan) was 2
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3.2. Structure of monomeric β-lactoglobulin in glycerol solution The secondary and tertiary structure of β-lactoglobulin in glycerol solution was measured by far-UV and near-UV CD, as shown in Fig. 2A and B, respectively. Taking into account the strong UV absorbance of hydroxyl group of glycerol in the vacuum ultraviolet region (λ < 200 nm) into consideration, the far-UV CD spectra were presented in a wavelength range from 200 to 260 nm. A typical β-sheet dominated spectra was observed with a broad negative band centered at 215 nm (Barteri, Gaudiano, Mei, & Rosato, 1998). The far-UV CD spectra showed little difference at different glycerol concentration. As shown in Table S1, the proportions of helix and sheet structure decreased while that of random coils increased in the presence of glycerol. However, they did not further change with glycerol concentration, indicating that the secondary structure of β-lactoglobulin was hardly influenced by the increase in glycerol concentration (Barbiroli et al., 2017). The tertiary structure of β-lactoglobulin in glycerol solution was characterized by near-UV CD (Fig. 2B). The spectra presented two sharp negative bands at 285 nm and 293 nm due to the vibrational fine structure of tryptophan (Barteri et al., 1998). With increasing glycerol, the bands strengthened and reached a minimum for the β-lactoglobulin in 50% glycerol solution, indicating changes in the exposure of tryptophan (Kelly, Jess, & Price, 2005). Stemming from aromatic groups the near-UV CD results indicated that the asymmetry of buried aromatic side chains improved because they are closer to hydrophobic environment. It suggests that the interior hydrophobic interactions might be strengthened with increasing glycerol concentration. Hydrophobic interaction at the interior of β-barrel structure contributed remarkably to the stability of β-lactoglobulin. Hence, the promotion of hydrophobic interaction by glycerol could be one of the factors which enhanced the stability of β-lactoglobulin. To investigate the micro-environment of tryptophan in the presence of glycerol, intrinsic fluorescence spectrum was used. Fig. 2C and D showed that the intrinsic fluorescence intensity of tryptophan quenched with glycerol concentration and reached a minimum (80% of the initial intensity) at 50% glycerol, with a 3 nm red shift. The Trp19 and Trp61 contribute to approximately 80% and 20% of the intrinsic fluorescence, respectively (Cho, Batt, & Sawyer, 1994). In 60% glycerol solution, approximate 80% intrinsic fluorescence of tryptophan remained probably owing to the quenching of Trp61 fluorescence, Trp61 being on the surface of β-lactoglobulin. A possible cause of the quenching and red shift of tryptophan fluorescence is its exposure to water (Viseu, Melo, Carvalho, Correia, & Sílvia, 2007), which suggests that the solvent accessibility to the hydrophobic area might increase. Besides, endogenous quenchers, such as proximity of Cys66-Cys160 to Trp61, might also result in the fluorescence quenching (Bao, Wang, Shi, Suying Dong, & Ma, 2007; Harvey, Bell, & Brancaleon, 2007).
Fig. 1. DSC thermograms of 5% β-lactoglobulin in glycerol solutions. The G10 to G60 represent 10% to 60% of glycerol concentration.
(Deighan, Bonomi, & Pfaendtner, 2012) and pressure was controlled by the Parrinello-Rahman algorithm (Parrinello & Rahman, 1981). Nonbonded interactions were calculated with a cut-off of 10 Å, and the coulomb interaction was treated by Particle mesh Ewald (PME). The bond length was constrained using the LINCS algorithm. (Hess, Bekker, Berendsen, & Fraaije, 1997) The root-mean-square fluctuation (RMSF), solvent accessible surface area (SASA), and mean-smallest distance (MSD) of residues were achieved from the MD results by GROMACS. Theoretical SAXS of βlactoglobulin was calculated from dimer (PDBID: 1beb) after MD simulation using a CRYSOL program (Svergun, 2010) in the package of ATSAS.
3. Results and discussion 3.1. Thermal stability of β-lactoglobulin in glycerol solution The thermal stability of 5% β-lactoglobulin was monitored in glycerol solution with concentration varying from 0 to 60% by micro-DSC. As shown in Fig. 1, the denaturation temperature of β-lactoglobulin increased with the glycerol concentration, which is in good agreement with not only our previous study in a high-protein content model system (Chen et al., 2019) but also with the work of Chanasattru (Chanasattru, Decker, & Mcclements, 2007). In water, the β-lactoglobulin denatured at 75.9 °C, consistent with the previous report under neutral condition (Huang, Skar, Vegarud, Langsurd, & Draget, 2009). Damodaran (2013) reported that the denaturation temperature of βlactoglobulin increased by 1.5 °C in the presence of 1 M glycerol (approximately 10%) while it was 1.3 °C in this study. Additionally, to consolidate the protective effects of glycerol, stability of 5% β-lactoglobulin in 70% and 80% glycerol solution was also measured, in which conditions the denaturation temperature was 3.7 and 7.1 °C higher than in 60% glycerol solution, respectively (data shown in Fig. S1). Unfortunately, glycerol at above high concentration affects some of the experimental methods used in this study. Thus, the structural characterization experiments were performed at glycerol concentration of 60% or below.
3.3. Structure of dimeric β-lactoglobulin in glycerol solution Synchrotron SAXS was used to monitor the steric structure of βlactoglobulin in glycerol solution. The Rg at 5 mg/mL was estimated in the Guinier region (Table 1). In water, the Rg of β-lactoglobulin was 20.7 ± 0.3 Å. As reported previously, the Rg of β-lactoglobulin under physiological condition was 21 Å, which is in good agreement with our experimental result (Panick, Malessa, & Winter, 1999). The Rg increased with glycerol concentration, particularly when glycerol was higher than 40%. The increase of the Rg of myoglobin was previously observed in the presence of 60% glycerol as well. If the overall conformation is unchanged, a lower value of Rg is expected owing to the density effect of glycerol (Barteri, Gaudiano, & Santucci, 1996). This confirms that glycerol influenced the conformation of β-lactoglobulin in solution. A little increase of the Rg without dramatic deformation of the spatial structure, to certain extents, indicated protein being in a more favorable environment (Li, Tan, Zhang, & Feng, 2010). The increase of 3
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Fig. 2. Structural characteristics of β-lactoglobulin in glycerol solutions. Changes in secondary (A) and tertiary (B) structure at β-lactoglobulin concentration of 0.1 mg/mL (in A) and 1 mg/mL (in B), respectively. Tryptophan intrinsic fluorescence (C) of 5 mg/mL β-lactoglobulin in glycerol solution. The decrease of the intrinsic fluorescence intensity peak vs the glycerol concentration (D). Error bars correspond to standard deviations. The G10 to G60 represent 10% to 60% of glycerol concentration.
and chain B are more distant in the case of 60% glycerol, e.g. the distance between stands I and CD loops in separate chains, as illustrated in Fig. 4B and D (marked by square No. 2 and 3), which might be also responsible for the changes in Rg and the surface accessible to the solvent. No dissociation was observed by MD simulation although the particular regions in between two individual β-lactoglobulin monomer became further in 60% glycerol. Moreover, as shown in Fig. 4B and D, the region marked by square No. 1 showed little changes, where the Asp33-Arg40 respectively from chain A and chain B are. The electrostatic attraction between Asp33-Arg40 in individual chain was considered as one of the major factors maintaining the dimer. According to previous report, the β-lactoglobulin dimer dissociated at pH < 3 which was the pKa of aspartic acid side chain (Mercadante et al., 2012). The pH of samples studied here is around 7.5 (Figure S2). As a result, the dimer was not expected to dissociate in 60% glycerol. Additionally, electrostatic interaction was one of long-range interaction, and glycerol decreases the dielectric constant of the solution, which is in favor of the
Rg will also results in an increase of the surface area accessible to the solvent, as obtained by the MD simulation using GROMACS (Fig. 3). The hydrophobic surface, where the amount of charge was less than 0.2, increased slightly with glycerol concentration while the hydrophilic surface grew with the increasing glycerol in the environment, possibly stemming from either the outward orientation of hydrophilic side chain or the increase of distance between two monomers. As seen in Fig. 4 A and C, an obvious expansion of the N-terminal indicated the likelihood of the residues located at flexible regions becoming more outward-oriented in 60% glycerol, contributing to the increase of molecular size and solvent accessible surface. On the other hand, the β-lactoglobulin existed as a dimer under neutral condition, which was stabilized particularly by Asp33-Arg40 interaction in the AB loop along with the antiparallel stands I (Sakurai & Goto, 2002). However, at a low salt concentration and low β-lactoglobulin concentration, the dissociation of the dimer occurs (Renard, Lefebvre, Griffin, & Griffin, 1998), indicating that the interactions of two monomers become weak. In the present system, the stands I in chain A Table 1 Radius of gyration (Rg) of β-lactoglobulin at 5 mg/mL in glycerol solution. Glycerol (%)
0
10
20
30
40
50
60
Rg (Å)
20.6 ± 0.3a
20.8 ± 0.1a
21.4 ± 0.2b
21.5 ± 0.3b
21.6 ± 0.2b
22.2 ± 1.4bc
23.4 ± 0.5c
Different letters represent the significant differences (p < 0.05). 4
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3.4. Conformational fluctuation of β-lactoglobulin in glycerol aqueous MD simulation was conducted to investigate the changes in conformation which was difficult to be well-characterized by current experimental methodology with respect of time and space scale. The equilibrated structure of β-lactoglobulin dimer after 100-ns MD simulation was extracted and compared with the SAXS result. As shown in Fig. 5A, the calculated SAXS profiles of simulated β-lactoglobulin structures under different glycerol concentrations matched those obtained from Synchrotron SAXS. The consistency of experimental and MD SAXS data was superior in higher glycerol concentrations, particularly in the q range from 0.01 to 0.15 Å−1, the range providing the information of macromolecules’ shape. Nevertheless, protein was considered as a weak scattering source. To guarantee a sufficient scattering difference between protein and solution, SAXS data were not collected under a higher glycerol concentration than 60%. The flexibility of protein was generally described by the root-meansquare fluctuation (RMSF) of residues from the crystal structure as shown in Fig. 5C and D. In water, RMSF of residues was lower in regular secondary structures than those which are not, e.g. β-sheet stands A, C, F, G, H, and I fluctuated less than 1 Å, as well as the helices other that the one close to C terminal. Nevertheless, residues distributed in the loop or random coil regions tended to be more flexible, with RMSF more than 2 Å or even higher, e.g. N-terminal, CD loop and EF loop. With the increase of glycerol concentration, the fluctuation of less flexible regions changed little, remaining at their low fluctuation level. The flexibility of residues in the loop among β-sheets apparently decreased, including the CD loop, EF loop and C-terminal helix, which
Fig. 3. Solvent accessible surface area of β-lactoglobulin dimer with increasing glycerol concentration obtained by MD simulation.
electrostatic interaction, e.g. Asp33-Arg40, thus, in favor of the stabilization of the dimer. The interaction between Asp33-Arg40 was briefly studied by quantum chemical calculation, as results shown in Fig. S3 and Table S1.
Fig. 4. Conformation of β-lactoglobulin dimer after 100 ns MD simulation in water (A) and 60% glycerol (C) and the mean smallest distance between residues of βlactoglobulin in water (B) and 60% glycerol (D) with residues in chain A given residue index from 1 to 162 and chain B from 163 to 324. 5
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Fig. 5. Conformation of β-lactoglobulin in glycerol solutions revealed by MD. Comparison of MD simulated structures (red lines) of β-lactoglobulin to SAXS data (black symbols) (A) in glycerol solution at concentration from 0 to 60%. The repaired integrity of β-lactoglobulin crystal (B). And root means squared fluctuation of residues in chain A (C) and chain B (D) of β-lactoglobulin in glycerol aqueous solutions. The regions highlighted in green stands for nine strands of β-sheet A to I. The pink regions stands for helix structures. The white regions stands for the loops between neighboring β-sheet strands or coil. The G20 to G60 represent 20% to 60% of glycerol concentration. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
greatly contribute to the stability enhancement of β-lactoglobulin. Noteworthy, the N-terminal residues remained to be the most flexible residue regardless of the glycerol concentration.
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