A method and system for predicting the monomolecular phase transition critical temperature of an elastin protein

Abstract

This invention discloses a method and system for predicting the single-molecule phase transition critical temperature of elastomeric proteins, belonging to the field of bioinformatics. The prediction method includes: obtaining the amino acid sequence of the elastomeric protein; performing motif identification on the amino acid sequence; and calculating the single-molecule phase transition critical temperature of the elastomeric protein. Starting from the amino acid sequence, this invention can directly predict the single-molecule phase transition critical temperature of elastomeric proteins based on the amino acid sequence, solving the problems faced by researchers in elastomeric studies, such as the lack of targeted analytical tools and the high cost and time-consuming nature of experimental determination of physicochemical properties. This improves the research efficiency of elastomeric proteins and reduces R&D costs.

Description

Technical Field

[0001] This invention belongs to the field of bioinformatics technology, specifically relating to a method and system for predicting the critical temperature of a single-molecule phase transition in an elastomeric protein. Background Technology

[0002] Elastomer proteins are a class of biomacromolecules with important applications. They are usually composed of repetitive sequences derived from the hydrophobic domains of elastin, such as VPGXG-type pentapeptide repeat units. In aqueous solutions, they typically exhibit temperature responsiveness, self-assembly, and reversible phase transition properties.

[0003] However, during the research and development process, there is a challenge in predicting the phase transition temperature, which significantly increases the experimental workload and research and development costs. McDaniel JR (A unified model for de novo design of elastin-like polypeptides with tunable inverse transition temperatures. Biomacromolecules. 2013 Aug 12;14(8):2866-2872) disclosed a simple predictive model for elastin, which uses the intrinsic phase transition temperature T tc Predicting the critical temperature T of phase transition t The current prediction model only considers the influence of amino acid residues A and V in elastin (VPGXG)n (single-factor). However, the phase transition temperature of elastin is not only affected by the content of amino acid residues A and V, and the amino acid sequence of elastin is not composed solely of the repeating sequence of (VPGXG)n. Therefore, this model has limited application scenarios and is difficult to apply in the research and development of elastin. Existing technology CN116102172A discloses a high-throughput computation-based method for predicting elastin, which can achieve high-throughput prediction of whether a protein belongs to elastin based on an algorithm. However, it can only achieve a qualitative judgment of whether a protein belongs to elastin based on its amino acid sequence, and still cannot predict the phase transition temperature of elastin. When facing the problem of screening elastins that meet the performance requirements, extensive experimental verification is still needed, which still consumes a lot of manpower and resources.

[0004] Therefore, there is an urgent need to develop a tool-based method that can directly identify motifs based on amino acid sequences and quantitatively calculate the critical temperature of phase transition during the candidate sequence design stage of elastomeric proteins, thereby providing a quantitative basis for the sequence design and high-throughput screening of elastomeric materials. Summary of the Invention

[0005] In order to overcome the above-mentioned shortcomings of the existing technology, the purpose of this invention is to provide a sequence-based method and system for predicting the features of elastomeric proteins.

[0006] The objective of this invention is achieved by at least one of the following technical solutions.

[0007] In a first aspect, the present invention provides a method for predicting the critical temperature of a single-molecule phase transition in an elastomeric protein, the method comprising:

[0008] S1. Count the total number of residues N and the absolute frequency count of residue type i in the amino acid sequence of the elastomer protein. i ;

[0009] S2. Obtain the contact free energy temperature response parameters of residue type i, and calculate the fundamental contact free energy ΔG between residues of residue type i under temperature variable T using the contact free energy temperature response parameters of residue type i. e (T) i ;

[0010] S3. Calculate the all-atom interaction energy G of the elastomer protein under temperature variable T. int_raw (T):

[0011] ,

[0012] Where, count i : Absolute frequency of residue type i; n c : Average coordination number, representing the average number of neighboring residues that are in physical contact with other residues in a fully collapsed, dense sphere state; N: Total number of residues in the amino acid sequence;

[0013] S4. Calculate the configurational entropy drag G of the elastomer protein under temperature variable T. conf (T):

[0014] ,

[0015] Among them, R gas α: Molar gas constant; α: Chain expansion / compressibility factor. , where R coil and R ext These are the radii of the collapsed state and the radii of the highly swollen and expanded state, respectively.

[0016] S5. Establish the single-chain collapse free energy function G with respect to the temperature variable T. total (T):

[0017] ,

[0018] Where, noffset : Pre-set calibration offset;

[0019] S6. Solve for the single-chain collapse free energy function G. total (T) The temperature at which the zero-point condition is met, and this temperature is determined as the single-molecule phase transition critical temperature T of the elastomer protein. c The zero-point condition is as follows:

[0020] .

[0021] Furthermore, the elastomeric proteins include natural elastomeric proteins, extrin, elastomeric polypeptides, natural elastomeric fragments or variants, extrin fragments or variants, elastomeric polypeptide variants, and fusion proteins containing elastomeric repeat sequences.

[0022] Furthermore, in step S2, the contact free energy temperature response parameter of residue type i includes the fundamental contact free energy ΔG between residues of residue type i at the reference temperature T0. e (T0) i Hydration free energy u h (T0) i and linear coupling coefficient (slope) k i ;

[0023] The fundamental contact free energy ΔG between residues of residue type i at a temperature variable T e (T) i Calculated using the following formula:

[0024] ,

[0025] Wherein, ΔG e (T) i : Fundamental contact free energy between residues of residue type i; ΔG e (T0) i The fundamental contact free energy between residues of residue type i at a reference temperature T0; u h (T) i : The solvation (hydration) free energy of residue type i at a temperature variable T; u h (T0) i : The fundamental solvation (hydration) free energy of residue type i at the reference temperature T0; k i The linear coupling coefficient (slope) of the residues.

[0026] Furthermore, the contact free energy temperature response parameter of residue type i also includes the enthalpy of hydration ΔH. h (T0) iHydration isobaric heat capacity change (ΔC) p ) i ;

[0027] Under a temperature variable T, the solvation (hydration) free energy u of residue type i h (T) i Calculated using the following formula:

[0028] ,

[0029] Where, ΔH h (T0) i At the reference temperature T0, the basic enthalpy of hydration of residue type i; (ΔC) p ) i : Hydration isobaric thermal capacity change of residue type i.

[0030] Furthermore, in step S315, the pre-set calibration offset is determined by having a known single-chain collapse critical temperature T. ref The baseline sequence is determined; Among them, G int_raw (T) ref The baseline sequence is at the known single-chain collapse critical temperature T. ref All-atomic interaction energy; G conf (T) ref The baseline sequence is at the known single-chain collapse critical temperature T. ref Configurational entropy resistance.

[0031] Furthermore, the reference sequence is a VGVPG sequence repeated 20 times, denoted as (VGVPG). 20 The baseline sequence (VGVPG) 20 The known single-chain collapse critical temperature T ref It is 305 K.

[0032] Furthermore, in step S6, the single-chain collapse free energy function G is solved. total (T) The temperature corresponding to the zero point condition is obtained by using the bisection method, secant method, Newton's iteration method or spline interpolation method.

[0033] Secondly, the present invention also provides a prediction system for sequence-based elastomer protein features, the system comprising:

[0034] The calculation module is used to calculate the critical temperature of the single-molecule phase transition of elastomeric proteins.

[0035] The single-molecule phase transition critical temperature T c Predict using the following steps:

[0036] S1. Count the total number of residues N and the absolute frequency count of residue type i in the amino acid sequence of the elastomer protein. i ;

[0037] S2. Obtain the contact free energy temperature response parameters of residue type i, and calculate the fundamental contact free energy ΔG between residues of residue type i under temperature variable T using the contact free energy temperature response parameters of residue type i. e (T) i ;

[0038] S3. Calculate the all-atom interaction energy G of the elastomer protein under temperature variable T. int_raw (T):

[0039] ,

[0040] Where, count i : Absolute frequency of residue type i; n c : Average coordination number; N: Total number of residues in the amino acid sequence;

[0041] S4. Calculate the configurational entropy drag G of the elastomer protein under temperature variable T. conf (T):

[0042] ,

[0043] Among them, R gas α: Molar gas constant; α: Chain expansion / compressibility factor. , where R coil and R ext These are the radii of the collapsed state and the radii of the highly swollen and expanded state, respectively.

[0044] S5. Establish the single-chain collapse free energy function G with respect to the temperature variable T. total (T):

[0045] ,

[0046] Where, n offset : A pre-set calibration offset; the pre-set calibration offset is determined by a known single-chain collapse critical temperature T. ref The baseline sequence is determined;

[0047] ,

[0048] G int_raw (T) ref The baseline sequence is at the known single-chain collapse critical temperature T. ref All-atomic interaction energy; G conf (T) refThe baseline sequence is at the known single-chain collapse critical temperature T. ref Configurational entropy resistance;

[0049] S6. Solve for the single-chain collapse free energy function G. total (T) The temperature at which the zero-point condition is met, and this temperature is determined as the single-molecule phase transition critical temperature T of the elastomer protein. c The zero-point condition is as follows:

[0050] .

[0051] Thirdly, the present invention also provides an apparatus comprising at least one storage medium and at least one processor, the at least one storage medium being used to store computer instructions, and the at least one processor being used to execute the computer instructions to implement the method for predicting the critical temperature of a single-molecule phase transition of an elastomeric protein as described in the first aspect.

[0052] Fourthly, the present invention also provides a computer-readable storage medium storing computer instructions that, when executed by a processor, implement the method for predicting the critical temperature of a single-molecule phase transition of an elastomeric protein as described in the first aspect.

[0053] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0054] This invention starts with the amino acid sequence to automatically identify the motifs related to elastomeric proteins, and further calculates the critical temperature of single phase transition. The critical temperature of single phase transition can be used as a reference value for the phase transition temperature of elastomeric proteins.

[0055] Current technology does not yet have the phase transition temperature T of elastin. t Reasonable prediction methods are difficult to implement due to the numerous influencing factors (such as protein concentration). This invention innovatively and directly uses the amino acid sequence of elastin to predict the single-molecule phase transition critical temperature T of elastin. c To make a prediction, T c The predicted value and T t The (minimum values) are similar, therefore T c The predicted value can be accurately used as T t By using a reference value (the minimum value), a large amount of experimental work can be avoided, and the phase transition temperature T can be predicted directly using the amino acid sequence of elastin. t This invention shortens the research and development cycle. It is suitable for high-throughput sequence design and virtual screening, reducing experiments such as expression, purification, and characterization of elastin, lowering research and development costs, improving screening efficiency, and enhancing the feasibility of subsequent research and development projects. Attached Figure Description

[0056] Figure 1Phase transition critical temperature prediction diagram (T) c_pre T represents c (Predicted value). Detailed Implementation

[0057] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0058] In this invention, "elastin-like polypeptide" refers to an engineered, recombinant, synthetic, or semi-synthetic polypeptide comprising one or more amino acid repeating sequence units derived from, mimicking, or modified from natural elastin or protoelastin. The elastin-like polypeptide includes, but is not limited to, elastin-like repeating polypeptides, elastin-like block polypeptides, silk-elastin-like polypeptides, collagen-elastin-like fusion polypeptides, elastin-like polypeptides containing cell adhesion sequences, elastin-like polypeptides containing enzyme cleavage sites, elastin-like polypeptides containing cross-linking sites, as well as fragments, mutants, variants, derivatives, fusions, covalently modified products, or cross-linked products of the above polypeptides. The elastin-like polypeptide preferably possesses at least one elastin-like characteristic, including elastic recovery, flexibility, reversible phase transition, temperature responsiveness, self-assembly, cross-linkability, biocompatibility, the ability to form an elastic network, or a combination thereof.

[0059] In this invention, "solvent-accessible surface area" refers to the total surface area of ​​a protein molecule that can be contacted by water molecules or solvent molecules.

[0060] In this invention, "hydrophobic solvent-accessible surface area" refers to the surface area of ​​a protein surface molecule that is accessible to a solvent, contributed by hydrophobic amino acid residues.

[0061] In this invention, the absolute frequency count of a specific type of amino acid (i) is used. i , which is dimensionless, is used to calculate the proportion of a specific amino acid in a sequence. This is used to calculate the weighted average of the contact energy.

[0062] In this invention, the average coordination number n c Unitless and dimensionless, it represents the average number of neighbors that each amino acid residue has in physical contact with other residues in a completely collapsed, dense sphere state.

[0063] In this invention, the total length N of the polypeptide chain (i.e., the total number of amino acid residues) is dimensionless and has no unit.

[0064] In this invention, ΔG e The fundamental contact free energy between residues, measured in kcal / mol, represents the difference in free energy between two amino acid residues in direct contact in a dense phase (collapsed phase) and those surrounded by water in a dilute phase (solvent-exposed state) at a given temperature.

[0065] In this invention, ΔH h (T0) is the basic enthalpy of hydration at 300 K, in kcal / mol; (ΔC) p ) i : Hydration isobaric thermal capacity change of residue type i, kcal / (mol·K).

[0066] In this invention, the molar gas constant R gas The value is 0.001987, and the unit is kcal / (mol·K).

[0067] In this invention, the chain expansion / compression factor α, being dimensionless and unitless, is used to quantify the degree of deformation of a polymer chain when it is "compressed" from an ideal swollen state to an extremely dense spherical state. The calculation method is as follows: , where R coil and R ext These are the radii of the collapsed state and the highly swollen expanded state, respectively, both in nm.

[0068] In this invention, the mean square end-to-end distance (Ree), measured in nm, is the root mean square distance of the straight line from the start point (N-terminus) to the end point (C-terminus) of the polymer chain in three-dimensional space, reflecting the degree of chain extension in the relaxed state. It is also calculated directly by the AFRC engine through the analysis of the input amino acid sequence.

[0069] This invention provides a method for predicting the critical temperature of a single-molecule phase transition in an elastomeric protein. This prediction method can be deployed on a server, personal computer, cloud computing platform, laboratory information management system, protein design platform, or local workstation and executed by one or more processors.

[0070] The prediction method includes:

[0071] S1. Count the total number of residues N and the absolute frequency count of residue type i in the amino acid sequence of the elastomer protein. i ;

[0072] S2. Obtain the contact free energy temperature response parameters of residue type i, and calculate the fundamental contact free energy ΔG between residues of residue type i under temperature variable T using the contact free energy temperature response parameters of residue type i. e (T) i ;

[0073] S313. Calculate the all-atom interaction energy G of the elastomer protein under temperature variable T. int_raw (T):

[0074] ,

[0075] Among them, the all-atom interaction energy G int_raw (T) is in kcal / mol; count i : Absolute frequency of residue type i; n c : Average coordination number; N: Total length of polypeptide chain (i.e., total number of amino acid residues).

[0076] S4. Calculate the configurational entropy drag G of the elastomer protein under temperature variable T. conf (T):

[0077] ,

[0078] Among them, configuration entropy drag G conf (T) is in kcal / mol; R gas : Molar gas constant, valued at 0.001987, unit is kcal / (mol·K); α: Chain expansion / compressibility factor, dimensionless, calculated as follows: , where R coil and R ext These are the radii of the collapsed state and the radii of the highly swollen expanded state, respectively, both in nm;

[0079] S315. Establish the single-chain collapse free energy function G with respect to the temperature variable T. total (T):

[0080] ,

[0081] Where, n offset : Pre-set calibration offset;

[0082] S6. Solve for the single-chain collapse free energy function G. total (T) The temperature at which the zero-point condition is met, and this temperature is determined as the single-molecule phase transition critical temperature T of the elastomer protein. c The zero-point condition is as follows:

[0083] .

[0084] In predicting the phase transition temperature of elastin, protein-protein interaction energies are typically obtained through all-atom molecular dynamics simulations, coarse-grained molecular dynamics simulations, residue pair interaction matrix calculations, or empirical regression methods based on hydrophobicity indices. While molecular dynamics simulations and residue pair interaction calculations can describe residue interactions in detail, they require computationally intensive molecular dynamics sampling over long periods or the construction of complete protein conformations and enumeration of residue contact pairs. On the other hand, empirical regression methods based solely on hydrophobicity indices fail to reflect the impact of temperature changes on residue interactions and hydration states.

[0085] This invention employs a simplified computational method that avoids direct full-atom simulation of elastomeric proteins and enumeration of interactions between all residue pairs in the sequence. It transforms the problem of calculating numerous different residue contact pairs into a low-dimensional computational problem based on amino acid type, the proportion of specific amino acid types in the elastomeric protein sequence, temperature-dependent basic contact free energy between residues, and the average coordination number of the collapsed state. This significantly reduces computational complexity while preserving key physical factors affecting elastomeric proteins. The simplified computational method of this application significantly improves the computational efficiency of phase transition temperature prediction, and the calculation results show good consistency with full-residue contact models / high-precision molecular dynamics simulations.

[0086] In some embodiments, the elastomeric proteins include natural elastomeric proteins, extrin, elastomeric polypeptides, fragments or variants of natural elastomeric proteins, fragments or variants of extrin, variants of elastomeric polypeptides, and fusion proteins containing repeating sequences of elastomeric proteins.

[0087] In some embodiments, residue type refers to a type of amino acid residue in the amino acid sequence of the elastomeric protein, and the symbol i is used to identify different residue types. The amino acid residue type includes native amino acid residues and / or modified amino acid residues.

[0088] In some preferred embodiments, residue type i is selected from any one of twenty natural amino acid residue types, including Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

[0089] In some embodiments, in step S2, the contact free energy temperature response parameter of residue type i includes the fundamental contact free energy ΔG between residues of residue type i at a reference temperature T0. e (T0) i Hydration free energy u h (T0)i and linear coupling coefficient (slope) k i (See Table 1);

[0090] The fundamental contact free energy ΔG between residues of residue type i at a temperature variable T e (T) i Calculated using the following formula:

[0091]

[0092] Wherein, ΔG e (T) i : The fundamental contact free energy between residues of residue type i, expressed in kcal / mol, represents the difference in free energy between two amino acid residues in direct contact in a dense phase (collapsed phase) compared to when they are surrounded by water in a dilute phase (solvent-exposed state) at a given temperature; ΔG e (T0) i The fundamental contact free energy between residues of residue type i at a reference temperature T0, expressed in kcal / mol; u h (T) i : The solvation (hydration) free energy of residue type i at a temperature variable T, in kcal / mol; u h (T0) i The fundamental solvation (hydration) free energy of residue type i at a reference temperature T0, expressed in kcal / mol; k i The linear coupling coefficient (slope) describes the modulation intensity of the direct contact energy between residues due to changes in the solvation environment, where k is the linear coupling coefficient for residue type i. i The values ​​are shown in Table 1, reference (Chen FK, Simulating Thermoresponsive Behavior of Disordered Proteins with Temperature-Dependent Coarse-Grained PotentialsDerived from Hydration Free Energies).

[0093] In some preferred embodiments, in step S1, the reference temperature T0 = 300 K.

[0094] Table 1 Critical temperature T of single-molecule phase transition c The prediction method involves the selection of parameter values ​​(T0=300 K).

[0095]

[0096] In some preferred embodiments, the contact free energy temperature response parameter of residue type i further includes enthalpy of hydration.

[0097] ΔH h (T0) i Hydration isobaric heat capacity change (ΔC) p ) i ;

[0098] Under a temperature variable T, the solvation (hydration) free energy u of residue type i h (T) i Calculated using the following formula:

[0099]

[0100] Where, ΔH h (T0) i The basic enthalpy of hydration of residue type i at the reference temperature T0, in kcal / mol;

[0101] (ΔC p ) i : Hydration isobaric thermal capacity change of residue type i, in units of cal / (mol·K), which is converted to kcal / (mol·K) by dividing by 1000 in the formula.

[0102] In step S3, the average coordination number n c It is the effective average coordination number after double counting correction, and its value is derived from the average contact number of the collapsed protein or coarse-grained bead model. In some embodiments, the average coordination number n c Calculated through user input, preset default values, or collapsed state conformation models.

[0103] In some preferred embodiments, the average coordination number n c The average coordination number n was calculated using a collapsed conformation model. This involved generating one or more collapsed conformations for the elastomeric protein, counting the number of non-bonded neighbors of each residue within the contact cutoff distance in each conformation, and averaging the results for all residues and all collapsed conformations. c (Tunyasuvunakool,K., Adler, J., Wu, Z. et al. Highly accurate protein structure prediction for the human proteome. Nature 596, 590-596 (2021)).

[0104] In some preferred embodiments, the average coordination number n c The default value range is 2-6.

[0105] In some preferred embodiments, the average coordination number n cThe default value is 4.

[0106] In some embodiments of the present invention, the collapse radius R coil The calculation method is as follows:

[0107] ,

[0108] Where, count i : Absolute frequency of residue type i in elastin proteins; V i,vdW : Van der Waals volume of residue type i.

[0109] First, calculate the volume V of the sphere in the final collapsed and compacted state. compact Then, according to the formula for the volume of a sphere The collapsed state radius R is obtained. coil Among them, the volume V of the sphere in the final collapsed and compacted state. compact Obtained through van der Waals accumulation of amino acid residues (assuming the collapsed compact state is a sphere, therefore the radius of the collapsed state is calculated):

[0110] ,

[0111] In this invention, the van der Waals volume V of residue type i i,vdW These are parameters well-known in the art. In specific calculations, this invention constructs a van der Waals volume mapping dictionary for amino acid residue types (see Table 1).

[0112] In some implementations, the radius R of the highly swollen expanded state ext The calculation method is as follows:

[0113]

[0114] For the radius R of the highly swollen expanded state ext This invention is based on the empirical scaling law of self-avoiding random walk (Dobay A, Dubochet J, Millett K, Sottas PE, Stasiak A. Scaling behavior of random knots. Proc Natl Acad Sci USA. 2003 May 13;100(10):5611-5615), using the recognized physical constant in condensed matter physics (ν≈0.588) (i.e., the Flory exponent) and the empirical scaling law of peptides to calibrate R0 (studies have shown that the equivalent characteristic step size factor R0 in typical disordered chains is generally 1.92 Å) as the radius R of the highly swollen unfolded state. ext The parameters used in the calculation.

[0115] In some implementations, in step S315, the preset calibration offset is determined by having a known single-chain collapse critical temperature T. ref The baseline sequence is determined;

[0116] ,

[0117] Among them, G int_raw (T) ref The baseline sequence is at the known single-chain collapse critical temperature T. ref All-atomic interaction energy;

[0118] G conf (T) ref The baseline sequence is at the known single-chain collapse critical temperature T. ref Configurational entropy resistance.

[0119] In some preferred embodiments, the reference sequence is a VGVPG sequence repeated 20 times, denoted as (VGVPG). 20 The baseline sequence (VGVPG) 20 The known single-chain collapse critical temperature T ref It is 305 K.

[0120] In some implementations, step S6 involves solving the single-chain collapse free energy function G. total (T) is the temperature at which the zero-point condition is met. Roots are found using the bisection method, secant method, Newton's iteration method, or spline interpolation method.

[0121] Example 1

[0122] This embodiment focuses on the single-molecule phase transition critical temperature T of elastin. c A prediction was made, and the predicted value obtained is T. c_pre and the predicted value T c_pre Compared with the experimental value of intrinsic phase transition temperature (T) tc Correlation analysis was performed (see) Figure 1 This embodiment investigated the single-molecule phase transition critical temperature T of elastin composed of VPGXG repeat sequences. c The prediction is that X is amino acid A or V, and the content ratio of residues involving A and V includes 1.0:0, 0.9:0.1, 0.8:0.2, 0.7:0.3, 0.6:0.4, 0.5:0.5, 0.2:0.8, and 0:1.0. The chain length of elastin (i.e., the total number of amino acid residues) includes 50, 500, 1000, and 2500.

[0123] according to Figure 1 It can be seen that, under the condition that the content ratio of A and V residues is the same, the longer the elastin chain, the higher its T content. c_preCompared with the experimental value of intrinsic phase transition temperature (T) tc The closer the predicted value is to the value of elastin chain length N, the better; for elastin chain length N, the predicted value T is between 50-2500. c_pre Compared with the experimental value of intrinsic phase transition temperature (T) tc The relationship exhibits a good linear relationship (coefficient of determination R). 2 =0.8831). This indicates that the critical temperature T for the unimolecular phase transition in this invention is 0.8831. c The predicted value can accurately reflect the phase transition temperature (T) of elastin. t (Minimum value)

[0124] Example 2

[0125] This embodiment predicts the single-molecule phase transition critical temperature T for elastin with different repeating sequence compositions, such as FPGVG and FPGV. c And the critical temperature T of the unimolecular phase transition c Predicted value vs. experimentally determined phase transition temperature (T) t A comparison was made (see Table 2).

[0126] Sequence 4: MHHHHHADEQEEKAKVRTELIQELAQGLGGFEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGFPGFGVGVGGIPGVAGVPGVGGVPGVGGVPGVGISPEAQAAAAAKAAKYGAAGAGVLGGLVPGAPGAVPGVPGTGGVPGVGTP AAAAAKAAAKAAQFGLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGIGEAQAAAAAKAAKYGAAGAGVLGGLVPGAPGAVPGVPGTGGVPGVGTPAAAAAKAAAKAAQFGLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGIG.

[0127] Table 2. Phase transition temperatures T of the same sequence t With the critical temperature of single-molecule phase transition T c Compare

[0128]

[0129] As shown in Table 2, for the same elastin sequence, the higher the concentration, the higher the experimentally determined phase transition temperature T. t The lower the concentration, the higher the experimentally determined phase transition temperature T. t The higher the temperature, the higher the phase transition temperature T of the elastin described in this embodiment.t It shows a negative correlation with its concentration, which is consistent with the characteristics of elastin. In this embodiment, the phase transition temperature T was determined experimentally using sequences 1-4. t The lowest values ​​were 293.7 K, 291.1 K, 290.2 K, and 307.2 K, respectively, and the critical temperature T for the unimolecular phase transition was... c The predicted values ​​are 283.2 K, 282.8 K, 282.6 K, and 304.4 K, respectively, indicating that the critical temperature T for the single-molecule phase transition is... c Predicted value vs. experimentally determined phase transition temperature T t The lowest values ​​are very close, indicating that the present invention is based on the sequence-predicted single-molecule phase transition critical temperature T. c The value can serve as a reference for the phase transition temperature of elastin. Although the repeating sequences and amino acid chain lengths contained in sequences 1-4 differ, this invention can provide a reliable single-molecule phase transition critical temperature T0. c Predicted value, and can be used as phase transition temperature T t The reference values ​​indicate that the method described in this invention is suitable for the phase transition temperature T of elastin with different chain lengths and sequence compositions. t The prediction.

[0130] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A method for predicting the critical temperature of a single-molecule phase transition in an elastomeric protein, characterized in that, The prediction method includes: S1. Count the total number of residues N and the absolute frequency count of residue type i in the amino acid sequence of the elastomer protein. i ; S2. Obtain the contact free energy temperature response parameters of residue type i, and calculate the fundamental contact free energy ΔG between residues of residue type i under temperature variable T using the contact free energy temperature response parameters of residue type i. e (T) i ; S3. Calculate the all-atom interaction energy G of the elastomer protein under temperature variable T. int_raw (T): ， Where, count i : Absolute frequency of residue type i; n c : Average coordination number; N: Total number of residues in the amino acid sequence; S4. Calculate the configurational entropy drag G of the elastomer protein under temperature variable T. conf (T): ， Among them, R gas α: Molar gas constant; α: Chain expansion / compressibility factor. , where R coil and R ext These are the radii of the collapsed state and the radii of the highly swollen and expanded state, respectively. S5. Establish the single-chain collapse free energy function G with respect to the temperature variable T. total (T): ， Where, n offset : A pre-set calibration offset; the pre-set calibration offset is determined by a known single-chain collapse critical temperature T. ref The baseline sequence is determined; , G int_raw (T) ref The baseline sequence is at the known single-chain collapse critical temperature T. ref All-atomic interaction energy; G conf (T) ref The baseline sequence is at the known single-chain collapse critical temperature T. ref Configurational entropy resistance; S6. Solve for the single-chain collapse free energy function G. total (T) The temperature at which the zero-point condition is met, and this temperature is determined as the single-molecule phase transition critical temperature T of the elastomer protein. c The zero-point condition is as follows: 。 2. The prediction method according to claim 1, characterized in that, The elastin proteins include natural elastin, extrin, elastin-like peptides, natural elastin fragments or variants, extrin fragments or variants, elastin-like peptide variants, and fusion proteins containing elastin-like repeat sequences.

3. The prediction method according to claim 1, characterized in that, In step S2, the contact free energy temperature response parameter of residue type i includes the fundamental contact free energy ΔG between residues of residue type i at a reference temperature T0 = 300 K. e (T0) i Hydration free energy u h (T0) i and linear coupling coefficient k i ; The fundamental contact free energy ΔG between residues of residue type i at a temperature variable T e (T) i Calculated using the following formula: , Among them, u h (T) i : The solvation free energy of residue type i at a temperature variable T; Under temperature variable T, the solvation free energy u of residue type i h (T) i Calculated using the following formula: ΔH h (T0) i Enthalpy of hydration, (ΔC) p ) i The isobaric thermal capacity change of the residues is due to hydration.

4. A system for predicting the critical temperature of a single-molecule phase transition in an elastomeric protein, characterized in that, The system includes: The calculation module is used to calculate the critical temperature of the single-molecule phase transition of elastomeric proteins. The critical temperature for the unimolecular phase transition is calculated using the following steps: S1. Count the total number of residues N and the absolute frequency count of residue type i in the amino acid sequence. i ; S2. Obtain the contact free energy temperature response parameters of residue type i, and calculate the fundamental contact free energy ΔG between residues of residue type i under temperature variable T using the contact free energy temperature response parameters of residue type i. e (T) i ; S3. Calculate the all-atom interaction energy G of the elastomer protein under temperature variable T. int_raw (T): ， Where, count i : Absolute frequency of residue type i; n c : Average coordination number; N: Total number of residues in the amino acid sequence; S4. Calculate the configurational entropy drag G of the elastomer protein under temperature variable T. conf (T): ， Among them, R gas α: Molar gas constant; α: Chain expansion / compressibility factor. , where R coil and R ext These are the radii of the collapsed state and the radii of the highly swollen and expanded state, respectively. S5. Establish the single-chain collapse free energy function G with respect to the temperature variable T. total (T): ， Where, n offset : A pre-set calibration offset; the pre-set calibration offset is determined by a known single-chain collapse critical temperature T. ref The baseline sequence is determined; , G int_raw (T) ref The baseline sequence is at the known single-chain collapse critical temperature T. ref All-atomic interaction energy; G conf (T) ref The baseline sequence is at the known single-chain collapse critical temperature T. ref Configurational entropy resistance; S6. Solve for the single-chain collapse free energy function G. total (T) The temperature at which the zero-point condition is met, and this temperature is determined as the single-molecule phase transition critical temperature T of the elastomer protein. c The zero-point condition is as follows: 。 5. The prediction system according to claim 4, characterized in that, In step S2, the contact free energy temperature response parameter of residue type i includes the fundamental contact free energy ΔG between residues of residue type i at a reference temperature T0 = 300 K. e (T0) i Hydration free energy u h (T0) i and linear coupling coefficient k i ; The fundamental contact free energy ΔG between residues of residue type i at a temperature variable T e (T) i Calculated using the following formula: , Among them, u h (T) i : The solvation free energy of residue type i at a temperature variable T; Under temperature variable T, the solvation free energy u of residue type i h (T) i Calculated using the following formula: ΔH h (T0) i Enthalpy of hydration, (ΔC) p ) i The isobaric thermal capacity change of the residues is due to hydration.

6. An apparatus, characterized in that, The device includes at least one storage medium and at least one processor, the at least one storage medium being used to store computer instructions, and the at least one processor being used to execute the computer instructions to implement the method for predicting the phase transition temperature of elastomeric proteins as described in any one of claims 1-3.

7. A computer-readable storage medium, characterized in that, The storage medium stores computer instructions, which, when executed by a processor, implement the method for predicting the phase transition temperature of elastomeric proteins as described in any one of claims 1-3.

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