Method and system for scoring collagen thermal stability

Abstract

The application discloses a collagen thermal stability scoring method and system, and the collagen thermal stability scoring method comprises the following steps: constructing a thermal stability scoring database; inputting a target sequence, judging the type of the target sequence according to the peptide chain composition of the target sequence; when the target sequence is a heterotrimer, performing permutation and combination on the target sequence to obtain all possible triple helix structures, and performing thermal stability scoring on the target sequence after permutation and combination based on the thermal stability scoring database; when the target sequence is a homotrimer, directly performing thermal stability scoring on the target sequence based on the thermal stability scoring database; and different scoring strategies are adopted for long and short target sequences in the thermal stability scoring process. The collagen thermal stability scoring method can be used for guiding the design of a recombinant collagen sequence, can shorten the research and development cycle of collagen products, and can provide more innovative choices and application possibilities for the medical material field.

Description

Technical Field

[0001] This application relates to the fields of bioinformatics and biocomputing science and technology, and in particular to a method and system for scoring the thermal stability of collagen. Background Technology

[0002] Collagen, a key structural protein in eukaryotes, plays a crucial role in many fields such as medicine and aesthetics due to its thermal stability. Current research methods primarily rely on extensive experimental data to predict thermal stability, which is not only costly but also extremely time-consuming. For heterotrimers, due to the complex interchain interactions, existing research methods based on homotrimers are not well-suited for effectively characterizing their thermal stability. Therefore, there is an urgent need for a simple, accurate, and efficient method for predicting collagen thermal stability applicable to different collagen types. Summary of the Invention

[0003] The purpose of this application is to overcome the shortcomings of the prior art and provide a method and system for scoring the thermal stability of collagen.

[0004] In a first aspect, this application provides a method for scoring the thermal stability of collagen, comprising the following steps: Construct a thermal stability scoring database; Input a target sequence, and determine the type of the target sequence based on its peptide chain composition; When the target sequence is a heterotrimer, the target sequence is permuted and combined to obtain all possible triple helix structures, and the thermal stability of the permuted and combined target sequences is scored based on the thermal stability scoring database. When the target sequence is a homotrimer, the thermal stability of the target sequence is directly scored based on the thermal stability scoring database. In the thermal stability scoring process, the target sequence includes: a short target sequence and a long target sequence; for the short target sequence, it is split into multiple YX and YX' motifs, and the above motifs are matched with the information in the thermal stability scoring database. According to the matched motifs, the corresponding scores are obtained and summed to obtain the thermal stability score of the short target sequence. For a long target sequence, a window is slid across the long target sequence, and the thermal stability scoring method described above for short target sequences is used to obtain the stability score of each window. The obtained thermal stability score is then designated as the local thermal stability score of the triplet at the center of the window.

[0005] Optionally, for the short target sequence, if the YXX' motif exists in the thermal stability scoring database, the score of the YXX' motif is used to replace the scores of the YX and YX' motifs.

[0006] Optionally, for the short target sequence, if there is a continuous salt bridge in the short target sequence, the score of the short target sequence obtained based on the thermal stability scoring database is used as the initial score. The difference between the initial score and the half score of the less stable motif between the previous YX' motif and the next YX motif in the continuous salt bridge is used as the final score of the short target sequence.

[0007] Optionally, the construction of the thermal stability scoring database includes: A virtual sequence was constructed, and temperature-scanning molecular dynamics simulations were performed on the virtual sequence. The average root mean square deviation of the main chain atoms in the temperature scanning molecular dynamics simulation was analyzed using analytical functions to obtain a scatter plot, and the unwinding temperature value of the temperature scanning molecular dynamics simulation was obtained by fitting. A thermal stability baseline sequence is determined, and site-directed mutagenesis is performed on the YXX' triplet in the virtual sequence to obtain a mutant. The melting temperature difference between the mutant and the thermal stability baseline sequence is calculated, and a thermal stability scoring database is obtained based on the melting temperature difference.

[0008] Optionally, the construction of the virtual sequence includes: using [(Gly-Pro-Hyp)] 10 Using the crystal structure of ]3 as a template, a capped collagen-like polypeptide structure was constructed, with N-terminus acetylation and C-terminus N-methylation to obtain the virtual sequence.

[0009] Optionally, the construction of virtual sequences further includes: truncating multiple GPO triplets in each virtual sequence, and each virtual sequence containing multiple sets of YXX' triplet mutations.

[0010] Optionally, performing temperature-scanning molecular dynamics simulations on the virtual sequence includes: using the last frame of the previous temperature-scanning molecular dynamics simulation as the starting structure for the temperature-scanning molecular dynamics simulation at the next temperature, and repeating the above-mentioned temperature-scanning molecular dynamics simulation multiple times for each structure.

[0011] Optionally, obtaining the unbreaking temperature value from the temperature scanning molecular dynamics simulation by fitting includes: obtaining the unbreaking temperature value from the temperature scanning molecular dynamics simulation by fitting a Boltzmann curve.

[0012] Secondly, this application also provides a collagen thermal stability scoring system, comprising: The thermal stability scoring database acquisition module is used to construct the thermal stability scoring database. A thermal stability scoring module is used to input a target sequence and determine the type of the target sequence based on its peptide chain composition. When the target sequence is a heterotrimer, the target sequence is permuted and combined to obtain all possible triple helix structures, and the thermal stability of the permuted and combined target sequences is scored based on the thermal stability scoring database. When the target sequence is a homotrimer, the thermal stability of the target sequence is directly scored based on the thermal stability scoring database. In the thermal stability scoring process, the target sequence includes: short target sequences and long target sequences. For short target sequences, they are split into multiple YX and YX' motifs, and these motifs are matched with information in the thermal stability scoring database. Based on the matched motifs, corresponding scores are obtained and summed to obtain the thermal stability score of the short target sequence. For long target sequences, a window is slid across the long target sequence, and the thermal stability scoring method for the short target sequence is used to obtain the stability score of each window. The obtained thermal stability score is designated as the local thermal stability score of the triplet at the center of the window.

[0013] This application provides a method and system for scoring the thermal stability of collagen. By constructing a thermal stability scoring database, it provides a foundation for scoring the thermal stability of different types of target sequences. By classifying and scoring thermal stability, it is possible to analyze the thermal stability characteristics of each type of collagen in detail. The overall thermal stability score and the local thermal stability score can quickly and accurately assess the thermal stability characteristics of collagen. The method and system of this application can provide strong support for the research and development of collagen products and greatly accelerate the optimization and commercialization process of collagen-related products.

[0014] To make the above-mentioned features and advantages of the invention more apparent and understandable, specific embodiments are described below, and detailed descriptions are provided in conjunction with the accompanying drawings. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments or related technologies of this application, the accompanying drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a flowchart of a collagen thermal stability scoring method provided in one embodiment of this application.

[0017] Figure 2 This is a flowchart of step S10 in a collagen thermal stability scoring method provided in one embodiment of this application.

[0018] Figure 3 This is a schematic diagram of a sequence pattern in a collagen thermal stability scoring method provided in one embodiment of this application.

[0019] Figure 4 This is a local thermal stability prediction diagram of a collagen thermal stability scoring method provided in another embodiment of this application.

[0020] Figure 5 This is a schematic diagram of the structure of a collagen thermal stability scoring system provided in another embodiment of this application. Detailed Implementation

[0021] To make the objectives and technical solutions of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the described embodiments of this application without creative effort are within the scope of protection of this application.

[0022] In one embodiment, see Figure 1 This application provides a method for scoring the thermal stability of collagen, which may include the following steps: S10~S60.

[0023] S10: Construct a thermal stability scoring database.

[0024] S20: Input the target sequence, and determine the type of the target sequence based on the peptide chain composition of the target sequence.

[0025] S30: When the target sequence is a heterotrimer, the target sequence is permuted and combined to obtain all possible triple helix structures, and the thermal stability of the permuted and combined target sequences is scored based on the thermal stability scoring database.

[0026] S40: When the target sequence is a homotrimer, the thermal stability of the target sequence is directly scored based on the thermal stability scoring database.

[0027] S50: In the thermal stability scoring process, the target sequence includes: a short target sequence and a long target sequence; for the short target sequence, it is split into multiple YX and YX' motifs, and the above motifs are matched with the information in the thermal stability scoring database. According to the matched motifs, the corresponding scores are obtained and summed to obtain the thermal stability score of the short target sequence.

[0028] S60: For a long target sequence, slide a window on the long target sequence, use the thermal stability scoring method of the short target sequence to obtain the stability score of each window, and designate the obtained thermal stability score as the local thermal stability score of the triplet at the center of the window.

[0029] In the collagen thermal stability scoring method of this application, a scoring database is constructed to provide rich data resources for subsequent thermal stability scoring of any target sequence; based on the thermal stability scoring database, the thermal stability of the target sequence is scored, which enables personalized thermal stability analysis for collagen sequences with different structural characteristics, and can comprehensively consider the influence of its sequence specificity on thermal stability.

[0030] In step S10, please refer to Figure 1 In step S10, a thermal stability scoring database is constructed.

[0031] For example, please refer to Figure 2 Step S10 may include the following steps: S11~S13.

[0032] S11: Construct a virtual sequence and perform temperature-scanning molecular dynamics simulations on the virtual sequence.

[0033] S12: Analyze the average root mean square deviation of the main chain atoms in the temperature-scanning molecular dynamics simulation using analytical functions to obtain a scatter plot, and obtain the unwinding temperature value of the temperature-scanning molecular dynamics simulation by fitting.

[0034] S13: Determine the thermal stability baseline sequence, perform site-directed mutagenesis on the YXX' triplet in the virtual sequence to obtain a mutant, calculate the melting temperature difference between the mutant and the thermal stability baseline sequence, and integrate the thermal stability scoring database based on the melting temperature difference.

[0035] As an example, in step S11, due to collagen's unique fibrous triple helix structure, interactions between residues separated by more than one or two triplets in sequence are negligible. Paired interactions in collagen are limited to two distinct interchain assembly structures. See also Figure 3 A peptide segment i Y residue at position (i.e. Figure 3 Y residues in each chain can interact with the left-hand adjacent chain. i The X residue at position -1 (i.e.) Figure 3 The X residues connected to the Y residues in the adjacent chain by the dashed line form a transverse interaction. Figure 3 (The dotted line in the middle is an illustration) and i +3 X residue (i.e. Figure 3The X residues connected to the Y residues in the adjacent chain by solid lines form axial interactions. Figure 3 (Solid lines in the diagram). Therefore, important interactions in collagen can often be characterized at the location of a YXX' triplet.

[0036] As an example, [(Gly-Pro-Hyp)] can be used... 10 Using the crystal structure of [3] (PDB code: 3B0S) as a template, a capped collagen-like polypeptide structure was constructed and chemically modified, with N-terminus acetylation and C-terminus N-methylation, to obtain the virtual sequence. Simultaneously, to reduce system size, each virtual sequence was truncated with multiple (e.g., 4) GPO triples; each sequence contained multiple sets (e.g., 3 sets) of YXX' triple mutations to increase the melting temperature. T m Distinctiveness, specific sequence patterns such as Figure 3 As shown.

[0037] Figure 3 In the sequence, ACE indicates acetylation modification at the N-terminus of the sequence, NME indicates N-methylation modification at the C-terminus of the sequence, and the middle part represents six short GPO triplets, with each sequence containing three sets of YXX' triplet mutations.

[0038] As an example, temperature-scanning molecular dynamics simulations can be performed in the Amber99sb*-ILDN force field of GROMACS2023, with water molecules using the TIP3P force field. Specifically, the constructed virtual sequence is placed in a dodecahedral box with periodic boundaries, and the distance from the box surface to the nearest protein atom is set to 10 Å. Solvation can be performed using the solvate module, and the necessary Na+ can be added via the -neutral command in the genion module. + or Cl -Ions maintain the overall system electroneutrality. Distances involving hydrogen bonding interactions are constrained, and the time step is set to 2 ps. Long-range electrostatic interactions are evaluated using the particle-grid Ewald method and interpolation with a sixth-order polynomial. The minimum distance threshold for electrostatic and van der Waals interactions can be set to 14 Å. System energy is minimized using the steepest descent method, and the system is equilibrated using 2 ns NVT and NPT simulations, maintaining the system temperature at 300 K during the equilibration simulations. In the NPT simulations, the pressure P = 1 bar is controlled using a weak coupling method. Subsequently, temperature-scanning molecular dynamics simulations are performed at a constant system volume, with the system temperature gradually increased from 300 K to 700 K in 20 K increments. MD simulations are run for 15 ns at each temperature, using the last frame of the previous temperature-scanning molecular dynamics simulation as the starting structure for the next temperature-scanning molecular dynamics simulation. Each structure is repeated at least three times (e.g., three times). The simulation results can intuitively reflect the dynamic changes of collagen molecules during the heating process, such as atomic displacement and conformational transformation, laying a solid foundation for accurately assessing thermal stability and making the study of thermal stability more scientific and reliable.

[0039] As an example, in step S12, after the simulation ends, a series of files recording atomic positions and motion information are generated. These files contain detailed data on the main chain atoms of the virtual sequence at different temperatures and time points. An analysis function specifically designed to calculate the average root mean square deviation (RMSD) is then called. During the function call, the input file can be specified as the atomic trajectory file generated by the simulation, and a reference structure can be specified, typically the initial structure. The function calculates the RMSD of the main chain atoms relative to the reference structure at each time step based on the input file, and then statistically averages these data to obtain the RMSD data of the main chain atoms throughout the entire simulation.

[0040] As an example, the analysis function can be implemented using molecular dynamics simulation software such as GROMACS and Amber.

[0041] Furthermore, the calculated root mean square deviation (RMSD) data and corresponding temperature values ​​are organized to analyze the RMSD of the main chain atoms in each simulation. A scatter plot is created using plotting software, with temperature on the x-axis and RMSD on the y-axis. The scatter plot data is then analyzed; a Boltzmann curve fitting method can be employed to find the curve parameters that best describe the data trend by fitting the scatter plot data to a Boltzmann curve model. The temperature value corresponding to the inflection point of the curve is the simulated unwinding temperature. T m The T mThe value represents the critical temperature at which the collagen structure begins to undergo significant delamination under simulated conditions. Each triad... T m The value is specified as the average of three simulations. By analyzing the root mean square deviation of the main chain atoms, the change of the virtual sequence main chain structure with temperature during temperature-scanning molecular dynamics simulations can be clearly understood; the simulated melting temperature value is obtained through fitting. T m This provides a precise quantitative indicator for the thermal stability of collagen.

[0042] As an example, in step S13, a representative collagen sequence is first selected as a thermal stability benchmark sequence. The benchmark sequence can be a relatively simple sequence that has been studied extensively. The benchmark sequence will serve as the basis for all subsequent comparisons of mutants.

[0043] As an example, the benchmark sequence can be AAA'.

[0044] Furthermore, using the constructed virtual sequence as the host peptide, site-directed mutagenesis was performed on the YXX' triplet within the virtual sequence. This allowed for the construction of up to 9261 different sequences. Specifically, a triplet composed of 20 natural amino acids and hydroxyproline, unique to collagen, could construct 21 × 21 × 21 = 9261 different sequences.

[0045] As an example, the site-directed mutagenesis can be performed using molecular biology techniques, such as virtual mutagenesis using PyMOL software.

[0046] Furthermore, temperature-scanning molecular dynamics simulations were performed on each mutant and AAA'. The mean root mean square deviation of the main chain atoms was analyzed using the GROMACS analysis function to obtain a scatter plot, and the simulated melting temperature values ​​for each mutant and AAA' were obtained by fitting the plot. T m Then, the melting temperature value for each mutant was... T m Subtracting the melting temperature value of AAA' from the melting temperature value yields the melting temperature difference ΔT. m The ΔT m The value represents the contribution of this mutant to thermal stability relative to AAA'. If ΔT m >0 indicates that the mutant's thermal stability is higher than AAA'; if ΔT m If the value is less than 0, it indicates that the thermal stability of the mutant is lower than that of AAA'. Finally, the thermal stability contribution ΔT of all mutants is calculated. m The corresponding mutation information (such as the location of the mutation, amino acid substitution, etc.) is organized and stored to form a thermal stability scoring database. The thermal stability scoring database is shown in Table 1.

[0047] Table 1. Partial data from the collagen thermal stability scoring database. In step S20, please refer to Figure 1 In step S20, the target sequence is input, and the type of the target sequence is determined based on the peptide chain composition of the target sequence.

[0048] As an example, based on the peptide chain composition of the input target sequence, collagen can be divided into homotrimers and heterotrimers. Homotrimers consist of three identical chains, while heterotrimer collagen consists of two (A2B heterotrimers) or three (ABC heterotrimers) different polypeptide chains. In the triple helix structure, individual residues of the peptide chains are misaligned. For binary mixtures of collagen, two... 3 =Eight unique triple helix structures, namely A3, B3, AAB, ABA, BAA, ABB, BAB, and BBA. Similarly, mixtures of ternary A, B, and C may form 3 3 =27 different triple helix structures. If the specific arrangement of the heterotrimers is not specified, the thermal stability of all possible structures will be evaluated.

[0049] As an example, in step S30, please refer to Figure 1 In step S30, when the target sequence is a heterotrimer, the target sequence is arranged and combined to obtain all possible triple helix structures, and the thermal stability of the arranged and combined target sequences is scored.

[0050] As an example, in step S40, please refer to Figure 1 In step S40, when the target sequence is a homotrimer, the thermal stability of the target sequence is directly scored.

[0051] As an example, firstly, based on the triple helix composition of the input target sequence, it is determined whether it is a homotrimer or a heterotrimer. When the input target sequence is a heterotrimer, it is permuted and combined to obtain all possible triple helix structures, and the thermal stability of the permuted and combined sequences is scored; when the input target sequence is a homotrimer, its thermal stability is scored directly.

[0052] As an example, in step S50, please refer to Figure 1In step S50 of the thermal stability scoring process, the target sequence includes: a short target sequence and a long target sequence; for the short target sequence, it is split into multiple YX and YX' motifs, and the motifs are matched with the information in the thermal stability scoring database. Based on the matched motifs, the corresponding scores are obtained and summed to obtain the overall thermal stability score.

[0053] As an example, for shorter sequences, the thermal stability of the entire sequence can be predicted directly. Based on the collagen trimer structure constructed from the input sequence, the sequence can be split into several YX motifs (i.e., Figure 3 The basis of Y and X connected by the dashed line) and the basis of YX' (i.e. Figure 3 The motifs of Y and X connected by solid lines are matched with information in the thermal stability scoring database, and scores are assigned to the corresponding motifs (i.e., YXA' and YAX' motifs) in the thermal stability scoring database. Finally, the scores of each motif are summed to obtain the overall thermal stability score of the collagen sequence.

[0054] As an example, if the thermal stability scoring database contains the YXX' motif, the score of the YXX' motif can be used to replace the score of the YX+YX' motif; that is, the thermal stability scores of the short target sequences split into the YX and YX' motifs can be the scores corresponding to the YXX' motif in the thermal stability scoring database. It should be noted that, in this case, the Y residues in the YX and YX' motifs are the same as the Y residues in the YXX' motif, the X residues in the YX motif are the same as the X residues in the YXX' motif, and the X' residues in the YX' motif are the same as the X' residues in the YXX' motif.

[0055] As an example, when Y, X, and X' are charged residues, the YX motif is a transverse salt bridge, the YX' motif is an axial salt bridge, and the YXX' motif is a complex salt bridge.

[0056] As an example, if a continuous salt bridge exists in the short target sequence, considering its contribution to thermal stability decay, the score of the short target sequence obtained based on the thermal stability scoring database is used as the initial score. The final score of the short target sequence is obtained by subtracting half the score of the less stable motif (between the preceding and following YX motifs) from the initial score. That is, when a continuous salt bridge exists in the short target sequence, after obtaining the initial score using the above method, the final score of the short target sequence is obtained by subtracting half the score of the less stable motif (between the preceding and following YX motifs) from the initial score. In this step, the scores of the preceding and following YX motifs in the continuous salt bridge are also obtained based on the aforementioned thermal stability scoring database. If a continuous salt bridge exists in the short target sequence, directly using the thermal stability scoring database as the final score will result in an inaccurate final score due to the acid-base contribution of the continuous salt bridge to thermal stability. A more accurate final score can be achieved by subtracting half the score of the less stable motif between the preceding and following YX motifs in the continuous salt bridge.

[0057] As an example, the length of the short target sequence can be 3 to 10 GPO triples; the length of the long target sequence can be more than 10 GPO triples.

[0058] As an example, in step S60, please refer to Figure 1 In step S60, for a long target sequence, a window is slid across the long target sequence, and the same measures as for the short target sequence are taken to obtain the stability score of each window, and the obtained thermal stability score is designated as the local thermal stability score of the triplet at the center of the window.

[0059] As an example, since the thermal stability of collagen decreases exponentially with increasing sequence length, longer collagen sequences can be broken down into multiple fragments, and their local thermal stability can be calculated separately. By sliding a window of length n triplets across the collagen sequence, the same steps as for predicting the thermal stability of short sequences are followed to predict the stability score of each window, and this score is designated as the local thermal stability of the central triplet within the window. Finally, the thermal stability score of the target sequence and the melting temperature obtained through fitting are output.

[0060] As an example, in practical applications, the length of the window sequence can be adjusted according to the requirements, but considering the asymmetry of collagen trimers, n should be n ≥ 3. For example, n can be 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0061] In one example, see Figure 4 , Figure 4 This is a prediction map of the local thermal stability of human type III collagen. The horizontal axis represents the sequence number, from 0 to 1029, indicating different locations within the collagen sequence; the vertical axis represents the score, ranging from 0 to 80, reflecting the degree of local thermal stability. The map shows that the scores fluctuate without a clear trend. This indicates that the local thermal stability of human type III collagen varies at different locations, with some locations exhibiting higher thermal stability (higher scores) and others lower thermal stability (lower scores).

[0062] It should be understood that although the steps in the flowcharts of the accompanying drawings are shown sequentially according to the arrows, these steps are not necessarily executed in the order indicated by the arrows. Unless explicitly stated herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least some of the steps in the accompanying drawings may include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily completed at the same time, but can be executed at different times. The execution order of these sub-steps or stages is not necessarily sequential, but can be performed alternately or in turn with other steps or at least some of the sub-steps or stages of other steps.

[0063] The collagen thermal stability scoring method of this application provides rich data resources for scoring the thermal stability of any target sequence by constructing a scoring database. Based on the database, the target sequence is scored, and personalized thermal stability analysis can be performed on collagen sequences with different structural characteristics. The method takes into account the influence of sequence specificity on thermal stability, which can provide strong support for the research and development of collagen products and greatly accelerate the optimization and commercialization process of collagen-related products.

[0064] In another embodiment, please refer to Figure 5This application also provides a collagen thermal stability scoring system, including: a thermal stability scoring database acquisition module 30 and a thermal stability scoring module 40. The thermal stability scoring database acquisition module 30 is used to construct a thermal stability scoring database; the thermal stability scoring module 40 is used to input a target sequence, determine the type of the target sequence based on its peptide chain composition; when the target sequence is a heterotrimer, the target sequence is permuted and combined to obtain all possible triple helix structures, and the thermal stability of the permuted and combined target sequences is scored based on the thermal stability scoring database; when the target sequence is a homotrimer, the thermal stability of the target sequence is directly scored based on the thermal stability scoring database; and the thermal stability of the target sequence is determined by the thermal stability scoring database. In the thermal stability scoring process, the target sequence includes: a short target sequence and a long target sequence. For the short target sequence, it is split into multiple YX and YX' motifs, and the motifs are matched with information in the thermal stability scoring database. Based on the matched motifs, the corresponding scores are obtained and summed to obtain the thermal stability score of the short target sequence. For the long target sequence, a window is slid across the long target sequence, and the thermal stability scoring method for the short target sequence is used to obtain the stability score of each window. The obtained thermal stability score is designated as the local thermal stability score of the triplet at the center of the window.

[0065] As an example, the collagen thermal stability scoring system further includes: a virtual sequence construction module 10 and a temperature scanning molecular dynamics simulation module 20. The virtual sequence construction module 10 is used to construct the virtual collagen sequence; the temperature scanning molecular dynamics simulation module 20 is used to analyze the average root mean square deviation of the main chain atoms in the temperature scanning molecular dynamics simulation using an analytical function, obtain a scatter plot, and obtain the simulated melting temperature value through fitting.

[0066] In the aforementioned collagen thermal stability scoring system, a virtual collagen sequence is constructed using a virtual sequence construction module 10, providing a research object for subsequent simulation and analysis. A temperature-scanning molecular dynamics simulation module 20 simulates the virtual sequence, comprehensively presenting the structural changes and dynamic behavior of collagen under different temperature conditions. A thermal stability scoring database acquisition module 30 stores thermal stability contribution values ​​and integrates key information such as the melting temperature difference between a large number of mutants and benchmark sequences, providing rich data resources for thermal stability scoring. A thermal stability scoring module 40, based on the type and characteristics of the target sequence and combined with the information from the thermal stability scoring database acquisition module 30, scores the thermal stability of the target sequence and fits the melting temperature value, achieving accurate evaluation of the thermal stability of different collagen sequences. Through the collaborative work of its modules, this system provides a comprehensive, efficient, and accurate analytical method for collagen thermal stability research, helping researchers gain a deeper understanding of the thermal stability mechanism of collagen under different conditions. It provides a scientific basis for the research and optimization of collagen-related products, accelerating the optimization and commercialization of collagen-related products.

[0067] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0068] Although this application has been disclosed above with reference to embodiments, it is not intended to limit this application. Anyone skilled in the art may make some modifications and refinements without departing from the spirit and scope of this application. Therefore, the scope of protection of this application shall be determined by the appended claims.

Claims

1. A method for scoring the thermal stability of collagen, characterized in that, Includes the following steps: Construct a thermal stability scoring database; Input a target sequence, and determine the type of the target sequence based on its peptide chain composition; When the target sequence is a heterotrimer, the target sequence is permuted and combined to obtain all possible triple helix structures, and the thermal stability of the permuted and combined target sequences is scored based on the thermal stability scoring database. When the target sequence is a homotrimer, the thermal stability of the target sequence is directly scored based on the thermal stability scoring database. In the thermal stability scoring process, the target sequence includes: a short target sequence and a long target sequence; for the short target sequence, it is split into multiple YX and YX' motifs, and the above motifs are matched with the information in the thermal stability scoring database. According to the matched motifs, the corresponding scores are obtained and summed to obtain the thermal stability score of the short target sequence. For a long target sequence, a window is slid across the long target sequence, and the thermal stability scoring method described above for short target sequences is used to obtain the stability score of each window. The obtained thermal stability score is then designated as the local thermal stability score of the triplet at the center of the window.

2. The method for scoring the thermal stability of collagen according to claim 1, characterized in that, For the short target sequence, if the thermal stability scoring database contains the YXX' motif, then the score of the YXX' motif is used to replace the scores of the YX and YX' motifs.

3. The method for scoring the thermal stability of collagen according to claim 1, characterized in that, For the short target sequence, if there is a continuous salt bridge in the short target sequence, the score of the short target sequence obtained based on the thermal stability scoring database is used as the initial score. The difference between the initial score and the half score of the less stable motif between the previous YX' motif and the next YX motif in the continuous salt bridge is used as the final score of the short target sequence.

4. The method for scoring the thermal stability of collagen according to claim 1, characterized in that, The construction of the thermal stability scoring database includes: A virtual sequence was constructed, and temperature-scanning molecular dynamics simulations were performed on the virtual sequence. The average root mean square deviation of the main chain atoms in the temperature scanning molecular dynamics simulation was analyzed using analytical functions to obtain a scatter plot, and the unwinding temperature value of the temperature scanning molecular dynamics simulation was obtained by fitting. A thermal stability baseline sequence is determined, and site-directed mutagenesis is performed on the YXX' triplet in the virtual sequence to obtain a mutant. The melting temperature difference between the mutant and the thermal stability baseline sequence is calculated, and a thermal stability scoring database is obtained based on the melting temperature difference.

5. The method for scoring the thermal stability of collagen according to claim 4, characterized in that, The construction of the virtual sequence includes: using [(Gly-Pro-Hyp)] 10 Using the crystal structure of ]3 as a template, a capped collagen-like polypeptide structure was constructed, with N-terminus acetylation and C-terminus N-methylation to obtain the virtual sequence.

6. The method for scoring the thermal stability of collagen according to claim 5, characterized in that, The construction of virtual sequences further includes: each virtual sequence truncating multiple GPO triplets, and each virtual sequence containing multiple sets of YXX' triplet mutations.

7. The method for scoring the thermal stability of collagen according to claim 4, characterized in that, The temperature-scanning molecular dynamics simulation of the virtual sequence includes: using the last frame of the previous temperature-scanning molecular dynamics simulation as the starting structure for the temperature-scanning molecular dynamics simulation at the next temperature, and repeating the above temperature-scanning molecular dynamics simulation multiple times for each structure.

8. The method for scoring the thermal stability of collagen according to claim 4, characterized in that, The step of obtaining the unbreaking temperature value from the temperature scanning molecular dynamics simulation by fitting includes: obtaining the unbreaking temperature value from the temperature scanning molecular dynamics simulation by fitting a Boltzmann curve.

9. A collagen thermal stability scoring system, characterized in that, The collagen thermal stability scoring system includes: The thermal stability scoring database acquisition module is used to construct the thermal stability scoring database. A thermal stability scoring module is used to input a target sequence and determine the type of the target sequence based on its peptide chain composition. When the target sequence is a heterotrimer, the target sequence is permuted and combined to obtain all possible triple helix structures, and the thermal stability of the permuted and combined target sequences is scored based on the thermal stability scoring database. When the target sequence is a homotrimer, the thermal stability of the target sequence is directly scored based on the thermal stability scoring database. In the thermal stability scoring process, the target sequence includes: short target sequences and long target sequences. For short target sequences, they are split into multiple YX and YX' motifs, and these motifs are matched with information in the thermal stability scoring database. Based on the matched motifs, corresponding scores are obtained and summed to obtain the thermal stability score of the short target sequence. For long target sequences, a window is slid across the long target sequence, and the thermal stability scoring method for the short target sequence is used to obtain the stability score of each window. The obtained thermal stability score is designated as the local thermal stability score of the triplet at the center of the window.

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