Method for detecting binding condition of ethylene receptor ETR1 and cuprous ions based on nuclear magnetic resonance technology

Abstract

The invention discloses a method for detecting the binding condition of an ethylene receptor ETR1 and cuprous ions based on a nuclear magnetic resonance technology. The method comprises the following steps: preparing an ethylene receptor key binding domain through genetic engineering; the method comprises the following steps: carrying out isotope labeling on an ethylene receptor key binding domain by using a 15N isotope, preparing BCA2-Cu < + >, titrating cuprous ions into the ethylene receptor key binding domain, detecting the binding of the ethylene receptor key binding domain and the cuprous ions by using a nuclear magnetic resonance technology, and comparing 1H-15N HSQC spectrograms when cuprous ions with different concentrations are added, it is proved that when the ethylene receptor key binding domain is combined with cuprous ions, chemical shift of amino acid in a spectrogram can change irregularly and remarkably, and it is judged that the ethylene receptor binding domain is combined with the cuprous ions. A binding system of the ethylene receptor key binding domain and the cuprous ions is constructed, and the binding condition of the ethylene receptor binding domain and the cuprous ions is verified.

Description

Technical Field

[0001] The present invention relates to a detection method, in particular to a method for detecting the binding between ethylene receptor ETR1 and cuprous ions based on nuclear magnetic resonance technology. Background Art

[0002] Ethylene is the simplest olefin gas and the first gaseous molecule proven to have hormonal effects. Ethylene is involved in nearly all regulatory processes for plant growth and development, affecting the expression of over 1,000 genes. Ethylene's effects include stimulating seed germination, regulating seedling growth in different environments, the rate and extent of leaf expansion, fruit maturation, aging, and shedding, regulating individual plant responses to external environmental stresses (drought, flooding, high salt levels, mechanical damage, viral infection, etc.), and post-harvest storage. Ethylene produced by different plant individuals can also influence each other; for example, ethylene produced by apples can accelerate the ripening of bananas. Furthermore, the regulatory mechanisms of ethylene are similar in different plants.

[0003] Ethylene receptor proteins are located in the endoplasmic reticulum and Golgi apparatus and negatively regulate ethylene responses. Five different types of ethylene receptors exist in Arabidopsis and many other plants: ethylene response 1 (ETR1), ethylene response 1 (ETR2), ethylene response sensor 1 (ERS1), ethylene response sensor 2 (ERS2), and ethylene insensitive 4 (EIN4). Each receptor contains an N-terminus, an ethylene-binding domain (transmembrane region), a GAF (cGMP-specific phophdiesterases, adenylyl cyclases, and FhlA) region, and a histidine kinase domain. ETR1, EIN4, and ETR2 also contain a receptor domain. Compared to other plant hormone receptors, ethylene receptors have more complex structures and functions, and their mechanisms are relatively understudied. ETR1 was the first ethylene receptor to be identified and has been the most thoroughly studied. In the absence of ethylene, ETR1 activates CTR1 (constitutive triple response 1, a Raf-like serine / threonine kinase); CTR1 then phosphorylates another Nramp-like membrane protein, EIN2 (ethylene insensitive 2), on the endoplasmic reticulum, inhibiting its ability to induce ethylene response, ultimately resulting in the inability of fruit to grow and mature normally. However, in the presence of ethylene, ethylene binds to ETR1, inhibiting CTR1 activity and inducing the hydrolysis of the EIN2 protein and the release of its C-terminal domain. This domain, upon translocation to the nucleus, activates the transcriptional cascade of EIN / EIN3 and ERF (ethylene response factor) transcription factors, leading to the appearance of relevant phenotypic traits and the normal development and maturation of the plant. Therefore, the binding of ethylene to the ethylene receptor is the starting point for ethylene regulation of plant growth and development.

[0004] In plants, ethylene receptors exist as stable dimers linked by two disulfide bonds at their N-termini. The cytoplasmic receptor domain of ETR1, a representative ethylene receptor, was deciphered in 1999. Using mutagenesis and a series of biochemical experiments, Professor Bleecker discovered that the key active sites for ethylene binding in ETR1-EBD are located on transmembrane helices 1 and 2. He also identified cuprous ions as cofactors for ETR1 binding to ethylene molecules. Cys65 in helix 2 of the ETR1-EBD is responsible for coordinating the cuprous ion. A C65Y mutation disables ETR1's ability to bind cuprous ions and ethylene. Furthermore, further mutational studies revealed a series of functionally important amino acid sites in the transmembrane region of ETR1-EBD. In addition to C65, six amino acids—D25, Y32, I35, P36, I62, and H69—play a crucial role in ethylene binding. Mutation of any of these amino acids resulted in ethylene binding efficiency less than 5% of the wild-type level. H69 and C65 are highly conserved, present in all receptors, and both participate in cuprous ion binding. Professor Gohlke further confirmed the importance of H69 and C65 for cuprous ion binding using X-ray absorption spectroscopy. Furthermore, earlier studies using co-purification methods revealed a 1:1 stoichiometric ratio between cuprous ions and ETR1 dimers, leading to the long-held assumption that each ETR1 dimer binds only one cuprous ion. However, recent experimental evidence suggests that each dimer may bind two cuprous ions, coordinated by helix 1 and helix 2, respectively. This model was constructed by integrating ab initio structure prediction and coevolutionary information, and the helical arrangement was explored using tryptophan scanning mutagenesis. All-atom molecular dynamics simulations of this dimer model also predicted how ethylene binds to the cuprous ion in the receptor and the initial stages of the ethylene sensing process. However, to date, the transmembrane structure of the ethylene binding region (ETR1-EBD) has not been resolved, and there is a lack of an intuitive system that can study the ethylene binding mechanism at the atomic level. Because the transmembrane region of the ethylene receptor is insoluble in water and is dynamic and changeable, it is very difficult to study the structure of ETR1 using crystallization methods. At the same time, the transmembrane portion of ETR1 has only more than 100 amino acids, and even if it forms a dimer, its molecular weight is still small. It is very challenging to use cryo-electron microscopy to analyze the structure of ETR1. Therefore, it is crucial to find a suitable method to explore the binding mechanism of ethylene receptors and cuprous ions.

[0005] Nuclear magnetic resonance technology (NMR) can intuitively identify the interactions between proteins and proteins, proteins and small molecule compounds, and can provide information on molecular interactions accurate to the level of amino acid residues or even single atoms, as well as other rich chemical reaction kinetic parameters. 1 H- 15N TROSY-HSQC (Heteronuclear Single Quantum Coherence) spectra can be used as a gold standard to determine whether the protein interacts with other molecules. The conformational and spatial changes caused by the binding of the target protein to other molecules can be intuitively and quickly reflected by the changes in chemical shifts in the HSQC spectrum. 1 H- 15 The changes in NHSQC spectra are closely related. 1 H- 15 N HSQC spectrum can reflect the chemical shift signal of the main chain or part of the side chain of the amino acid of the protein. 15 When ligands are gradually added to the N isotope-labeled protein sample, if the ligand interacts with the protein, the chemical environment of the amino acid residues at or near the binding site will change. 1 H- 15 The N HSQC spectrum shows a shift in the chemical shift of the corresponding amino acid residues, indicating a chemical shift perturbation (CSP). Therefore, TROSY-HSQC can be used to determine the binding of ETR1-EBD to cuprous ions and to demonstrate the biological activity of ETR1-EBD samples.

[0006] In the prior art, there are the following problems:

[0007] (1) Since the transmembrane region of the ethylene receptor is insoluble in water and is highly dynamic, ethylene gas molecules are difficult to retain in crystals. Therefore, it is very difficult to study the structure of ETR1 using crystallization methods. At the same time, the transmembrane region of ETR1 consists of only a few hundred amino acids. Even when forming a dimer, its molecular weight is still small. It is very challenging to use cryo-electron microscopy to analyze the structure of ETR1. Therefore, it is crucial to find a suitable method to explore the ethylene binding mechanism.

[0008] (2) There is still a lack of understanding of ETR1-EBD, and it is unknown what conformational changes occur after the receptor binds to ethylene, as well as what changes occur in the downstream kinase reaction and the binding of the receptor to other proteins. At the same time, cuprous ion is an important ligand for the binding of ethylene receptor to ethylene, and its binding mechanism with the ethylene receptor binding domain is still unclear.

[0009] (3) Cuprous compounds generally exhibit low water solubility. This is because cuprous compounds have low lattice energy and bond strength, high redox activity of Cu+ ions, and are easy to react with other components in the solution. These factors together lead to the fact that cuprous compounds generally exhibit low water solubility and make it difficult for cuprous ions to exist stably in solution. Summary of the Invention

[0010] The present invention overcomes the defects of the prior art and provides a method for detecting the binding of ethylene receptor ETR1 to cuprous ions based on nuclear magnetic resonance technology. The present invention uses an E. coli expression system to achieve large-scale expression of the key binding domain of the ethylene receptor and the use of 15 The binding domain is labeled with an N isotope. In the present invention, a binding system for the key binding domain of the ethylene receptor and cuprous ions is constructed to verify the binding of the ethylene receptor binding domain to cuprous ions. In the present invention, nuclear magnetic resonance technology is used to rapidly detect the binding of the key binding domain of the ethylene receptor and cuprous ions.

[0011] The technical solutions of the present invention are as follows.

[0012] A method for detecting the binding of ethylene receptor ETR1 to cuprous ions based on nuclear magnetic resonance technology, wherein the key binding domain of the ethylene receptor is expressed in large quantities using an Escherichia coli expression system through genetic engineering, and a high-purity key binding domain of the ethylene receptor is prepared through multi-step purification; 15 The key binding domain of ethylene receptor was labeled with N isotope and BCA2-Cu was prepared. + The cuprous ions are stabilized in the form of a solution, and finally the cuprous ions are titrated into the key binding domain of the ethylene receptor. The binding of the key binding domain of the ethylene receptor and the cuprous ions is detected by nuclear magnetic resonance technology. By comparing the addition of different concentrations of cuprous ions 1 H- 15 The N HSQC spectrum confirms that when the key binding domain of the ethylene receptor binds to the cuprous ion, the chemical shift of the amino acids in the spectrum will undergo irregular and significant changes, which can be used to determine that the ethylene receptor binding domain has bound to the cuprous ion.

[0013] Specifically: (1) preparing high-purity key binding domains of ethylene receptors through genetic engineering;

[0014] (2)Use 15 The key binding domain of ethylene receptor was labeled with N isotope and BCA2-Cu was prepared. + complex, and finally BCA2-Cu +The complex was titrated into the key binding domain of the ethylene receptor, and the binding of the key binding domain of the ethylene receptor to the cuprous ion was detected using nuclear magnetic resonance technology. When the key binding domain of the ethylene receptor bound to the cuprous ion, the amino acid chemical shift in the spectrum would undergo irregular and significant changes, indicating that the ethylene receptor binding domain bound to the cuprous ion.

[0015] Furthermore, step (1) is specifically as follows: by genetic engineering, the key binding domain of the ethylene receptor is expressed in large quantities using an E. coli expression system, and a high-purity key binding domain of the ethylene receptor is prepared through multi-step purification.

[0016] Furthermore, the method of expressing the key binding domain of ethylene receptor in large quantities by using E. coli expression system through genetic engineering is as follows: expressing the key binding domain of ethylene receptor in large quantities by using E. coli expression system through genetic engineering, and the expression conditions are: starting OD 600 :0.1, induced OD 600 :0.6-0.7, induction temperature 30-32 ° C, induction time 22-24h, preferably induced OD 600 :0.6, induction temperature 30℃, induction time 24h.

[0017] Furthermore, the multi-step purification is carried out through Dextrin beads affinity chromatography purification, TEV enzyme cleavage and high performance liquid chromatography to prepare a high-purity key binding domain of the ethylene receptor.

[0018] Furthermore, in step (2), the concentration of the BCA2-Cu+ solution is 0.25 mM to 8 mM.

[0019] Furthermore, in step (2), the BCA2-Cu + The amount of the complex added between the key binding domain of the ethylene receptor satisfies the following conditions: the ratio of the amount of protein to the amount of cuprous ion substance is 1:0 to 1:16.

[0020] Furthermore, in step (2), the BCA2-Cu + The amount of the complex added between the key binding domain of the ethylene receptor satisfies the following conditions: the ratio of protein to cuprous ion substance is 1:0, 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:16 and gradually increases.

[0021] Furthermore, in step (1), the amino acid sequence of the key binding domain of the ethylene receptor is: SEQ ID NO: 1.

[0022] Furthermore, the BCA2-Cu + The preparation method of the complex is:

[0023] (1) Weigh BCA, add Buffer G and fully dissolve it, then filter it with a nylon membrane to obtain a BCA solution;

[0024] (2) Weigh ascorbic acid and add Buffer G to fully dissolve;

[0025] (3) Weigh CuCl, add Buffer G to fully dissolve, then add ascorbic acid mother solution until the solution becomes clear and transparent, and then filter with a nylon membrane to obtain a CuCl solution;

[0026] (4) Take BCA solution and CuCl solution and mix them thoroughly to prepare BCA2-CuCl + , and store it at 4°C until use.

[0027] Furthermore, step (1) is: when the key binding domain of the ethylene receptor is expressed in large quantities using an E. coli expression system through genetic engineering, it is necessary to use 15 NH4Cl is used to isotope-label the target protein.

[0028] Furthermore, in step (2), before using nuclear magnetic resonance technology to detect the binding of the key binding domain of the ethylene receptor to the cuprous ion, it is necessary to use DPC to detect the binding of ETR1-EBD to the cuprous ion. 46-82 Proteins are assembled, that is, the membrane environment of proteins is reconstructed using the Micelle method.

[0029] Compared with the prior art, the advantages of the present invention are:

[0030] (1) Expression, purification and use of the key binding domain of the ethylene receptor 15 Method for labeling the key binding domain of ethylene receptor with N isotopes.

[0031] (2) A binding system between the key binding domain of the ethylene receptor and cuprous ions was constructed.

[0032] (3) Nuclear magnetic resonance technology was used to verify the binding of the ethylene receptor binding domain to cuprous ions. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the map of the expression vector pETM44;

[0034] Figure 2 This is the Dextrin beads affinity chromatography purification diagram;

[0035] Figure 3 Figure 1 shows (a) HPLC results of the purification and (b) SDS-PAGE results of the peak positions.

[0036] Figure 4 ETR1-EBD 46-82 Protein: cuprous ion = 1:0.

[0037] Figure 5 ETR1-EBD 46-82 Protein: cuprous ion = 1:0.5 and 1:0 superimposed comparison (green is 1:0.5);

[0038] Figure 6 ETR1-EBD 46-82 Protein: cuprous ion = 1:1 and 1:0 superimposed comparison (red is 1:1);

[0039] Figure 7 ETR1-EBD 46-82 Protein: cuprous ion = 1:2 and 1:1 superimposed comparison (purple is 1:2);

[0040] Figure 8 ETR1-EBD 46-82 Protein: cuprous ion = 1:4 and 1:2 superimposed comparison (yellow is 1:4);

[0041] Figure 9 ETR1-EBD 46-82 Protein: cuprous ion = 1:8 and 1:4 superimposed comparison (black represents 1:8);

[0042] Figure 10 ETR1-EBD 46-82 Protein: cuprous ion = 1:16 and 1:8 superimposed comparison (blue-cyan represents 1:16). DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below with reference to specific examples, but the embodiments of the present invention are not limited thereto. For process parameters not particularly noted, conventional techniques may be used.

[0044] Example 1. Construction of recombinant expression vector for the key binding domain of ethylene receptor

[0045] The second helix of the ethylene receptor binding domain (ETR1-EBD) in the model plant Arabidopsis thaliana, i.e., the gene sequence corresponding to its amino acid sequence 46-82 (SAVFPYRWVLVQFGAFIVLCGATHLINLWTFTTHSR), was selected for Escherichia coli codon optimization. Gene synthesis was then performed by GenScript, and the target gene sequence was inserted into the pETM44 expression vector through homologous recombination technology to encode pETM44-ETR1-EBD containing a 6×His-MBP-tag and a Tobacco Etch Virus protease (TEV) cleavage site at the N-terminus. 46-82 Protein, such as Figure 1 As shown, the recombinant plasmid can be obtained.

[0046] Example 2. ETR1-EBD 46-82 Protein expression

[0047] (1) Conversion

[0048] Use E.coli BL21 (DE3) competent cells for transformation, take out 1 μL of recombinant plasmid and add it to 100 μL of competent cells, gently pipet and mix, then let it stand on ice for 30 minutes, then heat shock in a metal bath at 42°C for 60 seconds, quickly transfer it to ice and continue to stand for 2 minutes, then add 500 μL LB liquid culture medium, gently pipet and mix, then transfer it to a 5 mL shaking tube, place the shaking tube in a shaker at 37°C and 220 rpm and culture for 60 minutes to express the corresponding resistance marker gene on the recombinant plasmid and allow the bacteria to recover. After the culture is completed, take out 30 μL of bacterial solution and add it to LB solid culture medium containing KanR resistance, and use sterile coated glass beads to evenly spread the cells on the plate. After the cells on the plate are evenly spread and dry, the plate is inverted and stored in a culture medium at 37°C for 12-14 hours.

[0049] (2) Small system cultivation

[0050] Pick a single transformed E. coli BL21 (DE3) colony and inoculate it into a 50 mL bottle of KanR-resistant LB liquid medium. Place it in a shaker at 37°C and 220 rpm for 12-16 hours.

[0051] (3) Large system cultivation

[0052] After the incubation time, measure the OD of the bacterial solution 600 When the OD of the bacterial solution 600 In the 3-5 range, the starting OD 600The culture was expanded under the condition of 0.1 and inoculated into 1L M9 liquid medium. Before inoculation, the bacterial liquid was centrifuged at 4°C and 5000×g for 30 minutes, the supernatant was discarded, and the bacterial pellet was resuspended in M9 liquid medium to complete the inoculation; the inoculated M9 medium was then placed in a shaker at 37°C and 220 rpm to culture until its OD 600 Reached 0.6.

[0053] (4) Induction

[0054] When the bacterial solution OD 600 After reaching 0.6, the bacterial solution was placed on ice to cool for 5-10 minutes, and then IPTG was added to it to a final concentration of 0.3 mM, and then it was placed in a shaker at 30°C and 220 rpm for 24 hours to induce the expression of the target protein.

[0055] (5) Collecting bacteria

[0056] After induction, the bacterial solution was collected and centrifuged at 4°C, 5000×g for 30 min. The supernatant was discarded and the bacterial pellet was retained for subsequent protein purification and stored at -20°C for a short period of time.

[0057] Example 3. ETR1-EBD 46-82 Protein purification

[0058] (1) Dextrin beads affinity chromatography purification

[0059] Resuspend the collected bacterial pellet in 30 mL of Buffer A and transfer it to a stainless steel beaker. Disrupt the pellet using an ultrasonic disruptor in an ice bath at 40% power, 1.5 seconds on, 1.5 seconds off, for a total of 12-18 minutes. Collect the disrupted bacterial suspension and centrifuge at 37,500 × g at 4°C for 30 minutes. Collect the supernatant and filter it through a 0.45 μm pore size sterile filter connected to a syringe. Store the filtered supernatant at 4°C. Next, take out 1mL of Dextrin beads filler and place it in a 20mL empty gravity column. When the liquid in the filler in the gravity column is about to drain out, add Buffer B for equilibrium. During equilibrium, add Buffer B twice, each time adding 5 column volumes (CV). When Buffer B is about to drain out in the gravity column, plug the gravity column with a plug. Take some of the supernatant after the previous membrane and add it to the gravity column to mix with the filler. After the supernatant and filler are evenly mixed, transfer this supernatant filler mixture to all the supernatant. Place the supernatant at 4℃ and rotate it up and down to allow the target protein in it to fully bind to the filler for 20-40 minutes. After the binding is completed, it is transferred to a 20mL empty gravity column. When the liquid in the gravity column is about to run dry, Buffer B is added to the gravity column to elute the impurities (Wash). During elution, Buffer B is added three times, 5CV each time. When Wash is about to run out, Buffer C is added to the gravity column to elute the target protein (Elute). During elution, Buffer C is added nine times, 1CV each time, and all Elute is collected at the same time. The entire process is carried out at 4°C. The purification results are shown in the figure. Figure 2 As shown. The target protein eluted from the Elute was collected, and then the background solution system of the target protein was replaced with TEV enzyme cleavage buffer Buffer D by dialysis. The specific dialysis operation was as follows: dialysis was performed at 4°C, using Buffer D as the dialysate, and dialyzed according to the ratio of collected target protein solution: dialysate = 1:100 (v / v). Dialysis was performed three times in total, with the first dialysis time being 2-3 hours, the second dialysis time being 3-4 hours, and the third dialysis time being overnight.

[0060] (2) TEV cleavage

[0061] TEV enzyme was added to the protein solution, and the enzymatic digestion conditions were: enzyme: protein concentration ratio was 1:1, enzymatic digestion time was 24 h, room temperature, and 25 rpm.

[0062] (3) HPLC purification

[0063] The protein solution after TEV cleavage was collected and further purified using high performance liquid chromatography (HPLC). An equal volume of 98% formic acid solution was added to the protein solution after TEV cleavage and vortexed thoroughly to a final concentration of 50% formic acid. The solution was then centrifuged at 12000 rpm for 3 minutes at room temperature, and filtered using a syringe connected to a PTFE filter. After filtration, the sample was loaded into a sample loop for purification. The A solution used in HPLC was Buffer E, and the B solution was Buffer F. The operating procedure was: flow rate 3 mL / min, 0-15 min-30% B, 15-55 min-30%-100% B, 55-60 min-100% B, and one tube was collected for every 3 mL. The purification results are shown in the figure. Figure 3 The target protein solution was collected and freeze-dried, and then stored at -80°C.

[0064] Example 4. BCA2-Cu + Preparation

[0065] (1) Preparation of 50 mM BCA: Weigh 0.096 g of BCA (bicinchoninic acid), add 4 mL of Buffer G, and vortex to dissolve it completely. Then, dilute the volume to 5 mL and filter it using a 0.22 μm nylon membrane.

[0066] (2) Prepare 1 M ascorbic acid stock solution: Weigh 0.176 g of ascorbic acid, add 500 μL of Buffer G, vortex to fully dissolve, and then adjust the volume to 1 mL.

[0067] (3) Preparation of 20 mM CuCl: Weigh 0.0198 g of CuCl, add 9 mL of Buffer G, and vortex to fully dissolve it. After the volume is adjusted to 10 mL, add 200 μL of 1 M ascorbic acid stock solution. After addition, gently invert the solution until the solution is clear and transparent, and then filter it with a 0.22 μm nylon membrane.

[0068] (4) Take 1 mL each of 50 mM BCA and 20 mM CuCl and mix them thoroughly to prepare 10 mM BCA2-CuCl. + , and store it at 4°C until use.

[0069] Example 5. ETR1-EBD 46-82 NMR Titration of Protein with Cuprous Ions

[0070] (1) Reconstruction of the ETR1-EBD46-82 protein membrane environment

[0071] Weigh 25 mg of Dodecylphosphocholine (DPC), dissolve it in 1 mL of Buffer I, add 10 μL of DTT stock solution, and centrifuge to defoam it after it is fully dissolved at 12000 rpm for 3 min at room temperature. 15 N-labeled ETR1-EBD 46-82 The protein powder was completely dissolved after thorough vortexing, and then centrifuged for defoaming at 12,000 rpm for 3 min at room temperature. The solution after dissolving the protein powder was transferred to a 3.5 kDa dialysis cassette, and the dialysis cassette was placed in 1 L of Buffer H for overnight dialysis. The next day, the dialysis cassette was transferred to a new 1 L of Buffer H and stirred for 4-5 h, using a 4 cm stirring bar at a stirring speed of 500 rpm. After dialysis, the solution in the dialysis cassette was aspirated, and centrifuged for defoaming again at 12,000 rpm for 3 min at room temperature. After defoaming, the solution was transferred to a 10 kDa ultrafiltration tube for concentration at 700 × g, 2,000 rpm at room temperature until it was concentrated to 300 uL. The concentrated solution was aspirated and transferred to a 1.5 mL centrifuge tube for further defoaming at 12,000 rpm for 3 min at room temperature. The defoamed solution was stored at 4 °C for later use.

[0072] (2) Nuclear magnetic titration

[0073] After reconstitution of the membrane environment, 0.5 mM, 300 μL 15 N ETR1-EBD 46-82 In the protein solution, 20 μL D2O was added as a lock solvent at a ratio of 1 / 15 (v / v), and then centrifuged for defoaming at 12000 rpm for 3 min at room temperature. Then, BCA2-Cu was added to the protein sample in the order of protein: cuprous ion concentration ratio of 1:0, 1:0.5, 1:1, 1:2, 1:4, 1:8, and 1:16. + , so that the volume of the total cuprous ion ligand added does not exceed 1 / 20-1 / 10 of the total protein volume (450 μL). After each addition of the ligand, mix it thoroughly and carefully transfer it to a 5 mm Shigemi NMR tube. Be careful to avoid bubbles during transfer. All NMR spectra were collected on a cryogenic probe ( 1 H( 13 C / 15 The results were recorded on an Agilent 800MHz NMR spectrometer equipped with a 5mm PFG Triple Resonance Probe, VT, 800NB, and the experimental temperature was 35°C.+ After 1 H- 15 N HSQC spectra. All NMR data were preprocessed for consistency and preliminary visualization using NMR pipe software, and the spectra were analyzed with the help of Sparky software.

[0074] (3) Analysis of NMR results

[0075] The results of NMR are as follows Figures 4 to 10 As shown, when no cuprous ions were added, ETR1-EBD 46-82 The protein spectrum has good resolution, regular and dispersed peaks, which indicates that ETR1-EBD 46-82 The protein has a good three-dimensional folding structure; then BCA2-Cu + In the titration experiment, after adding cuprous ions, when the protein: cuprous ion concentration ratio reached 1:0.5, the chemical shifts of all amino acids in the spectrum changed slightly compared to the spectrum without cuprous ions. The peaks of amino acids after adding cuprous ions basically coincided with the peaks without cuprous ions, with only a slight overall shift. When the protein: cuprous ion concentration ratio reached 1:1, the chemical shifts of amino acids in the spectrum changed significantly, and the overall amino acid peaks showed irregular shifts. This phenomenon proves that when the added cuprous ion and protein concentration ratio reaches 1:1, ETR1-EBD 46-82 The conformation of the protein has changed; as the concentration of cuprous ions gradually increases, the peaks of amino acids in the spectrum only show a slight overall shift, and no irregular changes occur. This means that the overall shift may be due to changes in the external environment, such as temperature, pH, etc., rather than changes in the conformation of the protein. This also shows that the added cuprous ions at this time are closely related to the ETR1-EBD 46-82 The protein binding has reached saturation.

[0076] Preparation of culture medium, reagents and buffer required in the above examples:

[0077] Buffer A: 100mM Tris-HCl, 200mM NaCl, pH 8.0, 2mM PMSF, Protease InhibitorCocktail

[0078] Buffer B: 20mM Tris-HCl, 200mM NaCl, 1mM EDTA, pH 7.4 Buffer C: 20mM Tris-HCl, 1mM EDTA, 10mM Maltose, pH 7.4 Buffer D: 50mM Tris-HCl, 0.5mM EDTA, 1mM DTT, pH8.0Buffer E: 25% ACN, 75% ddH2O, 0.1% TFA

[0079] Buffer F: 25% CAN, 75% IPA, 0.1% TFA

[0080] Buffer G: 50mM Tris, 200mM NaCl, pH 7.5

[0081] Buffer H: 25mM MES, 1mM DTT, pH 6.5

[0082] Buffer I: 6M Guanidine, 25mM MES, pH 6.5

[0083] Kanamycin antibiotic (40 mg / mL, 20 mL): Weigh 0.8 g of kanamycin sulfate powder and vortex with ultrapure water to dissolve. Add ultrapure water to 20 mL. Filter and sterilize using a syringe connected to a 0.22 μm pore size sterile filter. Aliquot into 1.5 mL centrifuge tubes and store at 4°C.

[0084] Glucose (20% (m / v)): Weigh 100 g of glucose powder and stir with a certain amount of ultrapure water. Once dissolved, continue adding ultrapure water to make up to 500 mL. Then, sterilize at high temperature and high pressure (121°C, 20 min) and store at 4°C.

[0085] Calcium chloride (CaCl2) (0.1M, 50mL): Weigh 0.555g of calcium chloride powder and stir with a certain amount of ultrapure water. Once dissolved, continue adding ultrapure water to make up to 50mL. Then use a syringe connected to a 0.22μm pore size sterile filter to filter and sterilize. Store at room temperature.

[0086] Magnesium sulfate (MgSO4) (1.0 M, 50 mL): Weigh 6.019 g of magnesium sulfate powder and stir with a certain amount of ultrapure water. After dissolution, continue to add ultrapure water to make up to 50 mL. Then use a syringe connected to a 0.22 μm pore size sterile filter to filter and sterilize. Store at room temperature.

[0087] Isopropyl-β-thiogalactopyranoside (IPTG) (0.3 mM): Weigh 0.072 g IPTG, dissolve it in 1 mL ultrapure water, and add it to 1 L M9 bacterial culture to induce expression.

[0088] DTT stock solution (1 M, 100 μL): Weigh 0.015 g of DTT powder, add 100 μL of ultrapure water and vortex to fully dissolve it.

[0089] 5×M9 liquid culture medium mother solution (2 L): Weigh 30 g anhydrous KH2PO4, 60 g anhydrous Na2HPO4, and 5 g NaCl, stir with a certain amount of ultrapure water, and continue to add ultrapure water to make up to 2 L after dissolution. Store at room temperature.

[0090] 1×M9 liquid medium (1 L): Measure 200 mL of 5×M9 liquid medium, add 800 mL of ultrapure water, and then sterilize at high temperature and high pressure (121°C, 20 min). After cooling, add 6 g of glucose (30 mL of 20% (m / v) glucose solution), 1 g 15 NH4Cl, 1mL 0.1M CaCl2, 2mL 1M MgSO4, and 1mL 40mg / mL kanamycin antibiotic are mixed and ready for use.

[0091] LB solid medium (40 g / L, 100 mL): Weigh 4 g of LB agar powder, add a small amount of ultrapure water, stir to mix, and then dilute to 100 mL. Then sterilize by high temperature and high pressure (121°C, 20 min). After cooling to 55-60°C, add kanamycin antibiotic at a final concentration of 40 mg / mL and shake to mix. Then, aliquot and pour into plates. After the LB medium cools and solidifies, store the plates upside down at 4°C.

[0092] LB liquid medium (25 g / L, 200 mL): Weigh 5 g of LB broth powder, add a small amount of ultrapure water, stir to mix, and then dilute to 200 mL. Then sterilize with high temperature and high pressure (121°C, 20 min) and store at 4°C.

Claims

1. A method for detecting the binding of ethylene receptor ETR1 to cuprous ions based on nuclear magnetic resonance technology, characterized in that: (1) Prepare high-purity key binding domains of ethylene receptors through genetic engineering; (2)Use 15 The key binding domain of ethylene receptor was labeled with N isotope and BCA2-Cu was prepared. + complex, and finally BCA2-Cu + The complex was titrated into the key binding domain of the ethylene receptor, and the binding of the key binding domain of the ethylene receptor to the cuprous ion was detected using nuclear magnetic resonance technology. When the key binding domain of the ethylene receptor bound to the cuprous ion, the chemical shift of the amino acids in the spectrum would change significantly and irregularly, indicating that the ethylene receptor binding domain bound to the cuprous ion.

2. The method for detecting the binding of ethylene receptor ETR1 to cuprous ions based on nuclear magnetic resonance technology according to claim 1, wherein: Step (1) specifically comprises: expressing the key binding domain of the ethylene receptor in large quantities by genetic engineering using an E. coli expression system, and preparing a high-purity key binding domain of the ethylene receptor through multi-step purification.

3. A method for detecting the binding of ethylene receptor ETR1 to cuprous ions based on nuclear magnetic resonance technology according to claim 2, characterized in that: The method of expressing the key binding domain of ethylene receptor in large quantities by using E. coli expression system through genetic engineering is as follows: expressing the key binding domain of ethylene receptor in large quantities by using E. coli expression system through genetic engineering, and the expression conditions are: starting OD 600 :0.1, induced OD 600 :0.6-0.7, induction temperature 30℃-32℃, induction time 22-24h.

4. A method for detecting the binding of ethylene receptor ETR1 to cuprous ions based on nuclear magnetic resonance technology according to claim 2, characterized in that: The multi-step purification steps include protein purification through Dextrin beads affinity chromatography purification, TEV enzyme cleavage and high performance liquid chromatography.

5. The method for detecting the binding of ethylene receptor ETR1 to cuprous ions based on nuclear magnetic resonance technology according to claim 1, wherein: In step (2), the concentration of the BCA2-Cu+ solution is 0.25mM to 8mM.

6. The method for detecting the binding of ethylene receptor ETR1 to cuprous ions based on nuclear magnetic resonance technology according to claim 1, wherein: In step (2), the BCA2-Cu + The amount of the complex added between the key binding domain of the ethylene receptor satisfies the following conditions: the ratio of the amount of protein to the amount of cuprous ion substance is 1:0 to 1:

16.

7. The method for detecting the binding of ethylene receptor ETR1 to cuprous ions based on nuclear magnetic resonance technology according to claim 1, wherein: In step (2), the BCA2-Cu + The amount of the complex added between the key binding domain of the ethylene receptor satisfies the following conditions: the ratio of protein to cuprous ion substance is 1:0, 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:16 and gradually increases.

8. The method for detecting the binding of ethylene receptor ETR1 to cuprous ions based on nuclear magnetic resonance technology according to claim 1, wherein: In step (1), the amino acid sequence of the key binding domain of the ethylene receptor is: SEQ ID NO:

1.

9. The method for detecting the binding of ethylene receptor ETR1 to cuprous ions based on nuclear magnetic resonance technology according to claim 1, characterized in that: The BCA2-Cu + The preparation method of the complex is: (1) Weigh BCA, add Buffer G and fully dissolve it, then filter it with a nylon membrane to obtain a BCA solution; (2) Weigh ascorbic acid and add Buffer G to fully dissolve; (3) Weigh CuCl, add Buffer G to fully dissolve, then add ascorbic acid mother solution until the solution becomes clear and transparent, and then filter with a nylon membrane to obtain a CuCl solution; (4) Take BCA solution and CuCl solution and mix them thoroughly to prepare BCA2-CuCl + , and store it at 4°C until use.

10. The method for detecting the binding of ethylene receptor ETR1 to cuprous ions based on nuclear magnetic resonance technology according to claim 1, characterized in that: In step (2), before using nuclear magnetic resonance technology to detect the binding of the key binding domain of the ethylene receptor to the cuprous ion, it is necessary to use DPC to detect the binding of ETR1-EBD to the cuprous ion. 46-82 Proteins are assembled, that is, the membrane environment of proteins is reconstructed using the Micelle method.

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