A method of analyzing protein structure under different ultrasonic environments by molecular dynamics simulation

Abstract

A method for simulating different ultrasonic environments by molecular dynamics and analyzing protein structures in the environments: 1) obtaining a crystal configuration of a protein from a protein database. 2) selecting a force field, creating a box. 3) adding ion neutralization system in the created box, and performing energy minimization in order to eliminate unreasonable energy barriers. 4) pre-equilibrating the system. 5) after the pre-equilibration, using GROMACS software to simulate the extracted protein under different ultrasonic conditions. 6) processing the trajectory file obtained after simulation by different software, and combining with visualization software to obtain data images related to the protein structure. By the above method, the simulation result is close to the experimental result, and the influence of ultrasonic waves on the protein structure and physicochemical properties is further explored.

Description

Technical Field

[0001] This invention relates to the field of molecular dynamics, and more specifically to a method for simulating different ultrasonic environments using molecular dynamics and resolving protein structures under those environments. Background Technology

[0002] Molecular dynamics (MD) is a computer simulation method for analyzing the physical motion of atoms and molecules in a system. It is the most important and widely used method in molecular mechanics. This method has advanced the development of dynamic models of biomolecules, demonstrating the influence of the relative motion of atoms within biomolecules and the corresponding morphological changes on their functional properties. Currently, due to limitations in experimental conditions, it is often difficult to directly observe the dynamic changes of biomolecules at the atomic level. Using molecular dynamics (MD) simulations to model the behavior of biomolecules can reveal structural and dynamic details that might otherwise be unavailable. Furthermore, by comparing and analyzing laboratory data with simulation data, experimental hypotheses can be more accurately verified, and experimental progress can be advanced. MD simulations have now become a powerful and versatile physics-based method for understanding the structure and dynamics of biomolecules.

[0003] Ultrasound, as a novel, green, innovative, and sustainable technology, has attracted widespread attention across various fields. Existing research has shown that ultrasound technology can alter the spatial structure of various plant proteins and enhance their functional properties, such as emulsifying, foaming, and gelling properties. However, the effects of ultrasound on storage proteins in root and tuber crops are rarely reported.

[0004] This invention utilizes molecular dynamics to simulate different ultrasonic environments, and analyzes the changes in the structure of yam protein under different environments, thereby analyzing the changes in the functional properties of yam protein. Summary of the Invention

[0005] To gain a deeper understanding of the effects of ultrasound on protein structure, this invention provides a method for simulating different ultrasound environments using molecular dynamics and resolving protein structures under those environments.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for simulating different ultrasonic environments using molecular dynamics and analyzing protein structures under those environments includes the following steps:

[0008] 1) Obtain the crystal configuration of a protein from a protein database.

[0009] 2) Select the force field and create a box.

[0010] 3) Add an ion neutralization system to the created box to minimize energy in order to eliminate unreasonable energy barriers.

[0011] 4) Perform pre-equilibrium on the system established above.

[0012] 5) After pre-equilibration, GROMACS software was used to simulate the proteins extracted under different ultrasonic conditions.

[0013] 6) Process the trajectory files obtained after simulation using different software, and then combine them with visualization software to obtain data images of protein structures.

[0014] In step 2), the extracted protein is placed in a periodic octahedral box using the OPLS-AA all-atom force field, and the minimum distance between the extracted protein and the box is set to 1.0 nm. The TIP3P water model is then added to the box for solvation.

[0015] In step 3), appropriate amounts of Na+ and Cl- ions are added to neutralize the system, maintaining an ionic strength of 0.10 mol / L. The system is then subjected to 8000-step energy minimization using the steepest descent algorithm. Long-range interactions are handled using the Particle Mesh Ewald (PME) method, and van der Waals interactions are handled using the cut-off method, with the distance cutoff value set to... A V-rescale thermostat was used to control the system temperature at 300K, and a Berendsen pressure regulator was used to control the pressure.

[0016] The specific method for pre-balancing the system in step 4) is as follows: Under the NVT ensemble, keep the number of atoms, system volume and temperature constant, and change the system pressure to balance the system for 100 ps with a step size of 2 fs; Under the NPT ensemble, keep the number of atoms, system pressure and temperature constant, and change the system volume to balance the system for 100 ps with a step size of 2 fs.

[0017] In step 5), the pressure changes of positive and negative square waves are used to simulate the ultrasonic environment. The frequency of the ultrasonic wave is changed by changing the period of the square wave, and the ultrasonic power is changed by controlling the pressure through a Berendsen constant pressure device.

[0018] The protein structure described is the Dioscorin protein structure.

[0019] This invention provides a method for simulating different ultrasonic environments using molecular dynamics and resolving protein structures under those environments, which has the following advantages:

[0020] 1. This invention provides a method for simulating ultrasonic conditions, which changes the frequency of the ultrasonic wave by changing the period of the square wave and changes the ultrasonic power by controlling the pressure using a Berendsen constant pressure device.

[0021] 2. The method for simulating ultrasound provided by this invention is easy to understand, and the simulation results are close to the experimental results.

[0022] 3. This invention utilizes molecular dynamics simulations to further explore the effects of ultrasound on the structure and physicochemical properties of Dioscorin. Attached Figure Description

[0023] Figure 1a This is a schematic diagram of a periodic square wave at 50 MPa in the method of the present invention;

[0024] Figure 1b This is a schematic diagram of a periodic square wave at 100 MPa in the method of the present invention;

[0025] Figure 2a This is a schematic diagram of a periodic square wave with a frequency of 400MHz in the method of the present invention;

[0026] Figure 2b This is a schematic diagram of a periodic square wave with a frequency of 250MHz in the method of the present invention;

[0027] Figure 3a The intrinsic fluorescence emission spectra of Dioscorin under different ultrasonic powers in the method of this invention;

[0028] Figure 3b The following are the Dioscorin UV-Vis spectra at different ultrasonic powers in the method of this invention;

[0029] Figure 4a These are the intrinsic fluorescence emission spectra of Dioscorin at different ultrasonic frequencies in the method of this invention;

[0030] Figure 4b These are the Dioscorin UV-Vis spectra at different ultrasonic frequencies in the method of this invention;

[0031] Figure 5a The above are SDS-PAGE images of Dioscorin at different ultrasonic powers in the method of this invention.

[0032] Figure 5b The above are SDS-PAGE images of Dioscorin at different ultrasonic powers in the method of this invention.

[0033] Figure 6a These are SDS-PAGE images of Dioscorin at different ultrasonic frequencies in the method of this invention.

[0034] Figure 6b These are SDS-PAGE images of Dioscorin at different ultrasonic frequencies in the method of this invention.

[0035] Figure 7 These are scanning electron microscope images of Dioscorin after processing with different ultrasonic powers in the method of this invention.

[0036] Figure 8 These are scanning electron microscope images of Dioscorin after processing with different ultrasonic frequencies in the method of this invention.

[0037] Figure 9a The RMSD of Dioscorin under different square wave frequencies in the method of this invention;

[0038] Figure 9b The RMSD of Dioscorin under different square wave pressure conditions in the method of this invention;

[0039] Figure 10a The RMSF of Dioscorin under different square wave frequencies in the method of this invention;

[0040] Figure 10b The RMSF of Dioscorin under different square wave pressure conditions in the method of this invention;

[0041] Figure 11a Rg of Dioscorin under different square wave frequencies in the method of this invention;

[0042] Figure 11b Rg of Dioscorin under different square wave pressure conditions in the method of this invention;

[0043] Figure 12a The number of hydrogen bonds in Dioscorin under different square wave frequencies in the method of this invention;

[0044] Figure 12b The number of hydrogen bonds in Dioscorin under different square wave pressure conditions in the method of this invention;

[0045] Figure 13a The SASA of Dioscorin at different square wave frequencies in the method of this invention;

[0046] Figure 13b The SASA of Dioscorin under different square wave pressures in the method of this invention;

[0047] Figure 14a This is a two-dimensional FEL plot of Dioscorin under blank conditions in the method of this invention;

[0048] Figure 14bThis is a three-dimensional FEL plot of Dioscorin under blank conditions in the method of this invention;

[0049] Figure 14c This is a two-dimensional FEL plot of Dioscorin under conditions of 250MHz and 50MPa in the method of this invention;

[0050] Figure 14d This is a three-dimensional FEL plot of Dioscorin under conditions of 250MHz and 50MPa in the method of this invention;

[0051] Figure 14e This is a two-dimensional FEL plot of Dioscorin under conditions of 400MHz and 50MPa in the method of this invention;

[0052] Figure 14f This is a three-dimensional FEL plot of Dioscorin under conditions of 400MHz and 50MPa in the method of this invention;

[0053] Figure 14g This is a two-dimensional FEL plot of Dioscorin under conditions of 250MHz and 100MPa in the method of this invention;

[0054] Figure 14h This is a three-dimensional FEL plot of Dioscorin under conditions of 250MHz and 100MPa in the method of this invention;

[0055] Figure 15 The method of this invention involves superimposing the Dioscorin structure with the original structure under different square wave conditions;

[0056] Figure 16a This refers to the content of secondary structure of Dioscorin under blank conditions in the method of this invention;

[0057] Figure 16b The content of secondary structure of Dioscorin under the conditions of 250MHz and 50MPa in the method of the present invention;

[0058] Figure 16c The content of secondary structure of Dioscorin under the conditions of 400MHz and 50MPa in the method of the present invention;

[0059] Figure 16d This refers to the secondary structure content of Dioscorin under conditions of 250MHz and 100MPa in the method of this invention. Detailed Implementation

[0060] A method for simulating different ultrasonic environments using molecular dynamics and analyzing protein structures under those environments includes the following steps:

[0061] 1) Obtain the crystal conformation of a protein from a protein database. The protein structure is the Dioscorin protein structure.

[0062] 2) Select force field and create box: Use OPLS-AA all-atom force field to place the extracted protein in a periodic octahedral box, and set the minimum distance between the extracted protein and the box to 1.0 nm, and add the TIP3P water model to the box for solvation.

[0063] 3) An ion neutralization system is added to the established box to minimize energy and eliminate unreasonable energy barriers; in step 3), appropriate amounts of Na+ and Cl- ions are added to neutralize the system, maintaining the ionic strength at 0.10 mol / L. The steepest descent algorithm is used to minimize the energy of the system over 8000 steps. Long-range interactions are handled using the particle mesh EwaldPME method, and van der Waals interactions are handled using the cut-off method, with the distance cutoff value set to... A V-rescale thermostat was used to control the system temperature at 300K, and a Berendsen pressure regulator was used to control the pressure.

[0064] 4) Pre-equilibrate the system established above: The specific method for pre-equilibrating the system is as follows: Under the NVT ensemble, keep the number of atoms, system volume and temperature constant, and change the system pressure to balance the system for 100 ps with a step size of 2 fs; Under the NPT ensemble, keep the number of atoms, system pressure and temperature constant, and change the system volume to balance the system for 100 ps with a step size of 2 fs.

[0065] 5) After pre-equilibration, GROMACS software was used to simulate the extracted proteins under different ultrasonic conditions: the ultrasonic environment was simulated by the pressure changes of positive and negative square waves, the frequency of the ultrasonic waves was changed by changing the period of the square waves, and the ultrasonic power was changed by controlling the pressure through a Berendsen constant pressure device.

[0066] 6) Process the trajectory files obtained after simulation using different software, and then combine them with visualization software to obtain data images of protein structures.

[0067] Example 1:

[0068] (I) Extraction, drying and ultrasonic treatment of Dioscorin

[0069] 1) Dioscorin extraction: Dioscorin was extracted from yam using an alkaline extraction and acid precipitation method, and the protein solution was dried using spray drying. The process flow is shown below:

[0070] Yam → Washing and peeling → Weighing and cutting into chunks → Color protection with 1% sodium bisulfite solution → Slurrying with a material-to-liquid ratio of 1:2 → Adjusting the pH of the slurry to 8.5 → Magnetic stirring → Centrifugation and preservation of the supernatant → Adjusting the pH of the supernatant to 3.5 → Magnetic stirring → Centrifugation to separate proteins → Rehydrating the protein with water and adjusting the pH of the protein solution to 7.0 → Spray drying

[0071] 2) Ultrasonic Treatment of Dioscorin: The extracted dioscorin was dissolved in deionized water to prepare a 1% protein solution, which was then magnetically stirred at 25°C for 2 hours. 0.01% potassium sorbate was added to the yam protein solution to prevent microbial growth and spoilage. The pH of the protein solution was adjusted to 7.0 using 1 mol / L NaOH. 16 mL of the protein solution was placed in a 25 mL beaker for ultrasonic treatment. The ultrasonic power was set to 200 W, the ultrasonic frequency to 25 kHz, and the ultrasonic treatment time to 30 min as fixed values. Ultrasonic treatment was performed on the protein solution under different ultrasonic conditions: ultrasonic power: 100, 150, 200, 250, 300 W; and ultrasonic frequency: 25, 33, 40, 59 kHz. The temperature was controlled below 30°C during the ultrasonic treatment process.

[0072] (II) Simulation of Ultrasonic Conditions

[0073] 1) Obtain the crystal configuration of Dioscorin from the protein database PDB ID: 4TWM.

[0074] 2) Select a force field and create a box: Use the OPLS-AA all-atomic force field to place Dioscorin in a periodic octahedral box, and set the minimum distance between Dioscorin and the box to 1.0 nm. Add the TIP3P water model to the box for solvation.

[0075] 3) Adding ions to neutralize the system and minimizing energy: Appropriate amounts of Na+ and Cl- ions were added to neutralize the system, maintaining the ionic strength at 0.10 mol / L. To eliminate unreasonable energy barriers, the steepest descent algorithm was used to minimize the system's energy over 8000 steps. Long-range interactions were handled using the particle mesh Ewald method, and van der Waals interactions were handled using the cut-off method, with the distance cutoff value set to... A V-rescale thermostat was used to control the system temperature at 300K, and a Berendsen pressure regulator was used to control the pressure at 50MPa and 100MPa.

[0076] 4) Pre-equilibrate the system: Use a two-step process to pre-equilibrate the system. Under the NVT ensemble, keep the number of atoms, system volume, and temperature constant, and change the system pressure to equilibrate for 100 ps with a step size of 2 fs. Under the NPT ensemble, keep the number of atoms, system pressure, and temperature constant, and change the system volume to equilibrate for 100 ps with a step size of 2 fs.

[0077] 5) The Dioscorin under different ultrasonic conditions was simulated using GROMACS software: the ultrasonic environment was simulated by the pressure change of positive and negative square waves, the frequency of the ultrasonic wave was changed by changing the period of the square wave, and the ultrasonic power was changed by controlling the pressure through a Berendsen constant pressure device. Figures 1 and 2 are schematic diagrams of different square wave conditions.

[0078] (II) Experimental Results

[0079]

[0080]

[0081] 1. Effects of ultrasonic power on the physicochemical properties and secondary structure of dioscorin

[0082] Table 1 shows the effects of different ultrasonic powers on the physicochemical properties and secondary structure of dioscorin. Figure 3a , 3b 5a, 5b, and Figure 7 The intrinsic fluorescence emission spectrum, UV-Vis spectrum, SDS-PAGE plot, and scanning electron microscope images of dioscorin at different ultrasonic powers are shown. With increasing ultrasonic power, ultrasonic cavitation intensifies, disrupting non-covalent bonds in dioscorin, such as hydrophobic interactions and hydrogen bonds. Large aggregates transform into smaller particles, dioscorin molecules unfold, some free SH is oxidized, and internal hydrophobic amino acids and positively charged amino acid groups are exposed on the dioscorin molecule surface, leading to an increase in Ho and a decrease in the absolute value of the Zeta-potential. Simultaneously, with the disruption of non-covalent bonds, the dioscorin structure changes, with a decrease in the content of α-helices and β-sheets, resulting in decreased thermal stability. However, when the power increases above 200 W, the thermal effect of ultrasound increases, and with increasing system temperature, dioscorin particles tend to aggregate, leading to an increase in Dh.

[0083] 2. Effects of ultrasonic frequency on the physicochemical properties and secondary structure of Dioscorin

[0084] Table 1 shows the effects of different ultrasonic frequencies on the physicochemical properties and secondary structure of Dioscorin. Figure 4a , 4b6a, 6b and Figure 8 The intrinsic fluorescence emission spectrum, UV-Vis spectrum, SDS-page diagram, and scanning electron microscope image of Dioscorin at different ultrasonic frequencies are shown. With increasing ultrasonic frequency, the cavitation threshold increases, and cavitation decreases. At lower ultrasonic frequencies, more non-covalent bonds in Dioscorin, such as hydrophobic interactions and hydrogen bonds, are broken, causing the Dioscorin molecule to unfold. Internal hydrophobic amino acids and positively charged amino acid groups are exposed on the Dioscorin molecule surface, resulting in a decrease in Dh, an increase in Ho, and a decrease in the absolute value of the Zeta-potential. Simultaneously, the structure of Dioscorin changes, with a decrease in the content of α-helices and β-sheets, leading to a decrease in thermal stability.

[0085] 3. Molecular dynamics simulation of ultrasonic conditions

[0086] (1) Root Mean Square Deviation (RMSD)

[0087] Figure 9a , 9b The changes in RMSD values ​​of Dioscorin under different square wave conditions are shown. Simulations revealed that the RMSD of Dioscorin increased after square wave pressure treatment under different conditions, indicating that square wave pressures of different frequencies and powers have a significant impact on the structure of Dioscorin. Furthermore, at low frequencies, the RMSD value of Dioscorin is larger, eventually stabilizing at around 0.21 nm. As the frequency increases to 400 MHz, the RMSD decreases slightly, stabilizing at around 0.17 nm, which is consistent with the experimental results for ultrasonic frequencies. With decreasing ultrasonic frequencies, the cavitation effect of ultrasound becomes stronger, resulting in a greater impact on the structure of Dioscorin. At lower square wave pressures, the RMSD value is higher, stabilizing at around 0.21 nm. As the square wave pressure increases to 100 MPa, the RMSD value decreases to around 0.16 nm. This result is because many residues exhibit greater variability throughout the low-pressure simulation compared to high-pressure simulations. Therefore, under lower square wave pressure, many residues of Dioscorin exhibit significant variability, resulting in substantial structural changes and a large RMSD under pressure. However, as pressure increases, the variability of residues weakens during simulation, making structural changes less likely, thus leading to a relative decrease in RMSD.

[0088] (2) Root Mean Square Fluctuation (RMSF)

[0089] Figure 10a , 10bThe changes in RMSF values ​​of Dioscorin under different square wave conditions are shown. Significant differences in RMSF values ​​were observed after square wave treatment under different conditions, indicating that square waves of different frequencies and pressures have a significant impact on the structure of Dioscorin. Simulations at different ultrasonic frequencies revealed that, compared to a high frequency of 400 MHz, the RMSF of Dioscorin's α-carbon atoms was generally larger at lower frequencies, only slightly lower at residues 20-40. This suggests that at lower frequencies, the residue variation of Dioscorin is relatively large, and structural changes will be greater, a result consistent with RMSD and experimental results. Furthermore, the RMSF value is relatively high at lower square wave pressures. With increasing square wave pressure, the RMSF of most α-carbon atoms of Dioscorin decreases, remaining similar to the RMSF of untreated Dioscorin, but the RMSF of residues 20-50 is higher. The fluidity of some globular proteins generally decreases with increasing pressure, a view that has become a widely observed experimental fact. Therefore, as pressure increases, the flexibility of many residues of Dioscorin decreases, and the RMSF decreases to near that of untreated Dioscorin within the last 2.5 ns time interval analyzed.

[0090] (3) Radius of gyration Rg

[0091] Figure 11a , 11bThe simulation results show the Rg values ​​as a function of simulation time under different square wave conditions. The simulation results reveal that the Rg of Dioscorin changes significantly after square wave processing under different conditions, indicating that square waves of different frequencies and pressures have a significant impact on the compactness of the Dioscorin structure. Furthermore, at low frequencies, the Rg value of Dioscorin is larger, eventually stabilizing at around 1.82 nm. As the frequency increases to 400 MHz, the Rg decreases slightly, stabilizing at around 1.79 nm, close to the Rg value of Dioscorin without square wave processing, which is consistent with experimental results on the effect of ultrasonic frequency on the Dioscorin structure. Under lower square wave pressures, the Rg value is higher, stabilizing at around 1.82 nm. However, as the square wave pressure increases to 100 MPa, the Rg value fluctuates significantly. It can be observed that the period of this fluctuation coincides with the period of the positive and negative square wave. At all pressures of 100 MPa, the Rg of Dioscorin remains stable at a relatively small value of approximately 1.78 nm. When the pressure is changed to -100 MPa, the Rg remains stable at a relatively large value of approximately 1.79 nm. This indicates that at lower square wave pressures, many residues of Dioscorin exhibit better flexibility. With changes in positive and negative wave pressures, the structure of Dioscorin changes, hence the Rg increases and then stabilizes. Therefore, both high positive and negative pressures can stabilize the residues of Dioscorin in one position, reducing flexibility and minimizing internal structural changes. However, when switching between higher positive and negative pressures, the Rg of Dioscorin also exhibits periodic fluctuations. Although the internal structural changes of Dioscorin are not significant, the overall compactness of the structure changes markedly.

[0092] (4) Number of hydrogen bonds

[0093] Figure 12a , 12b The changes in the number of hydrogen bonds in Dioscorin under different square wave conditions are shown. Simulations revealed that the number of hydrogen bonds in Dioscorin changed significantly after square wave treatment under different conditions, indicating that square waves of different frequencies and pressures have a significant impact on the structure of Dioscorin. At low frequencies, the number of hydrogen bonds in Dioscorin increases with increasing frequency up to 400 MHz. At lower square wave pressures, the number of hydrogen bonds is relatively small, but increases with increasing square wave pressure up to 100 MPa. The changes in the number of hydrogen bonds in Dioscorin during the simulation are consistent with the previous RMSD results. Figure 9a , 9bThe changes are consistent. Different ultrasonic frequencies were simulated by changing the square wave period. As the ultrasonic frequency decreases, the cavitation effect of the ultrasound is stronger, resulting in a greater impact on protein structure. Therefore, at lower square wave frequencies, the impact on Dioscorin structure is greater, hydrogen bonds are broken, the number of hydrogen bonds decreases, and the RMSD increases. As the frequency increases, the impact on Dioscorin structure decreases, the number of hydrogen bonds increases, and the RMSD decreases. Compared to high-pressure simulations, many residues exhibit greater variability throughout the low-pressure simulation. Therefore, at low square wave pressure, many Dioscorin residues have greater variability; under pressure, Dioscorin hydrogen bonds are broken, structural changes are significant, and the RMSD increases. However, as the pressure increases, the variability of residues during the simulation weakens, and structural changes are less likely. Therefore, the number of hydrogen bonds is higher and the RMSD decreases at relatively low pressures.

[0094] (5) Solvent-accessible surface area SASA

[0095] Figure 13a , 13b The study shows the variation of Dioscorin's SASA value under different square wave conditions. The effect of different square wave frequencies on Dioscorin was investigated, revealing that at low frequencies, Dioscorin exhibits the highest SASA value, slightly higher than the SASA of Dioscorin without a square wave. The SASA decreases significantly as the frequency increases to 400 MHz. The magnitude of the square wave pressure has a relatively small impact on Dioscorin's SASA; no significant differences in SASA were found in simulations under different square wave pressure magnitudes. However, it was observed that at 50 MPa, the SASA is slightly higher than the control group without square wave pressure, while at 100 MPa, the SASA value is almost identical to the control group. The simulation results are largely consistent with experimentally measured Ho data.

[0096] (6) Free energy morphology diagram FEL

[0097] Dioscorin's FEL 2D and 3D images, such as Figure 14a , 14bFigures 14c, 14d, 14e, 14f, 14g, and 14h are shown. It is clear from the figures that the FEL of Dioscorin without an applied square wave has the largest low-energy region. During the simulation, the main chain atoms of Dioscorin are mainly in low-energy structures, indicating that the structure of Dioscorin is more stable during the simulation. Compared to higher square wave frequencies and pressures, the FEL diagrams of Dioscorin at lower square wave frequencies and pressures have the smallest low-energy regions, indicating that the system is more unstable at lower square wave frequencies and pressures, and that the main chain atoms of Dioscorin have higher energies, making their structure more prone to change. This result is consistent with the previous kinetic results.

[0098] (7) Changes to the Dioscorin main chain

[0099] like Figure 15 As shown, the corresponding RMSDs are: 0.927 nm at 250 MHz and 50 MPa / blank, 0.815 nm at 400 MHz and 50 MPa / blank, and 0.640 nm at 250 MHz and 100 MPa / blank. It can be observed that the structure of Dioscorin changes under different square wave conditions. As the square wave frequency increases from 250 MHz to 400 MHz, the RMSD of the superimposed structure with the blank Dioscorin decreases from 0.927 nm to 0.815 nm. As the square wave pressure increases from 50 MPa to 100 MPa, the RMSD of the superimposed structure with the blank Dioscorin decreases from 0.927 nm to 0.640 nm. This indicates that the structural changes of Dioscorin are greater at lower frequencies and pressures. Increasing the frequency reduces the effect of pressure on the structure. However, increasing the pressure leads to a decrease in the flexibility of many residues, reducing the fluidity of Dioscorin, thus resulting in smaller structural changes. This result is consistent with the previous experimental and simulation results. Furthermore, at the circled location in the figure, a significant structural change can be observed under 250MHz, 100MPa square wave conditions, with the α-helix structure transforming into random coils. This is consistent with the secondary structure changes analyzed by the DSSP program.

[0100] (8) Changes in the secondary structure during the simulation process

[0101] Figure 16a , 16bFigures 16c and 16d show the changes in secondary structure over simulation time under different square wave conditions and without square wave application. It can be seen from the figures that the secondary structure of Dioscorin changes after applying a square wave, compared to the simulation without a square wave, indicating that the square wave can influence the secondary structure of Dioscorin during the simulation. The changes in the secondary structure of Dioscorin are more pronounced at lower square wave frequencies and pressures. The figures show that the α-helix structure of residues 10-20 and 20-45 changes to a β-turn structure, the β-sheet structure of residue 50-60 changes to a random coil, and the α-helix of residues 135-155 and 162-167 shows a tendency to transform into a random coil. As the square wave frequency increases to 400 MHz, the fluctuations in the secondary structure of Dioscorin decrease significantly, with only some β-sheet structures transforming into random coil structures observed at residues 50-60. With an increase of 100 MPa in square wave pressure, the fluctuations in the secondary structure of Dioscorin also decreased. It can be seen that the red β-sheet structure at residues 50-60 changes to a random coil structure. Additionally, the α-helix structure at residues 135-155 may show a tendency to transform into a β-turn. This indicates that at low frequencies and pressures, the fluctuations in the secondary structure of Dioscorin are greater. Increasing the square wave frequency effectively mitigates the influence of pressure on the structure, thus reducing secondary structure changes. However, increasing pressure leads to a decrease in the flexibility of many residues, reducing the fluidity of Dioscorin, resulting in smaller secondary structure changes. The changes in the secondary structure of Dioscorin with square wave frequency and pressure are basically consistent with the previous simulation and experimental data.

Claims

1. A method for simulating different ultrasonic environments using molecular dynamics and analyzing protein structures under those environments, characterized in that, Includes the following steps: 1) Obtain the crystal configuration of a protein from a protein database; 2) Select the force field and create a box; 3) Add an ion neutralization system to the created box to minimize energy in order to eliminate unreasonable energy barriers; 4) Perform pre-equilibrium on the system established above; 5) After pre-equilibration, GROMACS software was used to simulate the extracted proteins under different ultrasonic conditions: the pressure changes of positive and negative square waves were used to simulate the ultrasonic environment, the frequency of the ultrasonic waves was changed by changing the period of the square waves, and the ultrasonic power was changed by controlling the pressure through a Berendsen constant pressure device. 6) Process the trajectory files obtained after simulation using different software, and then combine them with visualization software to obtain data images of protein structures.

2. The method for simulating different ultrasonic environments using molecular dynamics and analyzing protein structures under those environments, as described in claim 1, is characterized in that: In step 2), the extracted protein is placed in a periodic octahedral box using the OPLS-AA all-atom force field, and the minimum distance between the extracted protein and the box is set to 1.0 nm. The TIP3P water model is then added to the box for solvation.

3. The method according to claim 1 for simulating different ultrasonic environments using molecular dynamics and analyzing protein structures under those environments, characterized in that: In step 3), an appropriate amount of Na+ and Cl- ions are added to neutralize the system, and the ionic strength is maintained at 0.10 mol / L; the system is minimized for 8000 steps using the steepest descent algorithm; long-range interactions are handled using the particle grid Ewald method, and van der Waals interactions are handled using the cut-off method, with the distance cutoff value set to 10 Å; A V-rescale thermostat was used to control the system temperature at 300 K, and a Berendsen pressure regulator was used to control the pressure.

4. The method according to claim 1 for simulating different ultrasonic environments using molecular dynamics and analyzing protein structures under those environments, characterized in that: The specific method for pre-balancing the system in step 4) is as follows: Under the NVT ensemble, keep the number of atoms, system volume and temperature constant, and change the system pressure to balance the system for 100 ps with a step size of 2 fs; Under the NPT ensemble, keep the number of atoms, system pressure and temperature constant, and change the system volume to balance the system for 100 ps with a step size of 2 fs.

5. The method according to claim 1 for simulating different ultrasonic environments using molecular dynamics and analyzing protein structures under those environments, characterized in that: The protein structure described is a Dioscorin structure.

Images