A method for predicting the change in the ratio of monomers and dimers after a single-site mutation of a PSI protein and a mutant of a PSI protein

Abstract

The present invention provides a method for predicting the change in the ratio of monomers and dimers after a single-point mutation of a PSI protein and a mutant of the PSI protein, belonging to the technical fields of computational biology and structural biology. The present invention calculates the change in the Gibbs free energy of the monomer conformation and dimer conformation of the protein after a single-point mutation of the wild-type PSI protein, screens out three mutant proteins with significant changes in monomer and dimer stability: G14A-PSI, W18A-PSI and V22R-PSI, and obtains the two-dimensional stability of each mutant at pH 3.0, pH 5.0 and pH 7.4, respectively, through liquid nuclear magnetic resonance experiments. 1 H‑ 15 N HSQC spectrum, the results showed that the dimer ratio of the G14A‑PSI mutant protein was significantly increased compared with the wild-type PSI.

Description

Technical Field

[0001] The present invention belongs to the technical fields of computational biology and structural biology, and particularly relates to a method for predicting the change in the ratio of monomers and dimers after a single-point mutation of a PSI protein and a mutant of the PSI protein. Background Art

[0002] In plants, Plant Specific Insert (PSI) is a unique insertion sequence of aspartic protease (AP) with antimicrobial activity. PSI often exists in plant cells in the form of AP proenzyme. After infection by exogenous pathogens, the proenzyme is transported to the vacuole and processed into mature AP hydrolase in the acidic environment of the vacuole. During this process, PSI separates from APs, interacts with the vacuole's biological membrane, and induces membrane fusion between the vacuole membrane and the cell membrane. PSI exists in solution as a monomer-dimer equilibrium. Single-site mutations in the PSI protein will change the ratio of monomers to dimers. How to use computational biology to predict the change in the monomer-dimer ratio after single-site mutations in the PSI protein to guide protein modification is a current problem that needs to be solved. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for predicting the change in the ratio of monomers and dimers after a single-point mutation of the PSI protein and a mutant of the PSI protein. The method of the present invention can accurately predict the change in the ratio of monomers and dimers after a single-point mutation of the PSI protein.

[0004] The present invention provides a method for predicting the change in the ratio of monomers and dimers after a single-site mutation of a PSI protein, comprising the following steps: collecting the Gibbs free energy change value of the monomer of the mutant after a single-site mutation of the PSI protein, and calculating it as ΔΔG 单 ; Collect the Gibbs free energy change value of the dimer of the mutant after the PSI protein single point mutation, calculated as ΔΔG 二 ; Relative stability is calculated according to the formula shown in formula 1; when relative stability is greater than 0, it is judged that the dimer ratio decreases and the monomer ratio increases after the single-point mutation of the PSI protein; when relative stability is less than 0, it indicates that the dimer ratio increases and the monomer ratio decreases after the single-point mutation of the PSI protein; relative stability = ΔΔG 单 -ΔΔG 二 Formula 1; the amino acid sequence of the PSI protein is shown in SEQ ID NO.1.

[0005] Preferably, the site of the PSI protein single point mutation is position 14, position 18 or position 22.

[0006] Preferably, the PSI protein single point mutation includes: glycine at position 14 mutated to alanine, tryptophan at position 18 mutated to alanine, or valine at position 22 mutated to arginine.

[0007] The present invention also provides PSI protein mutants, including G14A-PSI, W18A-PSI or V22R-PSI; the amino acid sequences of G14A-PSI, W18A-PSI and V22R-PSI are shown in SEQ ID NO.2 to SEQ ID NO.4, respectively.

[0008] The present invention also provides the coding gene of the PSI protein mutant described in the above scheme, the nucleotide sequence of the coding gene of the G14A-PSI is shown as SEQ ID NO.5; the nucleotide sequence of the coding gene of the W18A-PSI is shown as SEQ ID NO.6; the nucleotide sequence of the coding gene of the V22R-PSI is shown as SEQ ID NO.7.

[0009] The present invention also provides a recombinant plasmid into which the coding gene described in the above scheme is inserted.

[0010] The present invention also provides a recombinant bacterium comprising the recombinant plasmid described in the above scheme.

[0011] The present invention also provides the use of the PSI protein mutant, the encoding gene, the recombinant plasmid or the recombinant bacteria described in the above scheme in the preparation of tumor-targeted drugs and / or tumor diagnostic reagent carriers.

[0012] The present invention also provides a tumor-targeted drug and / or tumor diagnostic reagent carrier, comprising the PSI protein mutant, the encoding gene, the recombinant plasmid or the recombinant bacteria described in the above scheme.

[0013] The present invention also provides the application of W18A-PSI and V22R-PSI in the study of monomer conformation in protein structure; the amino acid sequences of the W18A-PSI and V22R-PSI are shown in SEQ ID NO.3 to SEQ ID NO.4, respectively.

[0014] The present invention provides a method for predicting the change in the ratio of monomers and dimers after a single-site mutation of the PSI protein. The method of the present invention collects the Gibbs free energy change value ΔΔG of the monomer after the single-site mutation. 单 and the Gibbs free energy change of the dimer ΔΔG 二 , compare the difference between the two, that is, relative stability (relative stability = ΔΔG 单 -ΔΔG 二 ). When the relative stability is greater than 0, it indicates that the monomer stability increases and the dimer stability decreases.1 H- 15 The N HSQC spectrum shows that the monomer ratio increases and the dimer ratio decreases; when the relative stability is less than 0, it indicates that the monomer stability decreases and the dimer stability increases. 1 H- 15 The NHSQC spectrum showed a decrease in the monomer ratio and an increase in the dimer ratio. The prediction results of the method of the present invention are consistent with the results of liquid nuclear magnetic resonance detection, which can prove the feasibility of the method of the present invention in accurately predicting the change in the monomer and dimer ratio after single-site mutation of PSI protein.

[0015] The present invention also provides mutants of PSI protein, including G14A-PSI, W18A-PSI, or V22R-PSI, wherein G14A-PSI is obtained by mutating the glycine at position 14 of the PSI protein to alanine, and its monomer stability is reduced and the dimer stability is improved compared with wild-type PSI; W18A-PSI is obtained by mutating the tryptophan at position 18 of the PSI protein to alanine; V22R-PSI is obtained by mutating the valine at position 22 of the PSI protein to arginine; compared with wild-type PSI, W18A-PSI and V22R-PSI have improved monomer stability and reduced dimer stability. The present invention combines computational biology and structural biology to predict and verify the ability of key amino acid single-point mutations to regulate protein conformational equilibrium, laying a solid foundation for further modification of functional PSI proteins in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 Schematic diagram of the pET32a(+)-G14A-PSI plasmid map;

[0018] Figure 2 Schematic diagram of the pET32a(+)-W18A-PSI plasmid map;

[0019] Figure 3 Schematic diagram of the pET32a(+)-V22R-PSI plasmid map;

[0020] Figure 4 This is the gel image of the dried sample of target protein detected by SDS-PAGE (with 15 N-G14A-PSI as an example);

[0021] Figure 5 for 1 H- 15 N HSQC characterization of the G40 region of wild-type PSI and its mutants;

[0022] Figure 6 for 1 H- 15 N HSQC characterization of HSQC 2D spectra of wild-type PSI and its mutants;

[0023] Figure 7 The monomer-to-dimer ratios of WT-PSI and its mutants at different pH conditions are shown. White represents pH 3.0, light gray represents pH 5.0, and dark gray represents pH 7.4. Diagonal squares represent monomers, and blank squares represent dimers. DETAILED DESCRIPTION

[0024] The present invention provides a method for predicting the change in the ratio of monomers and dimers after a single-site mutation of a PSI protein, comprising the following steps: collecting the Gibbs free energy change value of the monomer of the mutant after a single-site mutation of the PSI protein, and calculating it as ΔΔG 单 ; Collect the Gibbs free energy change value of the dimer of the mutant after the PSI protein single point mutation, calculated as ΔΔG 二 ; Relative stability is calculated according to the formula shown in formula 1; when relative stability is greater than 0, it is judged that the dimer ratio decreases and the monomer ratio increases after the single-point mutation of the PSI protein; when relative stability is less than 0, it indicates that the dimer ratio increases and the monomer ratio decreases after the single-point mutation of the PSI protein; relative stability = ΔΔG 单 -ΔΔG 二 Formula 1;

[0025] The amino acid sequence of the PSI protein is shown in SEQ ID NO.1.

[0026] The method of the present invention is based on the Gibbs free energy change (ΔΔG), which reflects the thermodynamic stability of proteins. It is a method for predicting changes in the monomer-dimer ratio of PSI proteins based on single amino acid mutations. The method of the present invention can predict mutants of the membrane fusion-inducing protein PSI that primarily form a highly active dimer.

[0027] In the specific implementation of the present invention, the Gibbs free energy is preferably collected through the protein Gibbs free energy calculation website https: / / biosig.lab.uq.edu.au / ddmut / .

[0028] The single-site mutation or alanine scanning mutagenesis function in the above website was used to calculate the Gibbs free energy change (ΔΔG) of the monomeric conformation of the target protein PSI after mutation.单 , ΔΔG WT→Mut(monomer) ); the multi-site mutation function of the above website was used to calculate the Gibbs free energy change value (ΔΔG 二 , ΔΔG WT→Mut(dimer) ). Then, the Gibbs free energy change value of the monomer conformation after mutation and the Gibbs free energy change value of the dimer conformation after mutation under the same mutation are subtracted to obtain a relative value. This relative value can reflect the relative stability of the monomer / dimer after the same mutation. The calculation formula is shown in Formula 2: Relative stability = ΔΔG WT→Mut(monomer) -ΔΔG WT→Mut(dimer) Equation 2. Alanine scanning mutagenesis is a single-site mutagenesis method, in which each amino acid is mutated one at a time, and each amino acid site is mutated to alanine in turn to measure the change in Gibbs free energy after mutation. This method is used to screen for significant sites in large quantities. Single-site mutagenesis is used for monomers, mutating one amino acid at a time. Multi-site mutagenesis is used for dimers, mutating the same amino acid site on both chains simultaneously.

[0029] In the present invention, the amino acid sequence shown in SEQ ID NO.1 is specifically: IVSMECKTIVSQY 14 G EMI 18 W DLL 22 V SGVRPDQVCSQAGLCFVDGAQHVSSNIKTVVERETEGSSVGEAPLCTACEEMAVVWMQNQLKQEGTKEKVLEYVNQLCEKIP.

[0030] In the present invention, the site of the PSI protein single point mutation is preferably position 14, 18 or 22. In the present invention, the PSI protein single point mutation preferably includes: glycine at position 14 mutated to alanine, tryptophan at position 18 mutated to alanine or valine at position 22 mutated to arginine.

[0031] The present invention also provides PSI protein mutants, including G14A-PSI, W18A-PSI or V22R-PSI; the amino acid sequences of G14A-PSI, W18A-PSI and V22R-PSI are shown in SEQ ID NO.2 to SEQ ID NO.4, respectively.

[0032] In the present invention, the amino acid sequence of G14A-PSI is shown in SEQ ID NO. 2; in the present invention, the amino acid sequence shown in SEQ ID NO. 2 is specifically:

[0033] IVSMECKTIVSQYAEMIWDLLVSGVRPDQVCSQAGLCFVDGAQHVSSNIKTVVERETEGSSVGEAPLCTACEEMAVVWMQNQLKQEGTKEKVLEYVNQLCEKIP.

[0034] Compared with wild-type PSI, the dimer ratio of G14A-PSI is significantly increased; specifically, under pH 3.0, pH 5.0 and pH 7.4 conditions, the dimer ratio of G14A-PSI is higher than that of wild-type PSI.

[0035] In the present invention, the amino acid sequence of W18A-PSI is shown in SEQ ID NO.3; in the present invention, the amino acid sequence shown in SEQ ID NO.3 is specifically: IVSMECKTIVSQYGEMIADLLVSGVRPDQVCSQAGLCFVDGAQHVSSNIKTVVERETEGSSVGEAPLCTACEMAVVWMQNQLKQEGTKEKVLEYVNQLCEKIP

[0036] In the present invention, the amino acid sequence of V22R-PSI is shown in SEQ ID NO.4; in the present invention, the amino acid sequence shown in SEQ ID NO.4 is specifically: IVSMECKTIVSQYGEMIWDLLRSGVRPDQVCSQAGLCFVDGAQHVSSNIKTVVERETEGSSVGEAPLCTACEMAVVWMQNQLKQEGTKEKVLEYVNQLCEKIP.

[0037] The present invention also provides the encoding gene of the PSI protein mutant described in the above scheme, the nucleotide sequence of the G14A-PSI encoding gene G14A-PSI is shown as SEQ ID NO.5; the nucleotide sequence of the W18A-PSI encoding gene W18A-PSI is shown as SEQ ID NO.6; the nucleotide sequence of the V22R-PSI encoding gene V22R-PSI is shown as SEQ ID NO.7.

[0038] In the present invention, the nucleotide sequence shown in SEQ ID NO.5 is specifically: ATAGTATCAATGGAATGTAAAACGATTGTCTCGCAATATGCCGAAATGATTTGGGACCTGTTGGTTTCTGGTGTTCGTCCGGATCAGGTATGTAGCCAAGCGGGTCTGTGCTTCGTGGACGGCGCGCAGCACGTGAGCTCCAACATCAAAACCGTTGTCGAGCGCGAGACTGAAGGCAGCAGCGTTGGTGAAGCACCGCTGTGCACCGCGTGTGAGATGGCTGTGGTGTGGATGCAAAACCAGTTGAAGCAGGAGGGCACCAAAGAAAAGGTGCTGGAATACGTTAATCAGTTATGCGAGAAGATCCCG。

[0039] In the present invention, the nucleotide sequence shown in SEQ ID NO.6 is specifically: ATCGTTTCCATGGAATGCAAGACTATTGTTTCTCAGTATGGTGAGATGATCGCAGACCTGCTGGTGTCTGGTGTGCGTCCGGACCAGGTGTGTTCTCAGGCAGGTCTGTGCTTCGTGGACGGCGCGCAGCACGTATCCTCCAACATCAAAACCGTTGTTGAGCGCGAGACTGAAGGTTCCAGCGTTGGCGAGGCCCCGCTGTGCACCGCATGCGAGATGGCCGTGGTTTGGATGCAGAACCAGCTGAAACAGGAGGGTACTAAGGAGAAAGTCCTGGAGTACGTTAACCAGCTGTGCGAAAAAATTCCG。

[0040] In the present invention, SEQ ID The nucleotide sequence shown in NO.7 is specifically: ATAGTATCAATGGAATGTAAAACGATTGTTTCCCAATATGGCGAGATGATTTGGGATTTGCTGCGTAGCGGTGTTCGTCCGGACCAGGTTTGTAGCCAAGCCGGTTTATGCTTCGTGGACGGCGCGCAGCACGTGAGCAGCAACATC AAAACCGTCGTGGAACGCGAAACCGAAGGCAGCTCTGTTGGTGAGGCTCCGCTGTGCACCGCGTGTGAGATGGCAGTTGTGTGGATGCAGAATCAACTGAAACAGGAGGGTACTAAGGAAAAGGTGCTGGAATACGTGAACCAGTTGTGCGAGAAGATCCCG.

[0041] In the present invention, the encoding gene is preferably produced by Nanjing GenScript Biotechnology Co., Ltd.

[0042] The present invention also provides a recombinant plasmid into which the coding gene described in the above scheme is inserted.

[0043] In the present invention, the backbone plasmid of the recombinant plasmid is preferably a pET32a(+) vector; the insertion site of the coding gene on the pET32a(+) vector is preferably after the Thrombin restriction site. In the present invention, the T7 promoter of the pET32a(+) vector is preferably followed by a TrxA solubility tag and a 6×His tag; the TrxA solubility tag is used to improve protein solubility and stability; the 6×His tag is used for subsequent purification; a Thrombin restriction site is added after the 6×His tag to remove the solubility tag and the His tag. In the present invention, the recombinant plasmid is preferably completed by Nanjing KingScript Biotechnology Co., Ltd.

[0044] The present invention also provides a recombinant bacterium comprising the recombinant plasmid described in the above scheme.

[0045] In the present invention, the original strain of the recombinant bacteria is preferably Escherichia coli; the E. coli is preferably E. coli competent cells Rosetta-gami B (DE3), pLysS. The present invention does not particularly limit the method for constructing the recombinant bacteria; conventional methods in the art can be employed. In the present invention, the recombinant plasmid can be stably expressed in the recombinant strain.

[0046] The present invention also provides a method for preparing a PSI protein mutant, comprising the following steps:

[0047] The recombinant bacteria described in the above scheme are induced to obtain an induced culture product; the induced culture product is purified to obtain a PSI protein mutant. In the present invention, the purification includes nickel column purification; the nickel column purification is simple and rapid.

[0048] The present invention also provides the use of the PSI protein mutant, the encoding gene, the recombinant plasmid or the recombinant bacteria described in the above scheme in the preparation of tumor-targeted drugs and / or tumor diagnostic reagent carriers.

[0049] In the present invention, the PSI protein mutant has tumor targeting properties.

[0050] The dimeric structure of the PSI protein is a key component for inducing membrane fusion activity, and increasing the dimer ratio is beneficial for enhancing membrane fusion activity. PSI protein has two major functions: targeting PS-rich phospholipid membranes under acidic conditions and inducing membrane fusion. Both the monomeric and dimeric structures of the PSI protein have targeting functions under acidic conditions, while only the dimer is the primary component for inducing membrane fusion. PSI protein exists primarily as a dimer in solution, capable of recognizing phosphatidylserine (PS) on the surface of tumor cells and targeting them to promote membrane fusion. Increasing the proportion of dimers, the active component of PSI, will facilitate the development of highly effective tumor-targeted drugs and / or tumor diagnostic reagent carriers. Therefore, G14A-PSI has the potential to be further developed as a highly effective tumor-targeted drug and / or tumor diagnostic reagent carrier.

[0051] The monomeric conformational stability of the W18A-PSI and V22R-PSI mutants is enhanced compared to the wild-type PSI protein, while the dimer stability is reduced. Increasing the proportion of monomeric structures and reducing the proportion of dimers can effectively weaken the protein's ability to induce membrane fusion, while retaining the protein's acidic targeting properties. This is more conducive to the future development of W18A-PSI and V22R-PSI mutants into tool carrier protein tools that specifically target the PS-rich phospholipid membranes of cancer cells. The above mutants only provide targeting functions and can be expanded to connect to numerous functional molecules without interfering with the activity of the protein itself. In addition, in terms of function, because the structural analysis of mixed conformations is difficult, the existence of a single PSI monomer structure is conducive to further guiding the analysis of the monomeric three-dimensional structure of the protein, providing more atomic information, and facilitating more refined molecular modifications in the future.

[0052] In the present invention, the pH value of the PSI protein mutant is preferably ≤5, more preferably 3-5.

[0053] The present invention also provides a tumor-targeted drug and / or tumor diagnostic reagent carrier, comprising the PSI protein mutant, the encoding gene, the recombinant plasmid or the recombinant bacteria described in the above scheme.

[0054] In the present invention, the tumor-targeting drug preferably further comprises an anti-tumor active ingredient.

[0055] In one embodiment of the present invention, the tumor-targeted drug comprises a liposome; the surface of the liposome is modified with the PSI protein mutant of the above scheme; and the liposome is encapsulated with an anti-tumor active ingredient.

[0056] In the present invention, the anti-tumor active ingredient includes a small molecule compound; the small molecule compound includes doxorubicin.

[0057] In the present invention, the dosage form of the tumor-targeted drug includes an injection.

[0058] The present invention also provides the application of W18A-PSI and V22R-PSI in the study of monomer conformation in protein structure; the amino acid sequences of the W18A-PSI and V22R-PSI are shown in SEQ ID NO.3 to SEQ ID NO.4, respectively.

[0059] To further illustrate the present invention, a method for predicting the change in the ratio of monomers and dimers after a single-site mutation of the PSI protein and a mutant of the PSI protein provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0060] Example 1

[0061] By collecting the Gibbs free energy change value ΔΔG of the monomer after the PSI protein single point mutation 单 and the Gibbs free energy change of the dimer ΔΔG 二 , compare ΔΔG 单 and ΔΔG 二 Relative stability (relative stability = ΔΔG 单 -ΔΔG 二 ). When the relative stability is greater than 0, the monomer stability is determined to be increased and the dimer stability is decreased. 1 H- 15 The N HSQC spectrum shows that the monomer ratio increases and the dimer ratio decreases; when the relative stability is less than 0, it is determined that the monomer stability decreases and the dimer stability increases. 1 H- 15 The N HSQC spectrum showed a decrease in the monomer ratio and an increase in the dimer ratio.

[0062] By simulating the change in Gibbs free energy for monomers and dimers before and after single-point mutations in the WT-PSI protein, the authors determined the effects of the mutations on the thermodynamic stability of the two conformations, identifying three amino acid sites that significantly influence conformational stability. Simulating alanine scanning mutagenesis, in which glycine at position 14 was mutated to alanine, tryptophan at position 18 to alanine, and valine at position 22 to arginine, reduced monomer stability in the G14A-PSI mutant and increased dimer stability in the W18A-PSI and V22R-PSI mutants.

[0063] Example 2

[0064] 1. Construction of recombinant plasmid

[0065] The DNA sequences of G14A-PSI, W18A-PSI and V22R-PSI were respectively inserted into the pET32a(+) vector after the Thrombin restriction site. Gene synthesis and plasmid construction were completed by Nanjing GenScript Biotechnology Co., Ltd. The G14A-PSI DNA sequence is shown in SEQ ID NO.5, and the G14A-PSI amino acid sequence is shown in SEQ ID NO.2; the W18A-PSI DNA sequence is shown in SEQ ID NO.6, and the W18A-PSI amino acid sequence is shown in SEQ ID NO.3; the V22R-PSI DNA sequence is shown in SEQ ID NO.7, and the V22R-PSI amino acid sequence is shown in SEQ ID NO.4. The plasmid map is shown in Figures 1 to 3 shown.

[0066] The following steps use G14A-PSI as an example. The subsequent operations are the same for W18A-PSI and V22R-PSI.

[0067] 2. Preparation of Rosetta-gami B(DE3)pLysS E. coli competent cells:

[0068] 500 μL of Rosetta-gami B (DE3) pLysS bacterial solution was inoculated into 50 mL of LB liquid medium containing 50 μg / mL kanamycin, 34 μg / mL chloramphenicol, and 12.5 μg / mL tetracycline. The culture was shaken at 37°C and 220 rpm for 4 h. 600=0.6-0.8. Transfer the bacterial suspension to a 50mL centrifuge tube, incubate on ice for 20 minutes, and then centrifuge at 4000 rpm for 10 minutes at 4°C to collect the cells. Resuspend the cells in 10mL of pre-chilled 0.1M CaCl2 solution and centrifuge at 4000 rpm for 10 minutes at 4°C to collect the cells. Repeat this process three times. Add 3mL of pre-chilled 0.1M CaCl2 to resuspend the cells, then add 10% glycerol to a final concentration and mix thoroughly. Aliquot 100μL into 1.5mL centrifuge tubes, snap-freeze in liquid nitrogen, and store at -80°C.

[0069] 3. E. coli transformation:

[0070] Pipette 100 ng of plasmid pET32a-G14A-PSI into 100 μL of Rosetta-gami B(DE3)pLysS competent cells, mix gently, and incubate on ice for 30 minutes. Heat shock the mixture at 42°C for 90 seconds, then immediately incubate on ice for 2 minutes. Add 1 mL of LB liquid medium containing 50 μg / mL kanamycin, 34 μg / mL chloramphenicol, and 12.5 μg / mL tetracycline, and incubate the cells at 37°C at 220 rpm for 60 minutes. After activation, centrifuge at 4°C at 12,000 rpm for 3 minutes, remove the supernatant, and resuspend the cells in the remaining 50 μL of supernatant. Spread the suspension evenly on LB solid medium containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, 34 μg / mL chloramphenicol, and 12.5 μg / mL tetracycline. Incubate the cells overnight at 37°C with an inverted rotation.

[0071] 4. Identification and preservation of positive monoclones:

[0072] The single clones obtained by resistance screening culture were picked, activated and cultured, and then plasmids were extracted and sequenced for verification.

[0073] Sequencing results were consistent with the pET32a(+)-G14A-PSI plasmid sequence, confirming successful isolation of positively transformed bacteria. The positively transformed bacteria were inoculated into LB liquid culture containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, 34 μg / mL chloramphenicol, and 12.5 μg / mL tetracycline and cultured at 37°C, 220 rpm, with shaking for 16 hours. After incubation, glycerol was added to a final concentration of 50%, mixed, and aliquoted, snap-frozen in liquid nitrogen, and stored at -80°C.

[0074] 5. Purification of target protein:

[0075] (1) Activation culture of transformed bacteria:

[0076] Take the -80℃ frozen glycerol bacteria, thaw in an ice bath, take 10μL and inoculate it into 100mL LB liquid culture medium containing 100μg / mL ampicillin, 50μg / mL kanamycin, 34μg / mL chloramphenicol and 12.5μg / mL tetracycline, and culture at 37℃, 220rpm, and shake for 14h. 100mL culture liquid was then inoculated into 1L LB liquid culture medium containing 100μg / mL ampicillin, 50μg / mL kanamycin, 34μg / mL chloramphenicol and 12.5μg / mL tetracycline, and culture at 37℃, 220rpm, and shake for 3h. The OD 600 =0.6~0.8, 4℃, 8000rpm, centrifuge for 10min to collect bacteria.

[0077] (2) Induction culture of transformed bacteria:

[0078] The activated cultured bacteria were resuspended in 1 L of M9 medium containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, 34 μg / mL chloramphenicol and 12.5 μg / mL tetracycline (the carbon source in the medium was 15 N-NH4Cl, the strain can be cultured using this medium to produce the target protein. 15 N-labeled) and incubated at 37°C, 220 rpm for 30 minutes. Immediately after the incubation period, incubate on ice for 5-10 minutes. Add IPTG to a final concentration of 500 μM and induce the culture at 30°C, 220 rpm for 18 hours. After the induction period, collect the cells by centrifugation at 4°C, 8000 rpm for 10 minutes.

[0079] (3) Nickel column purification:

[0080] Resuspend the induced bacteria in 50 mL of buffer (20 mM Tris, 100 mM NaCl, pH 8.0). Under low temperature conditions, use a high-pressure homogenizer to break the cells until the resuspension becomes clear, and centrifuge at 4 ° C, 12000 rpm for 30 minutes to collect the supernatant. The supernatant is combined with Ni-NTA filler at low temperature for more than 2 hours of oscillation. After the combination is completed, use a gravity column to collect the combined flow-through and the eluate after washing with 20 mMTris, 100 mM NaCl, pH 8.0 buffer containing 0 mM imidazole, 10 mM imidazole, 30 mM imidazole, 50 mM imidazole, 150 mM imidazole, and 300 mM imidazole. Sample the flow-through and the eluate of each gradient concentration of imidazole, and SDS-PAGE detection is used to determine the eluate where the target protein is located. The eluate containing the target protein (i.e., the eluate containing 150mM imidazole) was placed in a dialysis bag with a molecular weight cutoff of 7kDa and dialyzed against 20mM Tris, 150mM NaCl, pH 8.0 buffer. After the dialyzed protein, 0.5U of thrombin was added per 1mg of protein and the protein was digested overnight at 37°C. After the digestion was completed, the digestion system was combined with the Ni-NTA filler at low temperature and shaken for more than 2 hours. After the binding was completed, the flow-through was collected using a gravity column, as well as the eluate after washing with 20mM Tris, 100mM NaCl, pH 8.0 buffer containing 300mM imidazole. The flow-through and 300mM imidazole eluate were sampled and tested by SDS-PAGE to determine whether the target protein was present in the flow-through. The flow-through obtained in the above steps was placed in a dialysis bag with a molecular weight cut-off of 1 kDa and dialyzed in deionized water. After the dialysis, the protein solution was transferred to an ultrafiltration tube with a molecular weight cut-off of 3 kDa and concentrated to 30 mL. After the concentration was completed, the solution was divided and freeze-dried under low-temperature vacuum conditions. 15 The N-stable isotope-labeled G14A-PSI protein sample was dried, sealed, and stored at -80°C.

[0081] According to the above scheme, W18A-PSI protein dry sample and V22R-PSI protein dry sample were obtained in sequence.

[0082] 6. Liquid NMR:

[0083] The obtained G14A-PSI protein dry samples were dissolved in pH 3.0, pH 5.0, and pH 7.4 buffer solutions respectively; the obtained W18A-PSI protein dry samples were dissolved in pH 3.0, pH 5.0, and pH 7.4 buffer solutions respectively; the obtained V22R-PSI protein dry samples were dissolved in pH 3.0, pH 5.0, and pH 7.4 buffer solutions respectively. The protein samples obtained under the three pH conditions were subjected to 2D 1 H- 15N HSQC detection. 10% D2O was added to the above samples during the detection. All experiments were collected on a Bruker Avance 800MHz spectrometer. The chemical shift was calibrated by the chemical shift of the internal standard DSS. 15 N and 1 Indirect calibration using the ratio of the gyromagnetic ratio of H 15 The chemical shift of N, 15 N and 1 The gyromagnetic ratio of H is -0.10132905. The experimental temperature is 25℃, and the collected spectra are processed with Topspin 3.2 and analyzed with CARA software. Through literature research and two-dimensional NMR spectroscopy identification, it was found that the 40th amino acid glycine in the monomer and dimer of PSI has different chemical shifts. The chemical shift of G40 in the monomer is 1 H 7.44, 15 At N104, the chemical shift of G40 in the dimer is 1 H 7.53, 15 At N104, the cross-peak area of ​​residue G40 was used as a measure of the monomer-dimer ratio to determine the monomer-dimer ratio in the target protein. G40 peak integration: Import the 2D-HSQC spectrum obtained by liquid NMR scanning into CARA software. In the CARA interface, select the center of the target peak. Then, navigate to "File" → "Export" → "Planepeaks to Monoscope" in the toolbar. Mark the target peak and perform calibration. Navigate to the "Intergrator" toolbar → "Tune Peak Model" → Adjust "Width X" and "Width Y" so that the adjusted green and blue peaks overlap. Adjust "PeakTol.X" and "PeakTol.Y" to 65%. After calibration, perform integration. Navigate to the "Intergrator" toolbar → "Update All Amplitudes" → "Intergrator all" → Display the integrated area of ​​the target peak at "Amp."

[0084] Results see Figure 5 、 Figure 6 and Figure 7 . Figure 5 The peak diagrams of G40 of different mutants under three pH gradients are displayed, and the changes in the proportion of single dimers are judged according to the peak intensity, thereby verifying the correctness of the calculation. Figure 6 The HSQC two-dimensional spectra of different mutants under three pH gradients are shown. Figure 7Comparison of the G40 cross-peaks of mutant G14A-PSI and WT-PSI at different pH conditions reveals a significantly higher proportion of dimers in G14A-PSI compared to wild-type PSI. The monomer structure of this mutant even disappears at pH 5.0 and 3.0, indicating that mutation of glycine at position 14 of wild-type PSI to alanine enhances dimer stability and reduces monomer stability. W18A-PSI and V22R-PSI exhibit increased monomer stability and decreased dimer stability. Taking W18A as an example, comparison of the G40 cross-peaks of mutant W18A-PSI and WT-PSI at different pH conditions reveals a significantly higher proportion of monomers in W18A-PSI compared to wild-type PSI. At pH 7.4, 5.0, and 3.0, the dimer structure of this mutant disappears, leaving only monomers. This suggests that mutation of tryptophan at position 18 of wild-type PSI to alanine reduces dimer stability and enhances monomer stability. In addition, the G40 cross-peaks of mutants W18A-PSI and V22R-PSI under different pH solution conditions showed that the proportion of monomer conformation in the protein structure was increased, and the dimer conformation was greatly reduced under pH 7.4 and pH 5.0 conditions, indicating that the stability of the monomer conformation of the W18A-PSI and V22R-PSI mutants was enhanced compared with the wild-type PSI protein, while the dimer stability was reduced. These results are consistent with the results of protein thermodynamic stability calculation based on Gibbs free energy, demonstrating the feasibility of the protein single dimer regulation method based on thermodynamic stability simulation calculation in the present invention. The present invention combines computational biology and structural biology to predict and verify the regulatory ability of key amino acid single-point mutations on protein conformational equilibrium, laying a solid foundation for further modification of functional PSI proteins in the future.

[0085] In summary, the present invention simulates and calculates the change in Gibbs free energy of the monomer and dimer conformations of the wild-type PSI protein after single-point mutation, and screens out three mutant proteins with significant changes in monomer and dimer stability: G14A-PSI, W18A-PSI and V22R-PSI. The two-dimensional stability of each mutant at pH 3.0, pH 5.0 and pH 7.4 is obtained by liquid nuclear magnetic resonance experiments. 1 H- 15 NHSQC spectrum, the results showed that the dimer ratio of G14A-PSI mutant protein was significantly increased compared with wild-type PSI.

[0086] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for predicting the change in the ratio of monomers and dimers after a single-site mutation of a PSI protein, characterized in that: The following steps are involved: The Gibbs free energy change of the monomer of the mutant after the single-point mutation of the PSI protein is collected and calculated as ΔΔG 单 ; The Gibbs free energy change of the dimer of the mutant after the single-point mutation of the PSI protein is collected and calculated as ΔΔG 二 ; Calculate the relative stability according to the formula shown in Equation 1; When the relative stability is greater than 0, it indicates that the dimer ratio decreases and the monomer ratio increases after the PSI protein single point mutation; when the relative stability is less than 0, it indicates that the dimer ratio increases and the monomer ratio decreases after the PSI protein single point mutation; Relative stability = ΔΔG 单 -ΔΔG 二 Formula 1; The amino acid sequence of the PSI protein is shown in SEQ ID NO.

1.

2. The method according to claim 1, characterized in that The site of the PSI protein single point mutation is position 14, position 18 or position 22.

3. The method according to claim 2, characterized in that The PSI protein single point mutation includes: mutation of glycine at position 14 to alanine, mutation of tryptophan at position 18 to alanine, or mutation of valine at position 22 to arginine.

4. A PSI protein mutant, characterized in that It includes G14A-PSI, W18A-PSI or V22R-PSI; the amino acid sequences of G14A-PSI, W18A-PSI and V22R-PSI are shown in SEQ ID NO.2 to SEQ ID NO.4 respectively.

5. The gene encoding the PSI protein mutant according to claim 4, characterized in that The nucleotide sequence of the gene encoding the G14A-PSI is shown in SEQ ID NO.5; the nucleotide sequence of the gene encoding the W18A-PSI is shown in SEQ ID NO.6; and the nucleotide sequence of the gene encoding the V22R-PSI is shown in SEQ ID NO.

7.

6. A recombinant plasmid, characterized in that The coding gene according to claim 5 is inserted.

7. A recombinant bacterium, characterized in that Comprising the recombinant plasmid according to claim 6.

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