A molecular design method for improving the hydrophobicity of the catalytic center of a bacillus subtilis protease
By screening and mutating the amino acids in the catalytic center of Bacillus subtilis protease using the polarizable molecular force field method, the problem of precise design of protein hydrophobicity was solved, and the high hydrophobicity and water molecule expulsion of the catalytic center were achieved, which has broad application prospects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- EAST CHINA NORMAL UNIV
- Filing Date
- 2024-09-10
- Publication Date
- 2026-05-19
AI Technical Summary
Existing technologies struggle to accurately describe the charged states of proteins in solvent environments and to provide a true characterization of local hydrophobic regions, making it difficult to precisely calculate and design the ability of proteases to bind and transfer water molecules.
By employing the polarizable molecular force field method, potential mutation sites were screened and virtual mutations were performed by calculating the polar atomic charges of amino acids in the catalytic center of Bacillus subtilis protease, resulting in highly hydrophobic mutants and eliminating water molecules from the catalytic center.
It improves the hydrophobicity of the protease catalytic center and enhances the ability of water molecules to be expelled from the catalytic center, which has important application value in drug design, materials science and biomedical engineering.
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Figure CN119132455B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering, specifically relating to a rational design method for the binding capacity of water molecules within a charge characterization region. Background Technology
[0002] The term "hydrophobic" was coined in 1915 to specifically refer to the phenomenon that nonpolar compounds have very low solubility in water or tend to aggregate in water. Hydrophobic and hydrophilic effects have gradually been recognized as one of the dominant driving forces in biochemical processes, playing a crucial role in several key areas such as protein folding, nucleic acid stability, molecular recognition, and binding. These effects are fundamentally essential for understanding and researching the stability of molecular structures, functional realization, and disease mechanisms.
[0003] Hydrophobicity can be characterized on a macroscopic scale by measuring the contact angle of droplets, but it cannot be directly observed or accurately described at the microscopic scale of molecules and atoms. Whether it's the characterization method of the hydration index between water and amino groups proposed by Millard-Stafford, the suggestion by Tang et al. that protein hydrophobicity depends on the chemical properties of side chains and their positions within the protein, the method used by Gao et al. based on residue water density and single-chain clustering, or the method used by Rienzo et al. to describe the dynamic hydrogen bond network at the protein-solvent interface using atomic-level molecular dynamics simulations, all studies on protein hydrophobicity are based on the amino acid molecular level. More precise atomic-level analysis and design of protein hydrophobic regions remains a significant challenge.
[0004] This is because the local hydrophobic regions of proteins are not uniformly nonpolar, but rather exhibit polarity (charge changes) at the nanoscale due to the influence of surrounding amino acids under the influence of their geometric conformation. Therefore, it is necessary to comprehensively consider the influence of the environment of this local region and the interactions between several residues on the charge. However, the molecular force field parameters used in current traditional protein calculations fix the charge of each amino acid side chain atom, failing to account for the influence of environmental changes, making it difficult to achieve accurate calculation and design of local hydrophobic regions.
[0005] The polarizable force field method proposed by Shi et al. in 2015 promises to address this problem effectively. This is a variable molecular force field that can simulate local environmental changes, realistically reflecting changes in the dipole moment of water molecules, electrostatic polarization effects, and local structural rearrangements of proteins. Therefore, compared to traditional fixed-charge force fields, polarizable molecular force fields can more accurately describe the charged state of each atom in a protein within a solvent environment, thus accurately characterizing the overall polarity (i.e., hydrophobicity) of a specific region. Furthermore, a high-intensity, pervasive polarization-induced effect exists within proteases. Since water molecule fixation is primarily achieved through H bonds with surrounding polar atoms, the stronger the atom's polarity, the stronger the H bond, and consequently, the stronger the protease's ability to bind and transfer water molecules. For N and O atoms, a more negative polar charge indicates a higher electron cloud density and a higher degree of polarization, resulting in a stronger ability to form H bonds with water molecule H atoms; conversely, for H atoms on N and O atoms, a more positive polar charge makes it easier to form H bonds with other polar atoms, thus exhibiting high polarization intensity. In summary, selecting a precise polarizable molecular force field, "protein-specific polarization charge" (PPC), and calculating the polar charges of the N, O, and H atoms of proteases based on this, thereby characterizing their ability to bind water molecules, is of great significance for drug design, materials science, and biomedical engineering. A series of protein engineering techniques targeting the hydrophobicity of proteins, especially enzymes, have attracted the attention of scientists. Computational rational design methods are widely used due to their high efficiency, minimal experimental screening workload, and ability to quickly obtain superior mutants.
[0006] Therefore, current common hydrophobic computational designs mainly start from fixing the hydrophobic values of amino acid residues or fixing the charge force field parameters, and rarely start from the polarizable molecular force field method. This makes it difficult to directly observe and accurately describe the charged state of each atom of the protein in the solvent environment and the true characterization of the local hydrophobic region. This invention uses a series of computer rational calculations to perform targeted modification to improve the hydrophobicity of the Bacillus subtilis protease catalytic center, thereby achieving the expulsion of water molecules from the catalytic center. Summary of the Invention
[0007] To address the aforementioned problems in the prior art, this invention provides a molecular modification design method for improving the hydrophobicity of the catalytic center of Bacillus subtilis protease. This molecular modification design method can rapidly and effectively reduce water molecules in the catalytic center of Bacillus subtilis protease, thereby improving hydrophobicity. The water content of the catalytic center is confirmed by analyzing the crystal structures of Bacillus subtilis protease and its substrate, and the ability to exclude water molecules is further confirmed by calculating the binding free energy. Verification results show that this design method can rapidly and effectively improve the hydrophobicity of the catalytic center of Bacillus subtilis protease, and the enzyme modification method of this invention is reliable.
[0008] The purpose of this invention is to provide a molecular modification design method for improving the hydrophobicity of the catalytic center of Bacillus subtilis protease, comprising the following steps:
[0009] (1) Locate the resolved crystal structure of Bacillus subtilisin (BPN′) in the protein database PDB, and then... The Glide mode of Dinger software was used to dock the hexapeptide substrate to generate a subtilisin-substrate complex. The docking structure was simulated for 500 ns using MD. The subtilisin-substrate complex structure with the lowest potential energy during the simulation was selected as its analysis conformation. The amino acid sequence of the hexapeptide substrate is Suc-AAPFAF-amide.
[0010] (2) Based on the analytical conformation obtained in step (1), the polar charges of the polar atoms N, O and their bound H of each amino acid residue in the range of 10 Å of the amino acid His64 in the catalytic center of subtilisin are calculated by the polarized protein-specific charge PPC force field. The absolute values of the polar charges are summed and sorted from high to low. After excluding the amino acid residues that make up the subtilisin catalytic triplet, at least the first 10 amino acid residues with high absolute value sums are selected as potential mutation sites of subtilisin.
[0011] (3) The potential mutation sites of subtilisin obtained in step (2) are mutated into virtual single-point mutants with large steric hindrance nonpolar amino acids to obtain virtual subtilisin single-point mutants. The polar charges of polar atoms N, O and H bound to His64 amino acid in the catalytic center of the virtual subtilisin single-point mutant are calculated using the PPC force field. The absolute values of the polar charges are summed and sorted from low to high. At least the first 10 virtual subtilisin single-point mutants with low absolute value sums are selected for further expression. The above-mentioned large steric hindrance nonpolar amino acids are Val, Leu, Ile, Phe, Trp and Met.
[0012] (4) The virtual subtilisin single-point mutant selected in step (3) is expressed, and the successfully expressed mutant is iteratively mutated and expressed again to obtain the subtilisin mutant and verify its hydrophobicity.
[0013] Specifically, in step (2) above, the catalytic triplet of subtilisin is composed of serine at position 221, histidine at position 64, and aspartic acid at position 32. The key amino acids of the catalytic center of subtilisin are serine at position 221, histidine at position 64, and aspartic acid at position 32, which make up its catalytic triplet. The mutation point excludes the key amino acids of the catalytic center.
[0014] The hydrophobicity verification step in step (4) above is specifically as follows: the obtained subtilisin mutant is confirmed by crystal structure to determine whether there are water molecules of crystallization in the 4.5 Å range of the catalytic center, and the binding free energy between the mutant and the substrate Suc-AAPFAF-amide is calculated by MM / GBSA to verify the hydrophobicity of the subtilisin mutant.
[0015] The modified subtilisin mutant obtained through the molecular modification design method of this invention, confirmed by crystal structure, contains the catalytic center of the subtilisin mutant. No water molecules of crystallization are present inside; most water molecules are distributed in... In addition, the binding free energy of the subtilisin mutant and the original subtilisin to the substrate was calculated by MM / GBSA. The binding free energy of the subtilisin mutant to the substrate was significantly lower than that of the original protease, and the binding of the substrate to the subtilisin mutant was more "tight", excluding water molecules.
[0016] In the docking process of step (1) above, subtilisin was prepared in Protein Preparation Wizard, hydrogen bond network was optimized in OPLS2005 force field, substrate was prepared by LigPrep panel, the catalytic triplet of subtilisin was defined as the center of the grid frame, and docking was performed using the precision SP mode with default parameters in the receptor rigid docking RRD protocol.
[0017] The MD simulation in step (1) above includes the following steps:
[0018] (1) Simulation parameters were provided by the ff14SB force field of AMBER18, with a truncated periodic octahedral box of TTIP3P water molecules serving as the solvent environment, and a buffer distance of [missing information]. Add sodium ions as counterions;
[0019] (2) First, the solvent was optimized using the 5000-step steepest descent method and the 5000-step conjugate gradient method. The solute was subjected to... The system is constrained by the force constant; secondly, the entire system is optimized using the 25,000-step steepest descent method and the 25,000-step conjugate gradient method, without any constraints.
[0020] (3) The system was heated to 333K for 300 ps, and the solute was confined to the atmosphere. In the weak force constant, three MD simulations of 500 ns were performed for each system under the NPV set. Berendsen barostat and Langevin dynamics were used to control pressure and temperature, respectively. The SHAKE algorithm was used to constrain the bond lengths of all hydrogen atoms, and the cutoff distance for long-range interactions was set to [value missing].
[0021] The polar charges of the N, O, and H atoms of subtilisin are calculated using the PPC force field of the polarized protein specific charge in step (2) above, including the following steps:
[0022] (1) The subtilisin molecules were cleaved into amino acid fragments at the peptide bond by molecular differentiation and conjugated cap MFCC method. A pair of conjugated caps were added to each cleaved peptide bond position to complete the electronic structure. The electron density distribution of each amino acid fragment was calculated using Gaussian 16 at the B3LYP / 6-31G* level to obtain the electrostatic potential of the surface of each amino acid fragment.
[0023] (2) The atomic charge of the subtilisin molecule was fitted using the restrained electrostatic potential RESP program. The electrostatic dissolution energy and induced surface charge were obtained by solving the Poisson-Boltzmann (PB) equation. The induced surface charge and the atomic charge of other molecular fragments were used as background charges to repeatedly calculate the new charge density distribution. The solute and solvent were mutually polarized until the dissolution energy and the subtilisin charge tended to be consistent.
[0024] Compared with the prior art, the present invention:
[0025] (1) Based on the charge characterization region, the present invention calculates the charge of polar atoms N, O and H atoms bound by them according to molecular docking, full atom quantification and other methods, characterizes the polarization ability of amino acid residues in the 10 angstrom range of the catalytic center, that is, the ability to bind water molecules, virtually screens out mutant sites with potential improved hydrophobicity, and uses virtual mutation to mutate them into sterically hindered nonpolar amino acids (Val, Leu, Ile, Phe, Trp, Met), evaluates and screens out mutants with weak water-binding ability in the 10 angstrom range of amino acids in the catalytic center, selects the successfully expressed subtilisin single-point mutant, and then obtains the subtilisin mutant through iterative mutation and expression;
[0026] (2) Hydrophobicity verification revealed that the catalytic center of the subtilisin mutant... No water molecules of crystallization are present inside; most water molecules are distributed in... In addition, the binding free energy of the subtilisin mutant and the original subtilisin to the substrate was calculated using MM / GBSA. The binding free energy of the subtilisin mutant to the substrate was significantly lower than that of the original protease, indicating that the substrate was more "tightly" bound to the subtilisin mutant, excluding water molecules.
[0027] Therefore, the subtilisin mutant obtained by this design method has high hydrophobicity, enabling the expulsion of water molecules from the catalytic center, which will have extremely important value in drug design, materials science, and biomedical engineering. Attached Figure Description
[0028] Figure 1 This is a diagram showing the molecular docking between subtilisin and Suc-Ala-Ala-Pro-Phe-Ala-Phe-amide.
[0029] Figure 2 The RMSD value of subtilisin during MD simulation;
[0030] Figure 3 This diagram shows the binding capacity of amino acid residues within the 10 Å range of His64, the catalytic center of subtilisin, to water molecules.
[0031] Figure 4 Map showing the potential mutation site region within 10 Å of the His64 amino acid, the catalytic center of subtilisin;
[0032] Figure 5 SDS-PAGE gel images of purified subtilisin and mutant proteins;
[0033] Figure 6 Comparative diagram of local water molecule crystal structures of subtilisin and mutant. Detailed Implementation
[0034] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0035] The Bacillus subtilis protease of this invention is selected from Bacillus amyloliquefaciens.
[0036] Example 1: Molecular docking and MD simulation of Bacillus amyloliquefaciens protease
[0037] 1. Search the Protein Database (PDB) for the resolved crystal structure of Bacillus subtilisin BPN' (PDB ID: 1TO1).
[0038] 2. Use Dinger's Glide mode docks with the hexapeptide substrate Suc-AAPFAF-amide to generate an enzyme-substrate complex structure, such as... Figure 1As shown. During docking, the protein was prepared in the Protein Preparation Wizard, and the hydrogen bond network was optimized in the OPLS2005 force field to mitigate potential steric conflicts. Hexapeptide substrates were prepared via the LigPrep panel, and low-energy 3D structures were generated through tautomerism, stereoisomerism, and ring conformation. To predict the binding mode, the catalytic triplet in the protein (composed of serine at position 221, histidine at position 64, and aspartic acid at position 32) was defined as the center of the grid box, and docking was performed using the precision (SP) mode with default parameters in the RRD (receptor rigid docking) protocol.
[0039] 3. Further 500 ns MD simulations were performed on the docking structure to obtain stable results. The structure with the lowest potential energy during the simulation was selected as the stable subtilisin structure. The RMSD value of the subtilisin during the simulation is shown below. Figure 2 As shown.
[0040] The steps of MD simulation are as follows:
[0041] Simulation parameters were provided by the ff14S3B force field of AMBER18. A truncated periodic octahedral box of TTIP3P water molecules was used as the solvent environment, with a buffer distance of [missing information]. Sodium ions were added as counterions to maintain the electroneutrality of the entire system. The entire system was then optimized in two steps to avoid undesirable contact between solute and solvent molecules. First, the solvent was optimized using a 5000-step steepest descent method and a 5000-step conjugate gradient method, while the solute was subjected to… Force constant constraint. Next, the entire system was optimized using the 25,000-step steepest descent method and the 25,000-step conjugate gradient method, without any constraints. Then, the system was heated to 333K for 300 ps, while the solute was confined to... The weak force constant is determined. Finally, three MD simulations of the system were performed over 500 ns using the NPV set. Berendsen barostat and Langevin dynamics were used to control pressure and temperature, respectively. Simultaneously, the SHAKE algorithm was used to constrain all bond lengths involving hydrogen atoms, with the cutoff distance for long-range interactions set to [value missing].
[0042] Example 2: Screening for potential mutation sites within 10 angstroms of the amino acid catalytic center of subtilisin
[0043] Based on the stable enzyme-substrate complex structure obtained in Example 1, the charges of the polar atoms N, O, and their bound H atoms (protons H) of the residues within a 10 Å radius of the amino acid His64 at the catalytic center of the subtilisin were calculated using the polarized protein-specific charge (PPC) force field calculated by all-atom quantum mechanics. The binding affinity of the amino acid residues to water molecules (i.e., the sum of the absolute values of the polar charges of N, O, and their bound H atoms in the amino acid residues) was calculated using the following formula:
[0044]
[0045] Where C represents the binding ability of an amino acid to water molecules; the higher the C value, the higher the degree of polarization of the amino acid, and the stronger its ability to bind water molecules; e ppc,i This represents the charge of the i-th atom calculated using the PPC force field; n is the total number of polar atoms (N, O, H protons (H)) in this amino acid. The calculation results are as follows: Figure 3 As shown.
[0046] Amino acid residues within the 10 Å range of the His64 amino acid in the catalytic center of subtilisin were sorted from highest to lowest absolute value of their polar charge. After excluding the amino acid residues that make up the subtilisin catalytic triad, at least the top 10 amino acid residues with the highest absolute charge sum were selected as potential mutation sites for subtilisin. Potential mutation sites within the 10 Å range of the His64 amino acid in the catalytic center were virtually screened. Figure 4 As shown.
[0047] The steps for calculating the PPC force field are as follows:
[0048] All-atom quantum mechanical calculations were performed by cleaving peptide bonds into amino acid fragments using the molecular differentiation and conjugation cap (MFCC) method. A pair of conjugation caps was added to each cleaved peptide bond to complete the electronic structure. The electron density distribution of each fragment was then calculated using Gaussian 16 at the B3LYP / 6-31G* level. Simultaneously, the electrostatic potential of each fragment's surface was obtained. Next, the restrained electrostatic potential (RESP) procedure was used to fit the atomic charge of the entire protein. The electrostatic dissolution energy and induced surface charge were obtained by solving the Poisson-Boltzmann (PB) equation. The induced surface charge and the atomic charges of other fragments were used as background charges to iteratively calculate the new charge density distribution. Finally, the solute and solvent were mutually polarized until the dissolution energy and protein charge converged.
[0049] Example 3 Virtual screening of single-point mutants of subtilisin
[0050] Based on the stable enzyme-substrate complex structure obtained in Example 1, virtual mutations were performed on the potential mutation sites with high absolute values screened in Example 2. Each site was half-saturated with a sterically hindered nonpolar amino acid (Val, Leu, Ile, Phe, Trp, Met) to obtain virtual subtilisin single-point mutants. Virtual single-point mutants with weak water-binding capacity were selected. The virtual screening method used a PPC force field to calculate the water-binding capacity within a 10 Å region of the His64 amino acid residue in the catalytic center of the virtual single-point mutant. This was calculated by summing the absolute values of the polar charges of N, O, and their bound H atoms within a 10 Å region of the His64 amino acid residue in the catalytic center. The virtual subtilisin single-point mutants were sorted from low to high according to the sum of the absolute values of the polar charges within a 10 Å region of the His64 amino acid residue in the catalytic center. At least the top 10 virtual subtilisin single-point mutants with the lowest sums of polar charges were selected as virtual mutants for further expression. The water-binding capacity within a 10 Å region of the His64 amino acid residue in the catalytic center was calculated using the following formula:
[0051]
[0052] Among them, C t This refers to the water-binding capacity within the 10 Å region of the His64 amino acid residues in the catalytic center. The higher the carbon content (C), the higher the polarization of this region, and thus the stronger the water-binding capacity. i This represents the binding capacity of the i-th amino acid residue in the region to water molecules, calculated using the same method as in Example 2; n is the total number of amino acid residues in the region.
[0053] Example 4: Expression, purification, and iterative mutagenesis of subtilisin and mutants
[0054] 1. The Bacillus subtilis WB600 expression strain was cultured in 50 mL of 2xYT medium (containing 100 μg / ml Kanamycin, 5 mM CaCl2) (37℃, 200 rpm, 20-24 h), and the supernatant was collected by centrifugation (4000 g, 15 min, 4℃).
[0055] 2. Slowly add 3 volumes of pre-cooled 95% ethanol to the supernatant, stir at 4°C for 1-2 hours, centrifuge (18000g, 20min, 4°C), and dry the precipitate at room temperature. Resuspend the precipitate in equilibration buffer (20mM PBS, 500mM NaCl, 30mM imidazole, pH=7.4) using a 10m nickel column, centrifuge (18000g, 20min, 4°C), and retain the supernatant for protein purification using a HisTrap HP 1mL affinity chromatography column. Elute with a linear gradient of AKTA using nickel column elution buffer (20mM PBS, 500mM NaCl, 500mM imidazole) to obtain the purified protein. Concentrate the protein three times using a 10K centrifuge tube with buffer (20mM PBS, 500mM NaCl, pH 8.0) to remove imidazole and obtain a high-concentration protein.
[0056] 3. Select a successfully expressed subtilisin single-point mutant and perform targeted mutagenesis on its recombinant plasmid to obtain an iterative mutant plasmid. The mutation sites are the same as those of the other successfully expressed single-point mutants. Transform the iterative mutant plasmid into Bacillus subtilis WB600 for expression and purification again. Purity is detected by SDS-PAGE. The results for subtilisin and mutant detection are as follows: Figure 5 As shown.
[0057] Example 5: Comparison of crystal structures of subtilisin and mutant
[0058] A comparison of the water molecule crystal structures in local regions of Subtilisin and its mutant, such as... Figure 6 As shown in the figure, two real crystal structures are illustrated, allowing a direct visual representation of the number of water molecules at the catalytic center during the catalytic process. The number of red spheres represents the number of water molecules at the catalytic center in the aqualigase mutant, while the number of blue spheres represents the number of water molecules at the catalytic center in the wild type. Figure 6 It can be clearly observed that at a distance from the catalytic center Within the range, the aqualigase mutant (red) contains no water molecules, while the wild type (blue) contains three water molecules.
[0059] Crystal structure analysis confirmed that the mutant contains His64 and Ser221 in the catalytic center. No water molecules of crystallization are present inside; most water molecules are distributed in... In addition, this is related to the catalytic triplet in wild-type subtilisin (BPN'). The distribution of the three water molecules of crystallization is significantly different, indicating that the mutants obtained by the design method of this invention have high hydrophobicity.
[0060] Example 6: Determination of the binding free energy of subtilisin and mutant
[0061] The binding free energies of Subtilisin to the substrate Suc-AAPFAF-amide in mutant and wild-type samples were calculated using the MM / GBSA method.
[0062] The MM / GBSA method can be expressed by the following formula:
[0063] △G bind =△G gas +△G solv (1)
[0064] Where △G gas and △G solv These represent the gas phase energy and the dissolution free energy, respectively. Wherein, ΔG gas It consists of electrostatic interactions and van der Waals (vdW) interactions, as detailed below:
[0065] △G gas =△E ele +△E vdw (2)
[0066] △G solv It consists of two parts: polar and nonpolar dissolution free energy, as detailed below:
[0067] △G solv =△G gb +△G np (3)
[0068] Polar dissolution free energy ΔG gb The nonpolar dissolution free energy ΔG is calculated using the GB module. np Calculated in the following way:
[0069] △G np =γ×SASA+β(4)
[0070] SASA represents the solvent-accessible surface area, calculated by the MSMS program. γ and β are set to... and 0.00 kcal·mol -1 .
[0071] The measurement results are shown in the table below:
[0072]
[0073] The results showed that the binding free energies of the mutant and wild-type Subtilisin were -33.9 kcal / mol and -8.05 kcal / mol, respectively. The binding free energy was significantly lower than that of the wild-type by 25.8492 kcal / mol, indicating that the substrate and enzyme were bound more tightly, excluding water molecules. These data suggest that the mutant has relatively high hydrophobicity.
[0074] This invention uses Bacillus amyloliquefaciens as an example. Based on a rational design of the water-binding ability within the charge characterization region, the charges of polar atoms N, O, and H are calculated using methods such as molecular docking and full-atom quantification. The polarization ability (i.e., water-binding ability) of amino acid residues in the 10 Å range of the catalytic center amino acid His64 is characterized, and potential mutant sites that can improve hydrophobicity are virtually screened. After mutating these amino acids to sterically hindered nonpolar amino acids (Val, Leu, Ile, Phe, Trp, Met), virtual single-point mutants with weak water-binding ability within the 10 Å range of the catalytic center amino acid His64 are evaluated and screened. These single-point mutants are expressed, and iterative mutation and expression are performed to finally obtain mutants with high hydrophobicity at the catalytic center. The location of His64 and Ser221 at the catalytic center is confirmed by crystal structure analysis. No water molecules of crystallization are present inside; most water molecules are distributed in... In addition, it has a catalytic triplet with wild-type subtilisin (BPN'). The distribution of the three water molecules of crystallization is significantly different. The binding free energy of the mutant and wild-type Subtilisin with the substrate Suc-AAPFAF-amide was calculated by MM / GBSA. The binding free energy of the enzyme mutant with the substrate is significantly lower than that of the wild type, that is, the binding of the substrate with the enzyme mutant is more "tight", which excludes water molecules and achieves the expulsion of water molecules in the catalytic center. Finally, a mutant with high hydrophobicity in the catalytic center is obtained, which verifies that the molecular modification design method of the present invention is reliable and effective.
[0075] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention and should be defined by the claims.
Claims
1. A molecular modification design method for improving the hydrophobicity of the catalytic center of Bacillus subtilis protease, characterized in that, Includes the following steps, (1) Based on the analytical conformation obtained in step (1), the resolved crystal structure of Bacillus subtilis protease in the protein database PDB is searched using a polarized protein-specific charge (PPC) force field meter. The Glide mode of Dinger software was used to dock the hexapeptide substrate to generate a subtilisin-substrate complex, and its docking structure was simulated for 500 ns. The subtilisin-substrate complex structure with the lowest potential energy during the simulation was selected as its analysis conformation. The amino acid sequence of the hexapeptide substrate is Suc-AAPFAF-amide. (2) Based on the analytical conformation obtained in step (1), the polar charges of the polar atoms N, O and their bound H of each amino acid residue in the range of 10 Å of the amino acid His64 in the catalytic center of subtilisin are calculated by the polarized protein-specific charge PPC force field. The absolute values of the polar charges are summed and sorted from high to low. After excluding the amino acid residues that make up the subtilisin catalytic triplet, at least the first 10 amino acid residues with high absolute value sums are selected as potential mutation sites of subtilisin. (3) The potential mutation sites of the subtilisin obtained in step (2) are mutated into virtual single-point mutants with large steric hindrance nonpolar amino acids to obtain virtual subtilisin single-point mutants. The polar charges of the polar atoms N, O and their bound H in the 10 Å range of the amino acid His64 in the catalytic center of the virtual subtilisin single-point mutant are calculated using the PPC force field. The absolute values of the polar charges are summed and sorted from low to high. At least the first 10 virtual subtilisin single-point mutants with low absolute value sums are selected for further expression. The large steric hindrance nonpolar amino acids are Val, Leu, Ile, Phe, Trp and Met. (4) The virtual subtilisin single-point mutant selected in step (3) is expressed, and the successfully expressed mutant is iteratively mutated and expressed again to obtain the subtilisin mutant and verify its hydrophobicity.
2. The molecular modification design method for improving the hydrophobicity of the catalytic center of Bacillus subtilis protease according to claim 1, characterized in that, The catalytic triplet of subtilisin described in step (2) is composed of serine at position 221, histidine at position 64, and aspartic acid at position 32 of subtilisin.
3. The molecular modification design method for improving the hydrophobicity of the catalytic center of Bacillus subtilis protease according to claim 1 or 2, characterized in that, The hydrophobicity verification step in step (4) specifically involves confirming the presence of water of crystallization molecules within 4.5 Å of the catalytic center of the obtained subtilisin mutant through crystal structure analysis, and calculating the binding free energy between the mutant and the substrate Suc-AAPFAF-amide using MM / GBSA to verify the hydrophobicity of the subtilisin mutant.