Unravelling the Role of Hydroxyproline in Maintaining the Thermal

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Unravelling the Role of Hydroxyproline in Maintaining the Thermal Stability of the Collagen Triple Helix Structure Using Simulation Songcheng Xu, Min Gu, Kun Wu, and Guoying Li J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b05006 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

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The Journal of Physical Chemistry

Unravelling the Role of Hydroxyproline in Maintaining the Thermal Stability of the Collagen Triple Helix Structure Using Simulation

Songcheng Xua, Min Gub, Kun Wuc*, Guoying Li a,b*

aKey

Laboratory of Leather Chemistry and Engineering (Ministry of Education) Sichuan University, Chengdu 610065, PR China;

bNational

Engineering Laboratory for Clean Technology of Leather Manufacture, Sichuan University, Chengdu 610065, PR China;

c School

of Materials and Environmental Protection, Chengdu Textile College, Chengdu 610065, PR China;

*Corresponding author: Guoying Li E-mail address: [email protected] Kun Wu E-mail address: [email protected] Tel.: +86 028 85462568 Fax: +86 028 85405237

ACS Paragon Plus Environment

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Abstract The thermal stability of collagen has an important effect on its practical applications. Many believe that hydroxyproline (Hyp) improves the structural stability of collagen molecules. In this study, for the first time, a method of building natural collagen molecular models was described. We constructed a collagen model with typical triple helix structure and calculated the hydrogen bond energy between collagen alpha chains. The calculated hydrogen bond energy was consistent with the experimental results of differential scanning calorimetry (DSC). After the calculation simulation, we verified that the hydrogen bond energy between collagen chains was positively correlated with Hyp content in the models and an increased Hyp content in the model was beneficial in improving the thermal resistance of the structure. In addition, we found that thermal unfolding did not occur simultaneously along the entire molecule, but started in the regions with less Hyp content. This study provides a collagen model with a natural collagen amino acid sequence, which will be helpful for further investigation of the physical and chemical properties of natural collagen.

1. Introduction Collagen is the most predominant protein, representing approximately 30% of total proteins in the body. It is the major component of several connective tissues such as tendon, skin, bone, teeth, cartilage, and basement membranes.1-4 One of the defining features of collagen is its unique triple helical structure, consisting of three parallel left-handed polypeptide chains wound around a common axis to form a triple helix with a right-handed superhelical pitch. Due to supercoiling, the formation of a triple helix requires the presence of a repeated Gly-Xaa-Yaa sequence.5,

6

Xaa and Yaa

positions are often occupied by proline (Pro), and proline in the Yaa position is usually converted to hydroxyproline (Hyp) by enzymatic posttranslational hydroxylation.7 The triple helical structure of the collagen molecule is held together 2


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by hydrogen bonds between the N-H group of the glycyl residue and the C=O group of the Xaa position of the triple structure in the neighboring chains.8

Collagen is widely used in medicine, foods, cosmetics, and cell cultures due to its low immunogenicity, good biodegradability, and biological activity.9, 10 As a protein, the practical applications of collagen are based on its unique triple helical structure. At a temperature above a critical level, collagen chains separate and fold into random coils, resulting in the thermal denaturation of collagen into gelatin. Collagen is mainly extracted from terrestrial and aquatic animals, and the thermal stability of collagen from various sources has been found to be significantly different. According to previous reports, the denaturation temperature of collagen is closely related to the living environment of the source,11-16 and the thermal stability of mammalian collagens is higher than fish collagen. The amino acid composition of collagens showed that the content of imino acids (especially Hyp) in the collagen of terrestrial animals was significantly higher than that of aquatic animals, and it was speculated that Hyp could improve the thermal stability of collagen.17,18 Thus far, native collagen and collagen-like domains have not yielded to crystallization and structure determination, so the effect of Hyp on the stability of collagen cannot be directly studied. Fortunately, it has proved possible to solve the crystal structures of collagen-like peptides.19 In the 1970’s, researchers synthesized collagen-like peptides (Pro-Gly-Pro)n to study the factors that affect the stability of collagen.20,

21

Subsequently, high resolution collagen-like peptides have been reported to confirm the basic triple-helical model proposed from fiber diffraction studies, and elucidated in detail the hydrogen-bonding patterns.22-27 To further study the relationship between amino acids and the thermal stability of collagen, Persikov et al. used a systematic host-guest peptide approach to establish a propensity scale for all 20 possible amino acids in the Xaa and Yaa positions.28 After circular dichroism spectroscopy measurements, the denaturation temperatures of all samples were determined, and the 3



results showed that Pro was the most stabilizing residue in the Xaa position, and that the maximal stabilization at the Yaa position involved Hyp. In addition, Persikov’s group proposed an algorithm based on the triple helical stabilization propensities of individual residues and their intermolecular and intramolecular interactions, as quantitated by melting temperature values of host-guest peptides. Experimental melting temperature values of a number of triple helical peptides of varying length and sequence were successfully predicted by this algorithm.29 However, predicted Tm values were significantly higher than experimental values. So far, the investigators synthesized samples with Gly-Pro-Hyp repeats of the collagen alpha chain to study the thermal stability of collagen. Based on their results, they proposed that Hyp could improve the thermal stability of collagen. However, studies have focused on peptides with idealized sequences to elucidate the stability of the triple-helix conformation, and the amino acid sequence of natural collagen is not a regular (Gly-Pro-Hyp)n repetitive structure. Other amino acid residues may occupy the X and Y positions, thus affecting the thermal stability of collagen. Therefore, there were limitations in studying the thermal stability of collagen through synthetic non-natural collagen fragments.

Due to the complexity of the amino acid sequence of natural collagen peptides, it is not yet possible to directly study the relationship between the composition of collagen peptides and the thermal stability of collagen. In recent years, increasing attention has been directed to molecular modeling.30-34 Molecular modeling approaches can provide a detailed analysis of collagen structure at the atomic scale, and can characterize changes of molecules during the process of thermal denaturation.35 In the 1970’s, Gerig et al.36 investigated the conformations of cis- and trans-4-fluoro-L-proline in aqueous solution by computer calculations with a local version of the LAOCN3 program on an IBM 360/75 computer. Both molecules were found to be in envelope conformations. Panasik et al.37 synthesized the peptides of N-acetylproline methyester (1), N-acetyl-4(S)-hydroxyproline methylester (2), and N-acetyl-4(S)-fluoroproline 4


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methylester (3). The structures of these compounds were determined by X-ray diffraction analysis, and were also simulated by ab initio molecular orbital calculations at the RHF/3-21G level of theory. The results indicated that electron-withdrawing substituent in the 4-position of Pro residues had a significant influence on the structure of these residues. Subsequently, investigators studied the role of Pro in the structure of the triple-helix of collagen. Importa et al.38 presented a quantum mechanical/molecular mechanical study of (Pro-Pro-Gly)n polypeptides, to determine the influence of Pro puckering on the stability of the collagen triple helix. MM computations in vacuo had been performed using the AMBER6 package with the 1994 parameters and atomic charges. The results of these calculations showed that the thermal stability of the triple-helix increased if the imino acid in the X position adopted a down puckering and the imino acid in the Y position adopted an up puckering. However, collagen is a more complex system than a (Pro-Pro-Gly)nmodel. It is difficult to understand the factors affecting the thermal stability of collagen through simple modeling calculations. Jordi bella et al. determined the structure of a protein triple helix at 1.9 angstrom resolution by x-ray crystallographic. They found that the breaking of the repeating (Gly-Xaa-Yaa)n pattern by a Gly→Ala substitution results in a subtle alteration of the conformation, with a local untwisting of the triple helix.39In recent years, investigators have constructed fragments of collagen molecule to investigate problems that could not be solved by traditional studies. Singam et al. reported that the structural effect on the heterotrimeric models with G4G and G1G breaks present simultaneously in the constituent chains with difference in one residue chain staggering and found the structural parameters of hydrogen-bonding pattern differs significantly due to the difference in the staggering of chains.40Almora-Barrios et al.41 used the Materials Studio, version 4.0 package to construct three chains of [-OOC-(GLY-PRO-HYP)10-NH2+]3 folded into a triple-helix collagen fragment. By immersing the constructed collagen in a stoichiometric solution of Ca+, PO43-, and OH- ions, they observed the formation of calcium phosphate clusters at the collagen 5



template. Through computer simulation, they investigated the initial stages of nucleation and cluster formation of calcium phosphate for a collagen molecule fragment in aqueous solution. According to previous reports, the investigators have studied the thermal stability of collagen based on the model (Gly-Pro-Hyp)n, but ignored the effects of other amino acids on the structural stability of collagen. They only focused on the effects of amino acid composition on the thermal stability of the collagen with idealized sequences, whereas little information was available about how the collagen trimer whose peptides were formed by natural amino acid sequences changed during thermal denaturation. Meanwhile, whether the performance of the constructed model was consistent with natural collagen was not further verified. It is therefore necessary to construct collagen fragments with natural amino acid sequences and find a way to predict the properties of collagen molecules using the constructed collagen models.

This paper aimed to construct reasonable collagen models according to the amino acid sequence of natural collagen. The models were simulated and calculated to further explore the effect of Hyp content on molecular thermal stability and the mechanism of collagen in the process of thermal denaturation. In order to select the collagen fragments with different Hyp contents to construct models, we compared the amino acid sequences of the skin collagen of calf, pig, and other organisms, and found that the distribution of Hyp content in diverse regions in the amino acid sequence of grass carp (Ctenopharyngodon idellus) collagen met the requirements of modeling. Fragments with different Hyp content in the collagen of grass carp were selected to build models using the Materials Studio, version 6.0 package (Key Laboratory of the Ministry of Education for Advanced Catalysis Materials, Institute of Physical Chemistry, Zhejiang Normal University, Zhejiang, China). After structural optimization and energy minimization, the energy of hydrogen bonds of each model was calculated. In addition, Collagen extracted from grass carp skin was investigated 6


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by DSC, and the enthalpy change energy of collagen in the process of denaturation was determined. Based on the relationship between Hyp content and hydrogen bond energy of the model fragments, a mathematical model was proposed, and the hydrogen bond energy of the complete collagen molecule was predicted by the mathematical model. The value of hydrogen bond energy estimated by the mathematical model was compared with the experimental results of DSC to verify the rationality of the models. Successively, we selected a fragment from the collagen amino acid sequence of grass carp, which had multiple regions with different Hyp contents, to construct the model. The changes of triple helix in the constructed model during thermal denaturation were simulated by computer calculations.

2. Materials and methods 2.1. Construction of structural models Grass carp type I collagen was chosen to construct the models. The amino acid sequence of grass carp skin collagen was obtained from the UniProtKB (Entry name: E2GK07(α1), E2IPR2(α2)). The amino acid number of α1 and α2 is 1448 and 1352 respectively, and polypeptide chain is mainly composed of signal peptides, N-terminal, helical regions and C-terminal. In this paper, the amino acid sequence of the helical regions was chosen to construct the collagen models. Figure S1 showed the complete amino acid sequence of grass carp skin collagen, and the collagen molecule was divided into 33 fragments with the length of 30 amino acids in a single α chain. Pro in Yaa position is converted to Hyp followed by enzymatic posttranslational hydroxylation.7 According to the content of Hyp in different fragments, four fragments were selected to construct models. The amino acid sequences of the four fragments are listed in Table S1. With the increase of Hyp content, the collagen mimics were referred to as CM1, CM2, CM3, and CM4, sequentially. Collagen structures were modeled using the Materials Studio, version 6.0 package. Figure S2 showed the schematic diagram of model construction. Two α1 chains and 7



one α2 chain were constructed respectively according to the amino acid sequence of polypeptide chain, and the three chains were arranged parallel in a way that staggered longitudinally by one residue. The directions of NH groups of glycine residues and backbone CO groups of residues in Xaa position were adjusted to helix axis. The R groups of side chain amino acids extend outwards to the collagen molecule. Figure S3 showed the schematic diagram of top view in the direction of the helix axis. The structures were refined by energy minimization and the energy of hydrogen bonds was calculated. More details of the methods of model-construction and optimization are shown in the Supporting information.

2.2. Materials Fresh grass carp were purchased from a local market in Chengdu, Sichuan Province, China. The skins were manually removed with a filleting knife and then washed with cold distilled water. The cleaned skins were cut into small pieces (0.2 × 0.2 cm) manually and the skin pieces were treated with 0.1 M NaOH to remove non-collagenous proteins at a solid to solution ratio of 1:30 (w/v), for 12 h, with the solution changed every 4 h. Residual fat was removed in 0.5% (v/v) nonionic degreasing agent with a sample/solution ratio of 1:20 (w/v), for 24 h, with a change of solution each 12 h. To remove pigments more effectively, defatted skins were bleached with 3% H2O2 solution for 24 h and the solution was changed once. Collagen was extracted from the prepared fish skins following the method of Zhang et al.6 with a slight modification. All processes were performed at 4°C with gentle stirring. The prepared skins were washed with distilled water, followed by soaking in 0.5 M acetic acid containing 1% (w/w) pepsin (1:10,000; EC 3.4.23. 1; Sigma-Aldrich, St. Louis, MO, USA) at a sample: solution ratio of 1:30 (w/v) for 48 h. The suspensions were centrifuged at 9000 × g for 10 min at 4°C. The precipitates were salted out from the supernatants by adding NaCl to a final concentration of 0.7 M and the precipitate was dissolved in 0.5 M acetic acid. The resulting solutions were 8


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dialyzed against 0.05 M acetic acid for 3 days, with a change of solution every 12 h. Finally, the collagen solution was lyophilized using a freeze dryer (Labconco FreeZone (6 L); Ft. Scott, KS, USA) at -50°C for 2 days and stored in a desiccator until used. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to confirm purity and molecular weights of the extracted collagen.42 Grass carp skin collagen displayed two α bands (α1 and α2) and one β band in the electrophoretic patterns (as shown in Figure S4, Supporting information), which indicated that the extracted collagen had typical electrophoretic bands of type I collagen and preserved the original triple-helical structure.

2.3. Thermal stability measurements of lyophilized collagen DSC of grass carp collagen was run following the method of Foucat and Renou43 with a slight modification.(Netzsch DSC STA499C; Bavaria, Germany)Lyophilized collagen (5–10 mg) was accurately weighed into aluminium pans and sealed, and the pans were punctured. The samples were scanned over a temperature range of 20°– 60°C at a heating rate of 2 K/min in a nitrogen atmosphere. During the heating process, two thermostatic stages were set at 30°C and 35°C for 10 min to remove the water from the samples. An empty punctured aluminium pan was used as the reference. During the scanning process, the change of sample weight was monitored simultaneously. Total denaturation enthalpy was estimated by the area in the DSC thermogram.

2.4. Thermal stability measurements of collagen solution The thermal transition of collagen solution was determined by DSC. Dried collagen was dissolved in 0.5 M acetic acid to different concentrations (0.5 mg/mL, 2 mg/mL, 4 mg/mL, 6 mg/mL, and 8 mg/mL) (as shown in Figure S5, Supporting information), and ~10 mg samples were weighed in aluminium pans, sealed, and then scanned over a temperature range of 10°–50°C at a heating rate of 2 K/min in a nitrogen 9



atmosphere. A sealed aluminium pan was used as the reference. Total denaturation enthalpy was estimated by the area in the DSC thermogram. Dried collagen was dissolved in 0.5 M acetic acid to different concentrations, and 2 M guanidine hydrochloride of the same volume was added to mix with the collagen solution for 20 min at 4°C. The final collagen concentration in the solution was 0.5, 2, 4, 6, or 8 mg/mL. The thermal transition of the mixed solution was determined using DSC as described above.

2.5. Simple linear regression The relationship between the Hyp content and the hydrogen bond energy of the models can be expressed as: Y=aX+b Where a denotes the changing rate of hydrogen bond energy related to the Hyp content of models, and b is the intercept. By extending the regression equation between the Hyp content and the hydrogen bond energy of the models, the hydrogen bond energy of intact collagen molecules can be predicted. The regression equation was considered acceptable only if the determination coefficient (R2) was ≥ 0.95.44

2.6. Heating simulation of collagen models The four collagen models were used to simulate the relationship between Hyp content and thermal stability of collagen. Each model simulated 200 ps at the temperature of 298 K, 308 K, and 325 K, respectively, and the time step was 1 fs. The equilibration period was 200 ps, in which all the atoms were free to move in the NVT resemble (constant number of particles, volume and temperature). The temperature was fixed using the Velocity Scale thermostant(as shown in Table S4, Supporting information). For collagen structure optimization and heating simulation, the Dreiding force field was used. This force field was useful for predicting structures and dynamics of organic, biological, and main group inorganic molecules.45 After heating simulation, 10


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the hydrogen bond energy values of the models were calculated. All simulation processes were performed in triplicate.

2.7. Detection of hydrogen bonds in the heating simulation To understand the changes of the collagen triple helix during the process of thermal denaturation, we selected a fragment of 270 amino acid residues in the sequence of grass carp collagen, which has a significant difference in Hyp content in diverse regions (as shown in Table S5), to construct the model. From N-terminal to C-terminal, the content of Hyp in each 90 amino acid residue was 4.4%, 20%, and 11.1%. According to the modeling method mentioned in Section 2.1, the collagen fragment was constructed and the energy of structure was refined. The optimized model was placed at a temperature of 308 K for denaturation simulation under the Dreiding force field. The total simulation time was 100 ps and the time step was 0.1 fs. The calculation results were outputed every 5,000 steps. In the three regions with different Hyp content, seven hydrogen bonds were randomly selected, and the changes of hydrogen bond length during the process of thermal denaturation were calculated.

2.8. Statistical analysis The experimental data are expressed as the mean± standard deviation (SD), and the results of each independent experiment were based on repetitive samples (n = 3). One-way analysis of variance with Tukey’s multiple comparison tests were used to determine the statistical significance. Differences were considered significant at a value of P < 0.05.46

3. Results 3.1. Collagen models After structural optimization, four optimized collagen models were obtained. The 11



horizontal and longitudinal optimized models are shown in Figure 1 and Figure 2 respectively. Each model showed a typical triple helix structure. The models contained three polypeptide chains that exhibited a left-handed helix. The three chains were then wrapped around each other into a right-handed super-helix, showing a rope-like rod. The lengths of the four models were 91.15 ± 0.23, 91.55 ± 0.20, 91.43 ± 0.18, and 90.40 ± 0.28 Å (p > 0.05), respectively, which were similar to previously reported results.47 The data of calculated energy are shown in Table 1. With the increase of Hyp content, the total energies of the four optimized models were 1599.607, 1435.100, 1471.858, and 1747.020 kcal/mol, respectively. The total energy of each model was in a low state, which indicated that the optimized models were reasonable and close to the natural conformation of collagen molecules. In addition, the formation of hydrogen bonds between the chains of collagen reduced the energy of the system, which was conducive to the stability of the triple helix structure. The data from Table 1 showed that the hydrogen bond energy rose with the increase of Hyp content in the models, indicating that the Hyp residue could create additional hydrogen bonds between collagen chains to increase the thermal stability of collagen molecules.

12


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Figure 1. The optimized molecular models (horizontal)

13



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Figure 2. The optimized molecular models (longitudinal)

Table 1. The energy (kcal/mol) of the optimized models CM1

CM2

CM3

CM4

Total energy

1599.607

1435.100

1471.858

1747.020

Valence energy

895.136

774.886

829.671

1018.639

Bond

115.034

125.357

122.650

125.248

Angle

461.469

441.813

439.407

475.101

Torsion

301.622

202.891

258.396

399.895

Inversion

17.012

4.826

9.218

18.396

Non-bond energy

704.471

660.214

642.187

728.381

Hydrogen bond

-34.894

-37.896

-40.192

-42.850

van der Waals

404.649

370.455

364.420

416.364

Electrostatic

334.716

327.655

317.959

354.867

3.2. Thermal properties of dried collagen In order to obtain the hydrogen bond energy between collagen molecular chains, the thermal denaturation of collagen was analyzed by DSC and thermogravimetric analysis (TG), and the thermal denaturation temperature and calorimetric enthalpy changes (ΔH) were determined. The DSC and TG curves of lyophilized collagen are shown in Figure 3. From the TG curve, the weight of the sample decreased from 98.729% (point a) to 94.668% (point b) at the first thermostatic stage (stage 2), indicating that the water in the sample evaporated with the heating process. In the second thermostatic stage (stage 4), there was little change in sample weight [from 94.615% (point c) to 94.468% (point d)], indicating that the moisture in the sample had been removed. According to the DSC curve, in the final heating stage (stage 5), the endothermic peak value of collagen was 46.0°C and the ΔH of this peak was 18.38 J/g. 14


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Figure 3. TG and DSC thermograms of dry collagen. Point a, b, c and d represents the weight of the samples during the heating process.

3.3. Thermal properties of collagen solution The energy of hydrogen bonding between collagen molecular chains in the solution was determined. DSC results of collagen solutions with different concentrations showed that collagen concentration had no significant influence on ΔH (as shown in Figure S5, Supporting information). Figure 4 shows the DSC curves of the collagen solution, and shows that curve A is the thermogram of collagen solution with a concentration of 2 mg/mL, and curve B is the thermogram of collagen solution with the addition of guanidine hydrochloride. The endothermic peak, with maximum temperature, was observed for curve A, indicating that the triple helical structure of collagen in solution caused transformation into random coils at 32.6°C.After the collagen solutions were treated by guanidine hydrochloride, the triple helix of collagen was destroyed, so that there was no obvious endothermic peak on curve B. The calorimetric enthalpy change of collagen solution before and after denaturation was 17.98 J/g and 0 J/g, respectively, showing that the hydrogen bonding energy between collagen chains was 17.98 J/g. 15



Figure 4. DSC thermograms of collagen solution. Curve A represents the thermogram of collagen solution with a concentration of 2 mg/mL. Curve B represents the thermogram of collagen solution with the addition of guanidine hydrochloride.

3.4. Simple linear regression According to the calculations of hydrogen bond energies of the models in Table 1, the hydrogen bond energy values of the four models, 34.894, 37.896, 40.192, and 42.850 kcal/mol, were converted into 16.07, 17.45, 18.71, and 19.73 J/g, respectively. The linear relationship between Hyp content and hydrogen bond energy of the models is shown in Figure 5. The determination coefficient (R2) of the regression equation was 0.9709, suggesting that the model provided a good linear relationship between the Hyp content and hydrogen bond energy.48 The Hyp content in the collagen molecules of grass carp is about 8.95%. According to the regression equation, the energy of the hydrogen bonds between molecular chains was calculated to be 17.74 J/g.

16


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Figure 5. Linear regression of the Hyp content and hydrogen bond energy of fragments

3.5. Heating simulation of collagen models The results of heating simulation showed that when the molecular fragments were in a 298 K environment, there was no significant change in the hydrogen bond energy. When the models were in a 325 K temperature environment, the hydrogen bonds between molecular chains were almost all broken, so it was difficult to determine the effect of Hyp content on the structural stability of collagen. Based on these findings, the simulation models were carried out at a temperature close to the collagen denaturation temperature (308 K) (as shown in Table S4, Supporting information). The energy changes of hydrogen bonds after the heating simulation of collagen models are shown in Table 2. The Hyp contents of CM1, CM2, CM3, and CM4 were 4.4%, 7.8%, 11.1%, and 15.6%, respectively. With the increase of Hyp content in the models, the energies of hydrogen bonding were 34.894, 37.896, 40.192, and 42.850 kcal/mol, respectively. After heating simulation in the 308 K temperature 17



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environment, the hydrogen bond energy of the four models varied significantly (p < 0.05). The hydrogen bond energies of CM1, CM2, CM3, and CM4 were reduced by 14.251 ± 2.160, 10.352 ± 1.512, 7.970 ± 2.418, and 1.247 ±1.007 kcal/mol, respectively.

Table 2. Changes in hydrogen bond energy (kcal/mol) of the simulated models after heating Average H-bond energy after Energy of H-bond

Energy reduction heatinga

CM1

34.894

20.643±2.160

14.251±2.160

CM2

37.896

27.544±1.512

10.352±1.512

CM3

40.192

32.222±2.418

7.970±2.418

CM4

42.850

41.603±1.007

1.247±1.007

a: Data are represented as mean± SD (n=3, p

Unravelling the Role of Hydroxyproline in Maintaining the Thermal

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