Method for introducing disulfide bond in protein design process

Abstract

The invention relates to a method for introducing disulfide bonds in a protein design process, which comprises the following steps: firstly, designing main chain coordinates of all residues of a scaffold by using protein skeleton design software to enable the main chain coordinates to contain a given motif, and secondly, calculating candidate site pairs meeting disulfide bond forming conditions, then, redesigning all residues except the motif and the disulfide bond candidate sites by using protein sequence design software to obtain a protein sequence, carrying out structure prediction on the protein sequence by using protein structure prediction software to obtain a protein structure, and comparing the protein structure by using structure comparison software to obtain a protein structure; and screening out site pairs meeting conditions through calculation, and finishing the design if the site pairs meet the requirement on the number of disulfide bonds. Compared with the prior art, disulfide bonds are introduced in the design process of the protein motif scaffold, and the protein motif scaffold has diversity in the aspects of sequence length and residue distribution.

Description

Technical Field

[0001] The present invention relates to the technical field of protein design, and in particular to a method for introducing disulfide bonds in the process of protein design. Background Art

[0002] Proteins are one of the most important molecules in living organisms, and their structure and function are crucial to the maintenance and reproduction of life. Proteins catalyze chemical reactions, transduce signals in biological systems, provide structural elements in cells and extracellular matrices, act as messengers, etc. Stable folded structures are the basis of protein function. Disulfide bonds are a very common structure in proteins, which can increase the stability and functionality of proteins. Therefore, the introduction of disulfide bonds in protein engineering is very useful.

[0003] At present, the traditional method of introducing disulfide bonds in protein engineering is to analyze the sequence and structural characteristics of wild-type proteins, mutate the residues at potential sites to cysteine, thereby introducing possible disulfide bonds, obtaining new protein sequences, and finally screening them through experiments. This method has a very low success rate. Only point mutations are made, while other sites are not synergistically changed, resulting in the sequence after point mutation usually being far away from the distribution manifold and difficult to fold into a stable protein structure.

[0004] One of the main reasons for poor performance of proteins is aggregation. This is not only a problem in the laboratory, but also a problem in many diseases such as Alzheimer's disease. Aggregation is a particularly vexing problem when synthesizing designed proteins. As more proteins are designed or modified to be used as tools for studying biological systems or as more wild-type or modified proteins are used as therapeutic agents, there is a need to modify these proteins into more stable and / or aggregation-resistant systems.

[0005] A Chinese patent with publication number CN115331728A proposes a method for designing disulfide bonds from an initial polypeptide sequence. This method is limited by the initial polypeptide sequence and attempts to find sequences containing disulfide bonds within a small neighborhood of the initial sequence. It lacks diversity in sequence length and residue distribution, resulting in a low design success rate.

[0006] ProteinMPNN provides a method to redesign the residues in a specified region given a polypeptide main chain structure, and can specify that only certain given residues be designed. However, through multiple tests, it was found that even if the main chain structure contains a geometric structure suitable for forming disulfide bonds, ProteinMPNN will still not actively design cysteine, resulting in the redesigned sequence having no disulfide bonds. Summary of the invention

[0007] In response to the problems existing in the related art, the present invention provides a method for introducing disulfide bonds in the process of protein design, an electronic device and a computer-readable storage medium, which can effectively obtain preferred sequences, improve research and development efficiency and reduce research and development costs.

[0008] The first aspect of the present invention provides a method for introducing disulfide bonds in the process of protein design, comprising the following steps:

[0009] S1: Determine the number N of disulfide bonds to be introduced and the number x of scaffold segments to be designed, wherein N and x can be designed to be natural numbers not less than 1 as required;

[0010] S2: Set the length L of the zth segment of the bracket z (The length of the zth segment L z is the initial value of the number of residues in the zth segment), where z = 1, 2, ..., x; preferably, the length of each segment, i.e., L1, L2, ..., L x , set to the same initial value;

[0011] S3: Use protein backbone design software to design the main chain coordinates of all residues of the scaffold so that it contains the given motif, then analyze the main chain coordinates of all residues of the scaffold, calculate the number n of candidate site pairs that meet the disulfide bond formation conditions, and redesign if n is less than N; if n is not less than N or n obtained after redesign is not less than N, specify the residue type of the candidate site pair as cysteine, save the current protein structure as P0, and execute S4;

[0012] S4: Use protein sequence design software to redesign all residues outside the motif and disulfide bond candidate site pairs to obtain the protein sequence;

[0013] S5: using protein structure prediction software to perform structural prediction on the protein sequence to obtain the protein structure;

[0014] S6: Using the residue sites involved in the motif as alignment sites, the protein structure is aligned with P0 using a structural alignment software to obtain a root mean square deviation (RMSD);

[0015] S7: If RMSD is not less than 1, repeat S4-S7; if RMSD is still not less than 1 after repeated execution for more than R times, repeat S4-S7 after redesigning the protein skeleton; if RMSD is less than 1 or the RMSD obtained after repeated execution of S4-S7 is less than 1, execute S8;

[0016] S8: Calculate the distance between each set of disulfide bond candidate sites in P0 and the sulfur atoms of the corresponding residues in the protein structure. If the distance is The deviation is less than Then the site pair forms a disulfide bond in the protein structure. If the number of disulfide bond pairs is less than N, the protein skeleton is redesigned and S4-S8 is repeated; if the number of disulfide bond pairs is not less than N or the number of disulfide bond pairs obtained after repeating S4-S8 is not less than N, the design is terminated.

[0017] Preferably, step S3 is used to design a protein backbone, specifically:

[0018] S31: Use protein backbone design software to design the main chain coordinates of all residues of the scaffold so that it contains the given motif, then analyze the main chain coordinates of all residues of the scaffold, calculate the number n of candidate site pairs that meet the disulfide bond formation conditions, and if n is less than N, redesign; if n is not less than N or n obtained after redesign is not less than N, specify the residue type of the candidate site pair as cysteine, save the current protein structure as P0, and execute S4; if n is still less than N after redesign for more than M times, execute S32;

[0019] S32: after adding 1 to the value of the length L1 of the first scaffold, calculate n according to the step of S31, if n is less than N, redesign; if n is not less than N or n obtained after redesign is not less than N, specify the residue type of the candidate site pair as cysteine, save the current protein structure as P0, and execute S4; if n is still less than N after redesign times exceeding M times, execute S33;

[0020] S33: Set the length L of the y-th bracket y After adding 1 to the value of , calculate n according to the steps of S31. If n is less than N, redesign; if n is not less than N or n obtained after redesign is not less than N, specify the residue type of the candidate site pair as cysteine, save the current protein structure as P0, and execute S4; if n is still less than N after redesign times exceeding M times, replace y with y+1, and re-execute S33; wherein, 2≤y≤x-1, and y is an integer.

[0021] Among them, in steps S7 and S8, the redesign of the protein skeleton may only execute S33, or may execute any one or more steps of S31-S33.

[0022] Preferably, the L z The initial value of is a natural number greater than 4, M is a natural number greater than 4, and R is a natural number greater than 8.

[0023] Preferably, the disulfide bond formation condition refers to the distance between the carbon-α (CA) atoms of the two residues being less than

[0024] Preferably, the determination of the disulfide bond formation conditions comprises the following steps:

[0025] S3111: Filter the geometric information of disulfide bond residues from the protein structure database, calculate the local coordinate system transformation matrix, and construct a geometric hash table;

[0026] S3112: Calculate the local coordinate system transformation matrix for the residues of each set of disulfide bond candidate site pairs of P0;

[0027] S3113: If the transformation matrix obtained in S3112 obtains an index in the geometric hash table obtained in S3111, the disulfide bond formation condition is satisfied; otherwise, it is not satisfied.

[0028] Preferably, the method for calculating the number n of candidate site pairs that meet the disulfide bond formation conditions comprises the following steps:

[0029] S3121: Delete one of the two symmetrical groups of site pairs among all disulfide bond candidate site pairs; wherein, if the first site index value and the second site index value of a site pair are respectively the second site index value and the first site index value of another site pair, that is, the two symmetrical groups of site pairs, for example (1, 6) and (6, 1), at this time, if (1, 6) and (6, 1) both meet the conditions, delete one of the site pairs and retain the other site pair.

[0030] S3122: Screen out site pairs with a sequence distance greater than 4, where the sequence distance is calculated by subtracting the absolute value of the second site index value from the first site index value of the site pair;

[0031] S3123: add the first site pair to the candidate list;

[0032] S3124: For each remaining point pair, calculate the distance value between the point pair and each point pair in the candidate list. If the distance values ​​between the point pair and all the point pairs in the candidate list are greater than 2, add the point pair to the candidate list.

[0033] Preferably, the distance calculation method between the site pairs in step S3124 includes the following steps:

[0034] S31241: Calculate the absolute value of the difference between the first site index values ​​of two site pairs;

[0035] S31242: Calculate the absolute value of the difference between the second site index values ​​of the two site pairs;

[0036] S31243: Calculate the absolute value of the difference between the first point to the first point index value and the second point to the second point index value;

[0037] S31244: Calculate the absolute value of the difference between the first point index value to the second point index value and the second point index value to the first point index value;

[0038] S31245: The minimum value among the values ​​obtained in S31241, S31242, S31243 and S31244 is taken as the distance value between the two site pairs.

[0039] Preferably, the protein backbone design software includes one or more of RFdiffusion, SCUBA, and Rosetta, the protein sequence design software includes one or more of ProteinMPNN, RFDesign, and Rosetta, the protein structure prediction software includes one or more of AlphaFold, ColabFold, and RoseTTAFold, and the structure alignment software includes one or more of TM-align, US-align, and mTM-align.

[0040] A second aspect of the present invention provides an electronic device, comprising:

[0041] Processor; and

[0042] A memory having executable codes stored thereon, which, when executed by the processor, causes the processor to execute the method for introducing disulfide bonds in the design process of a protein motif scaffold as described in any one of the above items.

[0043] A third aspect of the present invention provides a computer-readable storage medium having executable code stored thereon. When the executable code is executed by a processor of an electronic device, the processor is caused to execute the method for introducing disulfide bonds in the design process of a protein motif scaffold as described in any of the above items.

[0044] The present invention includes the following beneficial effects:

[0045] Disulfide bonds were introduced during the design of protein motif scaffolds with diversity in sequence length and residue distribution. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The alignment results of 5TPN_3_1.pdb (green) and 5TPN_3.pdb (blue) in Example 1 are shown.

[0047] Figure 2 The three sets of disulfide bonds introduced in 5TPN_3_1.pdb in Example 1 are shown.

[0048] Figure 3The alignment results of 5IUS_4_1.pdb (green) and 5IUS_4.pdb (blue) in Example 2 are shown.

[0049] Figure 4 The four disulfide bonds introduced in 5IUS_4_1.pdb in Example 2 are shown.

[0050] Figure 5 The comparison results of 5YUI_4_1.pdb (green) and 5YUI_4.pdb (blue) in Example 3 are shown.

[0051] Figure 6 The four disulfide bonds introduced in 5YUI_4_1.pdb in Example 3 are shown. DETAILED DESCRIPTION

[0052] After extensive and in-depth research, a lot of screening and exploration, a method for introducing multiple pairs of disulfide bonds in the redesign process while maintaining protein function was proposed for the first time. This method redesigns a stably folded scaffold protein containing disulfide bonds for the functional motif of the protein. The structure of the designed protein sequence predicted by AlphaFold has little difference in the functional motif segment.

[0053] Terminology

[0054] The "RMSD" described in the present invention is the root mean square deviation, which is a commonly used analytical index in protein structure simulation and can be used to measure the degree of structural difference. In the basic dynamics simulation of protein structure or the molecular dynamics simulation of protein-ligand complex, the RMSD changes of protein skeleton and small molecule ligand are generally analyzed. It can be used to quickly determine whether the trajectory has reached equilibrium, so as to determine whether it is necessary to extend the simulation time or perform other analyses. Generally, if the RMSD reaches a periodic stable value, it can be considered that equilibrium has been reached.

[0055] The "protein backbone design software" mentioned in the present invention refers to software that only designs main chain coordinates but not side chain coordinates, such as RFdiffusion.

[0056] The "protein sequence design software" mentioned in the present invention refers to software for designing protein sequences based on protein main chain coordinates, such as ProteinMPNN.

[0057] The "protein structure prediction software" mentioned in the present invention refers to software that predicts protein structure based on protein sequence, such as Alphafold.

[0058] The "structure comparison software" of the present invention refers to software for comparing the tertiary structures of two proteins, such as TM-align, which can obtain the most direct result of a series of overlapping three-dimensional coordinates of the two proteins. The overlapping structures of the two proteins can be used to calculate the RMSD (root mean square deviation) value and other more complex similarity indicators. The RMSD value measures the degree of difference between the two structures.

[0059] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0060] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The following embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0061] Embodiment 1:

[0062] Three disulfide bonds were introduced during the design of the scaffold for the A163-181 functional motif of 5TPN:

[0063] The 5TPN.pdb file is available at www. Download rcsb.org website .

[0064] Call RFDiffusion to design a protein skeleton pdb file, named 5TPN_3.pdb;

[0065] Extract the site pairs that meet the disulfide bond formation conditions from 5TPN_3.pdb. The specific process is as follows:

[0066] First, the protein structure data file is input to extract the coordinates of the C, CA, and N atoms of each residue in the main chain of the protein structure;

[0067] Then, based on the coordinates of the C, CA, and N atoms of each residue, a 4 × 4 matrix is ​​calculated to represent the local coordinate system;

[0068] The residues are grouped into residue pairs, represented by site pairs, such as (1, 1), (1, 2), ..., (n, n), where n represents the number of residues in the input protein;

[0069] Filter out residue pairs with sequence distance less than 4. For example, if the residue pair is represented as (i, j), if the absolute value of the difference between i and j is less than 4, delete it.

[0070] Traverse the residue pairs remaining after filtering and extract the site pairs corresponding to all residue pairs that meet the disulfide bond formation conditions.

[0071] The site pairs are screened to finally obtain candidate site pairs, and the residue types of the sites involved in each candidate site pair are changed to cysteine.

[0072] For 5TPN_3.pdb, the sites corresponding to the fixed functional motifs and the sites involved in the candidate site pairs were used to redesign the scaffold sequences using ProteinMPNN;

[0073] After ProteinMPNN design, one sequence was obtained, named: 5TPN_3_1.fasta.

[0074] Use AlphaFold to predict the structure of the above sequence and obtain the predicted pdb file, named 5TPN_3_1.pdb;

[0075] The structure of 5TPN_3_1.pdb was compared with that of 5TPN_3.pdb for the sites involved in the functional motifs, and the RMSD was found to be less than 1. Figure 1 As shown, the results show that the structural differences are minimal, which is necessary to ensure protein function.

[0076] For 5TPN_3_1.pdb, calculate the sulfur atom distance of the corresponding candidate site to the involved residues, and compare the distance with Is the deviation value less than The site pairs that meet the conditions were screened and found to contain 3 groups of disulfide bond site pairs, such as Figure 2 shown.

[0077] Therefore, 5TPN_3_1.pdb can be finally used as the design result.

[0078] Embodiment 2:

[0079] Four disulfide bonds were introduced during the design of the scaffold for the A119-140 and A63-82 functional motifs of 5IUS:

[0080] The 5IUS.pdb file is available at www. Download rcsb.org website .

[0081] RFDiffusion was called to design a protein skeleton pdb file named 5IUS_4.pdb.

[0082] Site pairs that meet the conditions for disulfide bond formation were extracted from 5IUS_4.pdb, and the site pairs were screened to finally obtain candidate site pairs. The residue type of the site involved in each candidate site pair was changed to cysteine.

[0083] For 5IUS_4.pdb, the sites corresponding to the fixed functional motifs and the sites involved in the candidate site pairs were used to redesign the scaffold sequences using ProteinMPNN;

[0084] After ProteinMPNN design, one sequence was obtained, named: 5IUS_4_1.fasta.

[0085] Use AlphaFold to predict the structure of the above sequence and obtain the predicted pdb file, named 5IUS_4_1.pdb;

[0086] The structure of 5IUS_4_1.pdb was compared with the structure of 5IUS_4.pdb for the sites involved in the functional motifs, and it was found that the RMSD was less than 1. The comparison results are as follows Figure 3 As shown, the results show that the structural differences are minimal, which is necessary to ensure protein function.

[0087] For 5IUS_4_1.pdb, calculate the sulfur atom distance of the corresponding candidate site to the involved residues, and compare the distance with Is the deviation value less than The site pairs that meet the conditions were screened and found to contain 5 disulfide bond site pairs. Figure 4 As shown, Figure 4 There are 5 groups of disulfide bonds in total. The top 4 groups of disulfide bonds are formed by residues on the scaffold, and the bottom group of disulfide bonds is formed by residues on the scaffold and residues on the motif.

[0088] Therefore, 5IUS_4_1.pdb can be finally used as the design result.

[0089] Embodiment 3:

[0090] Four disulfide bonds were introduced during the design of scaffolds for the A93-97, A118-120 and A198-200 functional motifs of 5YUI:

[0091] The 5YUI.pdb file can be found at www. Download rcsb.org website .

[0092] RFDiffusion was called to design a protein skeleton pdb file, named 5YUI_4.pdb.

[0093] Site pairs that meet the conditions for disulfide bond formation were extracted from 5YUI_4.pdb, and the site pairs were screened to finally obtain candidate site pairs. The residue type of the site involved in each candidate site pair was changed to cysteine.

[0094] For 5YUI_4.pdb, the sites corresponding to the fixed functional motifs and the sites involved in the candidate site pairs were used to redesign the scaffold sequences using ProteinMPNN;

[0095] After ProteinMPNN design, one sequence was obtained, named: 5YUI_4_1.fasta.

[0096] Use AlphaFold to predict the structure of the above sequence and obtain the predicted pdb file, named 5YUI_4_1.pdb;

[0097] The structure of 5YUI_4_1.pdb was compared with the structure of 5YUI_4.pdb for the sites involved in the functional motifs, and it was found that the RMSD was less than 1. The comparison results are as follows Figure 5 As shown, the results show that the structural differences are minimal, which is necessary to ensure protein function.

[0098] For 5YUI_4_1.pdb, calculate the sulfur atom distance of the corresponding candidate site to the involved residues, and compare the distance with Is the deviation value less than The site pairs that meet the conditions were screened and found to contain 4 groups of disulfide bond site pairs, such as Figure 6 shown.

[0099] Therefore, 5YUI_4_1.pdb can be finally used as the design result.

[0100] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for introducing disulfide bonds in the process of protein design, characterized in that: The following steps are involved: S1: Determine the number of disulfide bonds N that need to be introduced and the number of scaffold segments x that need to be designed; S2: Set the length L of the zth segment of the bracket z The initial value of , where z = 1, 2, …, x; S3: Use protein backbone design software to design the main chain coordinates of all residues of the scaffold so that it contains the given motif, then analyze the main chain coordinates of all residues of the scaffold, calculate the number n of candidate site pairs that meet the disulfide bond formation conditions, and redesign if n is less than N; if n is not less than N or n obtained after redesign is not less than N, specify the residue type of the candidate site pair as cysteine, save the current protein structure as P0, and execute S4; S4: Use protein sequence design software to redesign all residues outside the motif and disulfide bond candidate site pairs to obtain the protein sequence; S5: using protein structure prediction software to perform structural prediction on the protein sequence to obtain the protein structure; S6: Using the residue sites involved in the motif as alignment sites, the protein structure is aligned with P0 using a structural alignment software to obtain a root mean square deviation (RMSD); S7: If RMSD is not less than 1, repeat S4-S7; if RMSD is still not less than 1 after repeated execution for more than R times, repeat S4-S7 after redesigning the protein skeleton; if RMSD is less than 1 or the RMSD obtained after repeated execution of S4-S7 is less than 1, execute S8; S8: Calculate the distance between each set of disulfide bond candidate sites in P0 and the sulfur atoms of the corresponding residues in the protein structure. If the distance is The deviation is less than Then the site pair forms a disulfide bond in the protein structure. If the number of disulfide bond pairs is less than N, the protein skeleton is redesigned and S4-S8 is repeated; if the number of disulfide bond pairs is not less than N or the number of disulfide bond pairs obtained after repeating S4-S8 is not less than N, the design is terminated.

2. A method for introducing disulfide bonds in a protein design process according to claim 1, characterized in that: Step S3 is used to design the protein skeleton, specifically: S31: using protein backbone design software to design the main chain coordinates of all residues of the scaffold so that it contains the given motif, then analyzing the main chain coordinates of all residues of the scaffold, and calculating the number n of candidate site pairs that meet the disulfide bond formation conditions, and if n is less than N, redesigning; If n is not less than N or n obtained after redesign is not less than N, the residue type of the candidate site pair is designated as cysteine, and the current protein structure is saved as P0, and S4 is executed; if n is still less than N after the number of redesigns exceeds M times, S32 is executed; S32: after adding 1 to the value of the length L1 of the first section of the bracket, calculate n according to the steps of S31, and if n is less than N, redesign; If n is not less than N or n obtained after redesign is not less than N, the residue type of the candidate site pair is designated as cysteine, and the current protein structure is saved as P0, and S4 is executed; if n is still less than N after the number of redesigns exceeds M times, S33 is executed; S33: Set the length L of the y-th bracket y After adding 1 to the value of , calculate n according to the steps of S31. If n is less than N, redesign; If n is not less than N or n obtained after redesign is not less than N, the residue type of the candidate site pair is designated as cysteine, and the structure of the current protein is saved as P0, and S4 is executed; if n is still less than N after the number of redesigns exceeds M times, y is replaced by y+1, and S33 is re-executed; wherein 2≤y≤x-1, and y is an integer.

3. A method for introducing disulfide bonds in the process of protein design according to claim 2, characterized in that: The L z The initial value of is a natural number greater than 4, M is a natural number greater than 4, and R is a natural number greater than 8.

4. A method for introducing disulfide bonds in the process of designing proteins according to any one of claims 1 to 3, characterized in that: The disulfide bond formation condition refers to the distance between the carbon-α (CA) atoms of the two residues being less than 5. A method for introducing disulfide bonds in the process of designing proteins according to any one of claims 1 to 3, characterized in that: The determination of the disulfide bond formation conditions comprises the following steps: S3111: Filter the geometric information of disulfide bond residues from the protein structure database, calculate the local coordinate system transformation matrix, and construct a geometric hash table; S3112: Calculate the local coordinate system transformation matrix for the residues of each set of disulfide bond candidate site pairs of P0; S3113: If the transformation matrix obtained in S3112 obtains an index in the geometric hash table obtained in S3111, the disulfide bond formation condition is satisfied; otherwise, it is not satisfied.

6. A method for introducing disulfide bonds in the process of protein design according to claim 5, characterized in that: The method for calculating the number n of candidate site pairs that meet the disulfide bond formation conditions comprises the following steps: S3121: Delete one of the two mutually symmetrical site pairs among all disulfide bond candidate site pairs; S3122: Screen out site pairs with a sequence distance greater than 4, where the sequence distance is calculated by subtracting the absolute value of the second site index value from the first site index value of the site pair; S3123: add the first site pair to the candidate list; S3124: For each remaining point pair, calculate the distance value between the point pair and each point pair in the candidate list. If the distance values ​​between the point pair and all the point pairs in the candidate list are greater than 2, add the point pair to the candidate list.

7. A method for introducing disulfide bonds in the process of designing proteins according to claim 6, characterized in that: The distance calculation method between the point pairs in step S3124 includes the following steps: S31241: Calculate the absolute value of the difference between the first site index values ​​of two site pairs; S31242: Calculate the absolute value of the difference between the second site index values ​​of the two site pairs; S31243: Calculate the absolute value of the difference between the first point to the first point index value and the second point to the second point index value; S31244: Calculate the absolute value of the difference between the first point index value to the second point index value and the second point index value to the first point index value; S31245: The minimum value among the values ​​obtained in S31241, S31242, S31243 and S31244 is taken as the distance value between the two site pairs.

8. A method for introducing disulfide bonds in the process of designing proteins according to any one of claims 1 to 3, characterized in that: The protein skeleton design software includes one or more of RFdiffusion, SCUBA, and Rosetta, the protein sequence design software includes one or more of ProteinMPNN, RFDesign, and Rosetta, the protein structure prediction software includes one or more of AlphaFold, ColabFold, and RoseTTAFold, and the structure alignment software includes one or more of TM-align, US-align, and mTM-align.

9. An electronic device, characterized in that: include: processor; as well as A memory having executable codes stored thereon, which, when executed by the processor, causes the processor to execute the method for introducing disulfide bonds in the design process of a protein motif scaffold according to any one of claims 1 to 8.

10. A computer-readable storage medium having executable codes stored thereon, which, when executed by a processor of an electronic device, causes the processor to execute the method for introducing disulfide bonds in the design process of a protein motif scaffold as described in any one of claims 1 to 8.

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