| Collagen is the main component of the raw hide used in the tanning process.As a biomaterial,the practical applications of collagen are based on its unique triple-helical structure.According to previous reports,the structural stability of collagen extracted from various organisms were greatly different,which was speculated to be closely related to the content of hydroxyproline and the composition of other amino acids.However,the existing experimental methods cannot further clarify the internal mechanism affecting the stability of collagen triple-helical structure.With the rapid development of computational simulation technology,researchers construct protein models to analyze the effects of amino acid residues on protein structure at the atomic scale.At present,researchers have synthesized collagen-like peptides based on the typical amino acid sequence of collagen(Gly-Pro-Hyp)n,combined with simulation to study the effect of single amino acid residues in the sequence on the structural stability of collagen-like peptides.However,the amino acid sequence of collagen-like peptides is quite different from that of natural collagen,and the sequence structure is not completely consistent with that of natural collagen,so the stability mechanism of natural collagen structure cannot be explained,such as the analysis of the relationship between hydroxyproline content and collagen thermal stability.If the model can be constructed based on the amino acid sequence of natural collagen molecule,it is expected to more effectively analyze and explore the stabilization mechanism of the triple-helical structure of natural collagen molecules.In the present work,the properties of mammalian and fish collagens were compared and the amino acid sequence and composition of the two collagens were statistically analyzed.Then,a triple-helical structure model based on the natural amino acid sequence of collagen was constructed using simulation and its rationality was verified.Based on this,traditional experiments and simulations were used to explore the internal mechanism of the influence of hydroxyproline content and other amino acid residues on the stability of collagen triple-helical structure.Combined with the practical application,the collagen model was introduced into the water solvent system to further improve the stabilization mechanism of the collagen triple-helical structure.Finally,the established model was used to explore the influence rules and mechanism of different alcohols on the properties of collagen materials,which will be beneficial for providing theoretical guidance and practical basis for collagenous biomaterials.Collagen was extracted from bovine skin,Southern catfish skin and Grass carp skin for performance testing.The results of ultraviolet and Fourier transform infrared spectra showed that the extracted collagens had high purity and complete triple-helical structure.The result of scanning electron microscopy showed that the average pore size of bovine skin collagen was 292.61± 33.53 μm,which was larger than that of fish skin collagens.In addition,the thermal stability of the three kinds of collagen was positively correlated with the hydroxyproline content.The thermal denaturation temperatures of bovine,Southern catfish and Grass carp skin collagen were 39.9 ℃,33.9 ℃ and 30.3℃,respectively.In order to explore the reasons for the difference in collagen properties,the amino acid sequences of collagen from bovine and Grass carp skin were statistically analyzed.The results showed that the two α chains of collagen were highly homologous and the amino acid residues in the same position were basically the same.In addition,the amino acids in the two kinds of collagen sequences have similar distribution.Imino acids and alanine were uniformly distributed in the whole sequence,and proline in the Yaa position was usually converted to hydroxyproline;the distribution of acidic and basic amino acids was concentrated,and the same coding position of three α chains was generally the same amino acid.Hydrophobic amino acids(such as phenylalanine and leucine) occurred only at Xaa and were concentrated to form a local hydrophobic environment.In addition,the difference between the two collagen amino acid sequences was mainly reflected in the proportion of some amino acids.For example,the content of imino acids in bovine skin collagen was significantly higher than that in Grass carp skin collagen,while the content of alanine in Grass carp skin collagen was higher than that in bovine skin collagen.The above differences may be the reasons for the different properties of collagen.In order to further study the influence of various amino acids in collagen molecular sequence on its properties,Materials Studio 6.0 software package was used to construct the model.Combined with the structural parameters of existing collagen molecules,the arrangement of three α chains of collagen and the hydrogen bond connection between the chains were determined.The results of the energy optimization algorithm showed that the Steepest Descent method consumed the least computing resources and the structure was the most stable.The screening results of charge charging method showed that the model energy was the most accurate when using Charge using Gasteiger.The optimization results of force fields showed that the output results of Dreiding was suitable for the structural stability analysis of the model.The triple-helical structure model of collagen with natural amino acid sequence was constructed by the optimized parameters,which provided the basis for the subsequent calculation and simulation experiments.To date,researchers generally believe that the structural stability of collagen molecules is related to hydroxyproline content,but the influencing mechanism remains unclear.Therefore,according to the proposed collagen modeling method and parameters,four groups of grass carp skin collagen models with hydroxyproline content of 4.4,7.8,11.1 and 15.6% were constructed,and the hydrogen bond energy of the collagen molecule was 17.74 J/g.Then,thermal-stability analysis indicated that the hydrogen bond breaking energies of lyophilized collagen and collagen solution were 18.38 J/g and 17.98 J/g,respectively,which verified the rationality of the established models.In addition,the kinetic simulation results of models showed that,with the increase of hydroxyproline content,the interchain hydrogen bond energy of the models at 308 K decreased by 14.251,10.352,7.970 and 1.247 kcal/mol,respectively,indicating that the thermal stability of the models increased gradually.The simulation result the of thermal denaturation of collagen model showed that the unwinding of triple-helical structures did not occur simultaneously in the molecular denaturation process of collagen,but the chain opening occurred first in the unstable region with less hydroxyproline content.Subsequently,10 groups of bovine collagen models were established to verify the results.The results showed that the interchain hydrogen bonding of the bovine collagen model was generally stronger than that of Grass carp skin collagen under the same hydroxyproline content.Therefore,the content of hydroxyproline was only one of the factors affecting the stability of collagen structure,and the content and distribution of other amino acids will also have an impact on the stability of collagen.In order to further explore the influence of various amino acids in the collagen molecular sequence on its structural stability,the distribution of 20 amino acids in Xaa and Yaa in the collagen tripeptide of bovine skin was statistically analyzed.According to the results,the first 20 tripeptides with high occurrence probability(62.9% in total)were selected,and 13 sequences were selected to build the model.The results of energy analysis showed that the existence of hydrophobic amino acids can increase the bond angular energy of the models and which was not conducive to the stability of the chain structure.The pyrrole ring in the imino acid was beneficial to the structural stability by improving the rigidity of the chain segment,and the hydrogen bonding between the model chains was positively correlated with the content of hydroxyproline.Similarly,the increase of acidic,basic and hydrophilic amino acid content can also strengthen the hydrogen bonding between model chains.In addition,the electrostatic interaction between the side chains of the model was also conducive to the stability of collagen structure,which was related to the number and distribution of acidic and basic amino acids in the sequence.Analysis of the structural stability mechanism in the microenvironment of tripeptides showed that the presence of acidic,basic or hydrophilic amino acids near hydroxyproline could stabilize the collagen structure by forming additional hydrogen bonds.When the amino acids were concentrated and the adjacent amino acids had opposite charges,the electrostatic interaction between the side chain R groups can improve the stability of the chain structure.However,the repulsion caused by R groups with the same charge was not conducive to structural stability.In addition,the heating results of the model further verify the above mechanism.The production,processing and preparation of collagen-based materials all involve the solution state of collagen.Therefore,the stabilization mechanism of collagen molecules in solution is particularly important.In the present work,the collagen model was introduced into the aqueous solution system.Based on the study of various amino acids on its structural stability,the influence mechanism of water molecules on the structural stability of collagen was studied.The results of the radial distribution function of water molecules showed that the tripeptide microenvironment affected the stability of collagen structure by disturbing the hydration layer.Serine was hydrophilic at Xaa while hydrophobic at Yaa.Both acidic and basic amino acids showed good hydrophilicity,and the hydrophilicity was better at the position of Xaa.However,continuous acidic or basic amino acids were not conducive to structural stability due to side chain repulsion.The hydrophobicity of alanine at the position of Yaa was stronger than that at the position of Xaa,and leucine and phenylalanine at Xaa have strong ability to repel water molecules.In addition,among the GPO tripeptides in collagen,proline and hydroxyproline showed hydrophobic and hydrophilic properties,respectively.The water molecules surrounding the GPO tripeptide could participate in the formation of water bridge bonds between the chains to improve the stability of the structure.The results showed that the hydrophilicity of microenvironment could improve the stability of the chain by stabilizing the hydrophobic layer,while the destruction of the hydrophobic layer was not conducive to the stability of the chains structure.Based on the constructed collagen model,the present work provided a systematic study of the effects of monohydric alcohols on collagen properties in solution,as well as the specific effect of the location and number of hydroxyl groups in polyhydric alcohols.The results of Ultra-sensitive differential scanning calorimetry showed that the denaturation temperature of collagen decreased from 42.43℃ to 37.85℃ with the increase of the alcohol molecular carbon chain.Polyhydric alcohols could increase the thermal denaturation temperature of collagen,and it was more obvious with the increase of the number of hydroxyl groups in alcohol molecules.It was worth noting that 1,2-propanediol(42.96℃)and 1,3-propanediol(42.25℃),which also have two hydroxyl groups,had opposite effects on the thermal denaturing temperature of collagen.The results of intrinsic fluorescence measurement and dynamic light scattering analysis showed that the aggregation behavior of collagen molecules in solution was affected by different alcohol systems and the influence rules was almost the same as the thermal stability.The results of the calculation of dissolution free energy confirmed the aggregation behavior of collagen molecules in different alcohol systems.The radial distribution function results showed that the g(x)of water molecule decreased from 1.40 to 0.92 with the increase of carbon chain of alcohol molecule,and with the increase of hydroxyl groups,g(x)increased from 1.00 to 1.42.The analysis of the interaction mechanism between alcohol and collagen molecules showed that the acetic acid roots,hydrogen ions,and alcohol molecules interact to form a complex network of bridge bonds between collagen molecules.This kind of bridge bond brought the two molecules closer,which facilitated the aggregation of collagen molecules,but led to a decrease in its structural stability.In addition,the special position of hydroxyl groups in the 1,3-propanediol molecule enabled it to cooperatively form chain-bridge bond structures between collagen molecules. |
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