Design method of methane hydrate small-molecule inhibitor based on antifreeze protein inhibition mechanism

Abstract

The invention discloses a design method of a methane hydrate small-molecule inhibitor based on an antifreeze protein inhibition mechanism, and the design process of an adsorption type target spot is as follows: HIS33, PHE59 and TYR71 are taken as templates, small molecules containing pyridine rings, benzene rings and phenolic hydroxyl groups as key characteristic structures are designed, hydrophobic groups and hydrogen bond donors / receptors are also contained, and the adsorption type target spot is prepared. Meanwhile, the cage spacing of methane hydrate is 0.742-0.990 nm, and spatial distribution of adsorption residues and the number of hydrogen bonds are used as geometric constraints of molecular design; the design process of the disturbance type target spot comprises the steps that molecules of a rigid multi-ring structure are designed on an ice bonding surface through a mechanism of disturbance inhibition of methane hydrate growth, and ordered arrangement of water molecules is disturbed through steric hindrance. The new insight is obtained through the mechanism difference between IBS and non-IBS, the mechanism of TmAFP for inhibiting methane hydrate growth can be further known, and help is provided for designing an anti-freezing material and a methane hydrate growth inhibitor with biological inspiration.

Description

Technical Field

[0001] The present invention belongs to the technical field of methane hydrate small molecule inhibitors of antifreeze protein binding surfaces, and particularly relates to a design method for methane hydrate small molecule inhibitors based on the antifreeze protein inhibition mechanism. Background Art

[0002] Antifreeze proteins (AFPs) are a special class of biomacromolecules that are widely found in polar fish, insects, microorganisms and some plants (Scholl, CL; Davies, PL Protein Engineering of Antifreeze Proteins Reveals that Their Activity Scales with the Area of the Ice-binding Site. FEBS Lett 2023, 597, 538–546; Melnik, B.; Finkelstein, A. Physical Basis of Functioning of Antifreeze Protein. Mol Biol (Mosk) 2022, 56, 297–305.). They protect body fluids from the effects of low temperatures by inhibiting the formation and growth of ice crystals in organisms ( P.;Elliott,KW;Watkins,SE;Sreter,JA;Jovic,K.;Lehner,IB;Baures,PW;Tsavalas,JG;Levy,DL;Varga,K. 2022,225,jeb243662.). This type of protein has special functional properties such as thermal hysteresis activity, modification of ice crystal growth morphology, and inhibition of ice crystal recrystallization (Deng, J.; Apfelbaum, E.; Drori, R. Ice Growth Acceleration by Antifreeze Proteins Leads to Higher Thermal Hysteresis Activity. J Phys Chem B 2020, 124, 11081–11088; Meister, K.; Moll, C.; Chakraborty, S.; Jana, B.; DeVries, A.; Due to their significant antifreeze effects, the antifreeze mechanism of AFPs has been extensively studied. Currently, the most widely accepted mechanism is the adsorption-inhibition mechanism proposed by Raymond and DeVries, which posits that the ice-binding site (IBS) of AFPs irreversibly adsorbs on the ice surface, leading to a local increase in the curvature of the ice surface. According to the Gibbs-Thomson effect, the increase in curvature leads to a decrease in the freezing point, which in turn inhibits the growth of ice crystals (Raymond, JA; DeVries, A. Adsorption Inhibition as a Mechanism of Freezing Resistance in Polar Fishes. Proc Natl Acad Sci USA 1977, 74, 2589–2593.). In recent years, with the comprehensive understanding and in-depth exploration of the antifreeze mechanism of AFPs, their potential application value has gradually emerged.For example, researchers have found that AFPs have potential application prospects in food processing, cryopreservation, cryosurgery, fisheries and agriculture, and the petroleum industry (Mahatabuddin, S.; Tsuda, S. Applications of Antifreeze Proteins: Practical Use of the Quality Products from Japanese Fishes. Adv Exp Med Biol 2018, 1081, 321–337; Wu, X.; Yao, F.; Zhang, H.; Li, J. Antifreeze Proteins and Their Biomimetics for Cell Cryopreservation: Mechanism, Function and Application-A Review. Int J Biol Macromol 2021, 192, 1276–1291; Xiang, H.; Yang, X.; Ke, L.; Hu, Y. The Properties, Biotechnologies, and Applications of Antifreeze Proteins. Int J Biol Macromol 2020,153,661–675; Ekpo, MD; Xie, J.; Hu, Y.; Liu, X.; Liu, F.; Xiang, J.; Zhao, R.; Wang, B.; Tan, S. 2022,23,2639; Naing, AH; Kim, CKA Brief Review of Applications of Antifreeze Proteins in Cryopreservation and Metabolic Genetic Engineering. 3Biotech 2019, 9, 329; Gharib, G.; Saeidiharzand, S.; Sadaghiani, AK;. A. Antifreeze Proteins: A Tale of Evolution from Origin to Energy Applications. Front Bioeng Biotechnol 2022, 9, 770588.). Particularly in the petroleum industry, the potential application of antifreeze proteins in inhibiting the growth of natural gas hydrates has attracted widespread attention due to the high solubility and excellent biodegradability of AFPs (Gordienko, R.; Ohno, H.; Singh, VK; Jia, Z.; Ripmeester, JA; Walker, VK. Towards a Green Hydrate Inhibitor: Imaging Antifreeze Proteins on Clathrates. PloS one 2010, 5, e8953.).

[0003] Natural gas hydrates are solid inclusion compounds formed by encapsulating gas molecules within water cages under low temperatures and high pressures. Methane is the most common guest molecule in natural gas hydrates, adopting an sI structure (Wang, J.; Tie, Y.; Liu, Z.; Zhang, L.; Jiang, H.; Guo, P. Effects of Different Factors on Methane Hydrate Formation Using a Visual Wellbore Simulator. ACS Omega 2022, 7, 23147–23155.). During oil and gas extraction and transportation, the formation of methane hydrates can cause pipeline blockage, leading to a large pressure drop, which may in turn cause serious safety accidents and ultimately cause significant economic losses and casualties (Cao, P.; Li, T.; Ning, F.; Wu, J. Mechanical Instability of Methane Hydrate–mineral Interface Systems. ACS Appl Mater Interfaces 2021, 13, 46043–46054; Farhadian, A.; Varfolomeev, MA; Shaabani, A.; Nasiri, S.; Vakhitov, I.; Zaripova, YF; Yarkovoi, VV; Sukhov, AV Sulfonated Chitosan as Green and High Cloud Point Kinetic Methane Hydrate and Corrosion Inhibitor: Experimental and Theoretical Studies. CarbohydrPolym 2020, 236, 116035.).Therefore, how to effectively prevent the formation of natural gas hydrates represented by methane to avoid flow safety accidents caused by natural gas hydrate blockage is an important issue that needs to be urgently addressed in the entire oil and gas transportation field (Semenov, AP; Mendgaziev, RI; Stoporev, AS. Dataset for the Experimental Study of Dimethyl Sulfoxide as a Thermodynamic Inhibitor of Methane Hydrate Formation. Data Brief 2023, 48, 109283; Effendi, AD; Md Yusof, MA; Abd Mutalib, NF; Sia, CWAmidated Pectic Polysaccharides (Pectin) as Methane Hydrate Inhibitor at Constant Cooling and Isobaric Condition. Polymers (Basel) 2023, 15, 2080.).

[0004] Related research (Effendi, AD; Md Yusof, MA; Abd Mutalib, NF; Sia, CWA midatedPectic Polysaccharides (Pectin) as Methane Hydrate Inhibitor at Constant Cooling and Isobaric Condition.Polymers(Basel)2023,15,2080;Bagherzadeh,SA;Alavi,S.;Ripmeester,JA;Englezos,P.Why Ice-binding Type I Antifreeze ProteinActs as a Gas Hydrate Crystal Inhibitor.Phys Chem Chem Phys 2015,17,9984–9990;Sun,T.;Davies,PL;Walker,VKStructural Basis for the Inhibition of GasHydrates byα-helical Antifreeze Proteins.Biophys J 2015,109,1698–1705; Maddah,M.; Maddah,M.; Peyvandi,K.The Influence of a Type III Antifreeze Protein and Its Mutants on Methane Hydrate Adsorption-inhibition:A Molecular Dynamics Simulation Study.Phys Chem Chem Phys 2019,21,21836–2184.) showed that AFPs can bind to the crystal surface of methane hydrate and effectively inhibit the formation and growth of methane hydrate by changing its growth kinetics.Bagherzadeh et al. (Bagherzadeh, SA; Alavi, S.; Ripmeester, JA; Englezos, P. Why Ice-binding Type I Antifreeze Protein Acts as a Gas Hydrate Crystal Inhibitor. PhysChem Chem Phys 2015, 17, 9984–9990.) explored the mechanism by which winter flounder antifreeze protein (wf-AFP) inhibits the growth of methane hydrate through MD simulation. The results showed that the THR-(i), ALA-(i+4) and ALA-(i+7) methyl side chains in wf-AFP can bind to the semi-cage structure of the methane hydrate growth interface, thereby effectively preventing its further growth. Sun et al. (Sun, T.; Davies, PL; Walker, VK Structural Basis for the Inhibition of GasHydrates by α-helical Antifreeze Proteins. Biophys J 2015, 109, 1698–1705.) studied the inhibition mechanism of fish antifreeze protein Maxi and tetrahydrofuran sII type hydrate. The simulation results showed that the internal cage-shaped water network of Maxi extends to the outer surface, which helps its binding with sII hydrate. Subsequently, Maddah et al. (Maddah, M.; Maddah, M.; Peyvandi, K. The Influence of a Type III Antifreeze Protein and Its Mutants on Methane Hydrate Adsorption-inhibition: A Molecular Dynamics Simulation Study. Phys Chem Chem Phys 2019, 21, 21836–21846.) used MD simulation to explore the adsorption-inhibition mechanism of type III antifreeze protein and its mutants on methane hydrate. The results showed that type III antifreeze protein mainly relies on hydrophobic interactions with methane hydrate, and hydrogen bonds are not an indispensable force.Most of these studies are based on fish antifreeze proteins, while insect-derived antifreeze proteins exhibit more significant thermal hysteresis activity than fish antifreeze proteins (Hudait, A.; Qiu, Y.; Odendahl, N.; Molinero, V. Hydrogen-bonding and Hydrophobic Groups Contribute Equally to the Binding of Hyperactive Antifreeze and Ice-nucleating Proteins to Ice. J Am Chem Soc 2019, 141, 7887–7898.). In addition, these two types of antifreeze proteins with different activities also show significant differences in inhibiting the growth of methane hydrates. Chen et al. (Chen, C.; Zhang, Y.; Sun, J.; Liu, Y.; Qin, Y.; Ling, Z.; Liu, W.; Li, W. The Roles of Functional Groups of Antifreeze Protein in Inhibition of Hydrate Growth. Fuel 2022, 327, 125060.) described the inhibitory effect of Tenebrio molitor antifreeze protein (TmAFP) on methane hydrate growth and its molecular mechanism. The study showed that TmAFP adsorbed on the surface of methane hydrate through residues on the non-ice-binding surface (non-IBS) and destroyed the hydrogen bonds formed by water molecules around the methane hydrate before adsorption. The study by Liu et al. (Zhang, N.; Du, YT; Yao, PQ; Huang, HY; Zhang, LR; Zhang, FS; Liu, JJ Synergistic Effect of Hyperactive Antifreeze Protein on Inhibition of Gas-Hydrate Growth by Hydrophobic and Hydrophilic Groups. J Phys Chem B 2023, 127, 10469–10477.) revealed that the residues on the ice-binding surface (IBS) of TmAFP bind to methane hydrate, and the hydrophobic and hydrophilic groups of the threonine residues on the IBS play a synergistic role in the adsorption of methane hydrate.Huard et al. (Huard, DJE; Johnson, AM; Fan, Z.; Kenney, LG; Xu, M.; Drori, R.; Gumbart, JC; Dai, S.; Lieberman, RL; Glass, JB) studied bacterial methane clathrate-binding proteins (CbpAs). Their results showed that CbpAs are selective for inclusion complexes on ice, with the TxxxAxxxAxx motif shared with AFPs being buried and not involved in inclusion complex binding. In summary, both the IBS and non-IBS of TmAFP contribute to the inhibition of methane hydrate growth, but the differences in their inhibitory properties remain unclear. Summary of the Invention

[0005] The present invention aims to provide a method for designing small-molecule methane hydrate inhibitors based on the antifreeze protein (AFP) inhibition mechanism. This method constructs models in which the ice-binding and non-ice-binding surfaces of the initial TmAFP face the methane hydrate, respectively. The method also compares the effects of ubiquitin, which lacks antifreeze activity, and a system without AFP, in order to systematically explore the differences in the methane hydrate growth inhibition effects of different TmAFP ice-binding surfaces. Molecular dynamics simulation (MD) is a common and effective method for revealing the interaction mechanisms between molecular objects at the atomic level. This method employs MD simulations to systematically elucidate the dynamics of the interaction between TmAFP and methane hydrate, thereby providing a deeper understanding of the inhibitory mechanism. The results provide valuable theoretical guidance for the design of novel, green, and highly effective hydrate growth inhibitors.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned purpose:

[0007] A design method for methane hydrate small molecule inhibitors based on the antifreeze protein inhibition mechanism is characterized by the following process: using HIS33, PHE59 and TYR71 as templates, small molecules containing pyridine rings, benzene rings and phenolic hydroxyl groups as key characteristic structures are designed, and hydrophobic groups and hydrogen bond donors / acceptors are also included. At the same time, the methane hydrate cage spacing of 0.742-0.990nm, the spatial distribution of adsorbed residues and the number of hydrogen bonds are used as geometric constraints for molecular design.

[0008] Furthermore, the pyridine ring is used to simulate the imidazole group of HIS, the benzene ring is used to simulate the hydrophobic group of PHE, and the phenolic hydroxyl group is used to simulate the phenolic hydroxyl group of TYR; the hydrophobic group is an aromatic ring or a methyl group; the spatial distribution of the adsorption residues and the number of hydrogen bonds are specifically designed to form an average of 3-5 hydrogen bonds when the non-IBS binds to the surface.

[0009] A method for designing small molecule inhibitors of methane hydrate based on the antifreeze protein inhibition mechanism is characterized in that the design process of the perturbation-type target is as follows: the ice-binding surface inhibits the growth of methane hydrate by perturbing the mechanism, and molecules with rigid multi-ring structures are designed to interfere with the orderly arrangement of water molecules through steric hindrance.

[0010] Furthermore, the molecule having a rigid polycyclic structure is specifically an adamantane derivative or a naphthalene derivative.

[0011] Furthermore, the adamantane derivative is specifically adamantane alcohol, and the naphthalene derivative is specifically naphthol.

[0012] The present invention has the following advantages and beneficial effects: The present invention systematically studies the difference in the mechanism of inhibition of methane hydrate (001) plane growth by the ice-binding surface and the non-ice-binding surface of the super-active antifreeze protein TmAFP through molecular dynamics simulation method. The research results show that although both the non-ice-binding surface and the ice-binding surface of TmAFP have an inhibitory effect on methane hydrate, there are significant differences in the mechanism of action between the two. The non-ice-binding surface adsorbs on the surface of methane hydrate through its specific amino acid residues, especially the three residues HIS33, PHE59 and TYR71, significantly inhibiting the growth of methane hydrate. This adsorption effect mainly depends on hydrogen bonds and hydrophobic interactions. In contrast, the ice-binding surface mainly relies on perturbation rather than direct adsorption. The present invention found that due to the distance limitation of the THR residues on the IBS, the residues on the IBS cannot serve as adsorption sites. In addition, the residues on the ice-binding surface are mainly distributed on the β-sheet, and it is difficult for these residues to deform to bind to the newly formed hydrate cage. The new insights gained from the mechanistic differences between IBS and non-IBS will help further understand the mechanism by which TmAFP inhibits methane hydrate growth and provide assistance for the design of bio-inspired antifreeze materials and methane hydrate growth inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1A shows the assembly of TmAFP_IBS, methane hydrate, and methane / water molecules (system 1); B shows the assembly of TmAFP_non-IBS, methane hydrate, and methane / water molecules (system 2); C shows the assembly of ubiquitin, methane hydrate, and methane / water molecules (system 3); and D shows the assembly of no TmAFP, methane hydrate, and methane / water molecules (system 4). Proteins are displayed by NewCartoon. Methane molecules are represented in blue using VDW. Water and methane hydrate are represented by chemical bonds and hydrogen bonds, respectively.

[0014] Figure 2 A is the water P in the hydrogen bond structure of the specified area of simulation system 1, 2, 3 and simulation system 4. A2D2 The temporal evolution of the TmAFP content. B shows the RMSD of TmAFP in simulation system 1. C shows the RMSD of TmAFP in simulation system 1. The data in A are the average of multiple runs. The number of runs for each simulation system is shown in Tables 2 and 3; the data in B and C are obtained from a single simulation trajectory.

[0015] Figure 3 In the hydrogen bond structure of A in the designated area of simulation system S1 and S2, water P A2D2 The content of B is the evolution of P in the hydrogen bond structure of the specified area of the simulation system S3 and S4. A2D2 Evolution of water content over time.

[0016] Figure 4 A and B are the RMSD of TmAFP parallel simulation in simulation system 1.

[0017] Figure 5 A and B are the RMSD of TmAFP parallel simulation in simulation system 2.

[0018] Figure 6 A is the RMSF of TmAFP in simulation system 1; B is the RMSF of TmAFP in simulation system 2. The data in A and B are the results obtained from one simulation.

[0019] Figure 7 For the big 5 12 6 2 Cage and small 5 12 The evolution of the cage over time in simulation system 1 (A), simulation system 2 (B), simulation system 3 (C), and simulation system 4 (D). The data in the figure are the average of multiple simulation results.

[0020] Figure 8 For the big 5 12 6 2 Cage and small 5 12The evolution of the cage over time in simulation system S1 (A), simulation system S2 (B), simulation system S3 (C) and simulation system S4 (D).

[0021] Figure 9 The evolution of F4 over time in simulation system 1 (A), simulation system 2 (B), simulation system 3 (C) and simulation system 4 (D) respectively. The data in the figure are the average values of multiple simulation results.

[0022] Figure 10 The evolution of F4 over time in simulation system S1 (A), simulation system S2 (B), simulation system S3 (C) and simulation system S4 (D) respectively.

[0023] Figure 11 The hydrogen bonds between amino acids on IBS and surrounding water molecules in simulation system 1 and the hydrogen bonds between amino acids on non-IBS and surrounding water molecules in simulation system 2 evolve with time.

[0024] Figure 12 Figure 1 shows the structure of non-IBS adsorbed on the methane hydrate surface. A non-IBS is adsorbed on the methane hydrate surface; B the amino acids on the non-IBS are located above an empty half-cage on the adjacent methane hydrate surface; and C residues HIS33, PHE59, and TYR71 are encapsulated in an empty half-cage at the methane hydrate / water interface.

[0025] Figure 13 The three key residues of non-IBS Evolution over time.

[0026] Figure 14 A is the distribution of THR residues selected for distance statistics; B is the change of the distance between different THR residues in IBS over time.

[0027] Figure 15 To simulate the THR4 (A, D), THR27 (B, E) and THR29 (C, F) in system 1 and Evolution over simulation time.

[0028] Figure 16 To simulate the THR4 (A, D), THR27 (B, E) and THR29 (C, F) in system 2 and Evolution over simulation time. DETAILED DESCRIPTION

[0029] The above contents of the present invention are further described in detail below through examples, but this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0030] Example

[0031] 1. Methods

[0032] 1.1 Molecular model and simulation design

[0033] In this study, all MD simulations were performed using GROMACS 2020.2 (Abraham, MJ; Gready, JEOptimization of Parameters for Molecular Dynamics Simulation Using SmoothParticle-mesh Ewald in GROMACS 4.5. J Comput Chem 2011, 32, 2031–2040.) using the TIP4P / ICE water model (Mahoney, MW; Jorgensen, WLA Five-site Model for Liquid Water and the Reproduction of the Density Anomaly by Rigid, Nonpolarizable Potential Functions. J Chem Phys 2000, 112, 8910–8922.) and the CHARMM27 force field (Sapay, N.; Tieleman, DPCombination of the CHARMM27 Force Field with United-atom Lipid ForceFields. J Comput Chem 2011, 32, 1400–1410. (See Table 1). In the TIP4P / ICE water model, the melting temperature of methane hydrate is 302 K, which is in good agreement with the experimental observation of 297.23 K (Conde, M.; Vega, C. Determining the Three-phase Coexistence Line in Methane Hydrates Using Computer Simulations. J Chem Phys 2010, 133.064507.). Previous studies have shown that the CHARMM force field can be used to fully describe AFPs (Lee, H. Structures, Dynamics, and Hydrogen-bond Interactions of Antifreeze Proteins in TIP4P / Ice Water and Their Dependence on Force Fields. PLoS One 2018, 13, e0198887.).The combination of CHARMM27 and TIP4P / ICE was shown to be suitable for describing the adsorption process of AFPs (Mochizuki, K.; Molinero, V. Antifreeze Glycoproteins Bind Reversibly to Ice via Hydrophobic Groups. J Am Chem Soc 2018, 140, 4803–4811.). The temperature and pressure during the simulation were coupled at 250 K and 45 MPa via a V-rescale thermostat (Bussi, G.; Donadio, D.; Parrinello, M. Canonical Sampling through Velocity Rescaling. J Chem Phys 2007, 126.014101.) and a Parrinello-Rahman gas pressure regulator (Martoňák, R.; Laio, A.; Parrinello, M. Predicting Crystal Structures: the Parrinello-Rahman Method Revisited. Phys Rev Lett 2003, 90, 075503.), respectively. All covalent bonds involving hydrogen atoms were constrained to their equilibrium lengths using the LINCS algorithm (Hess, B.; Bekker, H.; Berendsen, HJ; Fraaije, JG LINCS: A Linear Constraint Solver for Molecular Simulations. J Comput Chem 1997, 18, 1463–1472.) The equations of motion were integrated with a time step of 2 fs. During the simulation, we used the Particle Mesh Ewald (PME) algorithm to perform a detailed evaluation of the long-range electrostatic interactions (Chen, L.; Cruz, A.; Roe, DR; Simmonett, AC; Wickstrom, L.; Deng, N.; Kurtzman, T. Thermodynamic Decomposition of Solvation Free Energies with Particle Mesh Ewald and Long-range Lennard-Jones Interactions in Grid Inhomogeneous Solvation Theory. J Chem Theory Comput 2021, 17, 2714–2724.).To truncate short-range electrostatic and van der Waals interactions, a smooth 1.2 nm sphere was applied. The initial methane hydrate layer was generated using the GenIce program (Matsumoto, M.; Yagasaki, T.; Tanaka, H. GenIce: Hydrogen Disordered Ice Generator. J Comput Chem. 2018, 39, 61-64). VMD 1.9.4 was used to analyze and display the MD trajectories. To explore the differences in the growth inhibition mechanisms of methane hydrates between ice-bound and non-ice-bound surfaces, we simulated a hyperactive AFP, TmAFP. Initial models were constructed with the ice-bound and non-ice-bound surfaces of TmAFP facing the methane hydrate, and comparisons were made with a system without TmAFP and a ubiquitin system without antifreeze activity. Throughout the simulation, the center of mass of the protein was not constrained to its initial position. Ubiquitin does not have antifreeze properties (Kumari, S.; Muthachikavil, AV; Tiwari, JK; Punnathanam, SNComputational Study of Differences between Antifreeze Activity of Type-III Antifreeze Protein from Ocean Pout and Its Mutant. Langmuir 2020, 36, 2439-2448.), and its size is similar to TmAFP (Duboué-Dijon, E.; Laage, D. Comparative Study of Hydration Shell Dynamics around a Hyperactive Antifreeze Protein and around Ubiquitin. J Chem Phys 2014, 141.055103.). Therefore, it is often used as a reference for AFPs. In this article, we designed the following four different types of molecular assemblies for simulation studies. All simulation details are shown in Table 2. In addition, non-(001) planes were also studied, and all simulation details are shown in Table 3.

[0034] Table 1 Force fields, water models, and software used in the study

[0035]

[0036] Table 2 Molecular assemblies involved in the study

[0037]

[0038]

[0039] a: number of independent simulations

[0040] b: The IBS of TmAFP faces the methane hydrate growth plane

[0041] c: The non-IBS of TmAFP faces the methane hydrate growth plane

[0042] d: The initial surface of ubiquitin was chosen arbitrarily

[0043] Table 3 Non-001 face molecular assemblies involved in the study

[0044]

[0045] a: number of independent simulations

[0046] b: IBS of TmAFP faces the (010) surface of methane hydrate

[0047] c: Non-IBS of TmAFP faces the (010) surface of methane hydrate

[0048] d: IBS of TmAFP faces the (100) surface of methane hydrate

[0049] f: non-IBS of TmAFP faces the (100) surface of methane hydrate

[0050] 1.2 Protein-methane hydrate assembly system

[0051] The three-dimensional structures of wild-type TmAFP and ubiquitin (PDB IDs 1EZG and 1UBQ, respectively) were obtained from the Protein Data Bank. Figure 1 Middle A (Assembly System 1), Figure 1 Middle B (Assembly System 2) and Figure 1As shown in Figure C (assembly system 3). By manually adjusting the TmAFP conformation, the IBS and non-IBS were oriented toward the growth surface of the methane hydrate. The optimal growth surface (001) reported in the literature was chosen as the growth surface of the methane hydrate. This plane was chosen for the AFP binding simulation because it is stable, has the most complete cage structure on the outer layer, and has relatively few "overhanging" water molecules (Bagherzadeh, SA; Alavi, S.; Ripmeester, JA; Englezos, P. Why Ice-binding Type I Antifreeze Protein Acts as a Gas Hydrate Crystal Inhibitor. Phys Chem Chem Phys 2015, 17, 9984–9990.). The binding of AFP to the (001) plane provides a potential site for inhibiting hydrate growth (Zhang, N.; Du, YT; Yao, PQ; Huang, HY; Zhang, LR; Zhang, FS; Liu, JJ Synergistic Effect of Hyperactive Antifreeze Protein on Inhibition of Gas-Hydrate Growth by Hydrophobic and Hydrophilic Groups. J Phys Chem B 2023, 127, 10469–10477.). The initial conformation of the methane / water liquid phase was generated using the Packmol software package (Schott-Verdugo, S.; Gohlke, H. Packmol-memgen: A Simple-to-use, Generalized Workflow for Membrane-protein–lipid-bilayer System Building. J Chem Inf Model 2019, 59, 2522-2528.). Specifically, the assembly system contains 1483 methane molecules and 8539 water molecules, with a molar ratio of water to methane of 46:8, which is consistent with the conditions for methane hydrate formation at 100% cage occupancy (Sloan, ED; Koh, CA, Clathrate Hydrates of Natural Gases, 3rd ed.; CRC Press: Boca Raton, FL, 2007.). + The ions make the system electrically neutral and use a force constant of 1000 kJ·mol-1 nm -2 It was confined to the edge of the box to prevent it from affecting the growth of methane hydrate. For comparison, a methane hydrate-methane / water molecule system of the same size but without TmAFP was constructed (assembly system 4). Figure 1 In order to prevent the dissolution of the initial methane hydrate, 1000 kJ·mol -1 nm -2 Force constants were used to constrain the system to its initial position. Each solvated system was initially minimized for 50,000 steps using the steepest descent algorithm. Subsequently, a 100-ps simulation was performed in the canonical (NVT) ensemble, followed by another 100-ps simulation in the isobaric isothermal (NPT) ensemble. Finally, a 300-ns MD simulation was performed on the resulting molecular assemblies.

[0052] 2. Results and Discussion

[0053] 2.1 Statistics of methane hydrate growth rate

[0054] In order to statistically analyze the differences in the growth rates of methane hydrate in the four systems, three independent 300ns simulations were performed for the system with the ice-binding surface (assembly system 1) and the non-ice-binding surface (assembly system 2) of TmAFP facing the methane hydrate, ubiquitin (assembly system 3), and the system without AFP (assembly system 4). The evolution of the number of ordered water molecules between the IBS and non-IBS and the methane hydrate surface over the simulation time was monitored, and the average results were compared with the average results of the ubiquitin and non-AFP systems in the same area. Figure 2 As shown in A.

[0055] The hydrogen bond structure of a water molecule is represented by AmDn, where m and n represent the number of hydrogen bonds formed with O (proton acceptor, denoted as A) and H (proton donor, denoted as D), respectively. For each water molecule, m and n = 0, 1, or 2. Therefore, nine types of water molecules or hydrogen bond structures were determined (Tan, J.; Sun, Y.; Ma, L.; Feng, H.; Guo, Y.; Cai, W.; Shao, X. Knowledge-based Genetic Algorithm for Resolving the Near-infrared Spectrum and Understanding the Water Structures in Aqueous Solution. Chemometr Intell Lab Syst 2020, 206, 104150.). A2D2 Indicates the proportion of water molecules that form 4 hydrogen bonds among the 9 types of water molecules. A2D2The higher the value, the higher the methane hydrate content. Figure 2 Middle A Middle P A2D2 The slope of reflects the growth rate of methane hydrate. A2D2 The amino acids on the IBS disrupt the order of water molecules on the surface of methane hydrate, making it more difficult to form stably bound methane hydrate. A2D2 Basically, it reaches a plateau after 150ns. However, in simulation system 3 and simulation system 4, P A2D2 It has been increasing throughout the simulation time. It can be seen that both IBS and non-IBS have a significant inhibitory effect on the growth of methane hydrate. In contrast, simulation system 3 and simulation system 4 have almost no inhibitory effect on the growth of methane hydrate. At the same time, the P of four different molecular components on the non-(001) plane is A2D2 The same statistical analysis was performed on the values ( Figure 3 In the statistical results for non-(001) planes, there was no significant difference in inhibition between IBS and non-IBS. Therefore, this result strongly demonstrates that TmAFP's IBS is more effective than non-IBS in inhibiting the growth of methane hydrate on the (001) plane, but the reasons for this need further exploration.

[0056] In order to analyze the conformational changes of different binding surface models of TmAFP during the simulation process, the protein RMSD in simulation system 1 and simulation system 2 were statistically analyzed, such as Figure 2 The RMSD values of the parallel simulation results are shown in B and C. Figure 4 and Figure 5 As shown. The results show that during the simulation process, the conformation of TmAFP in simulation system 1 changed significantly, and the RMSD value fluctuated between 0.10 and 0.30. However, the conformation of TmAFP in simulation system 2 was relatively more stable, and the RMSD value was limited to a narrow range of 0.10 to 0.15. The RMSF calculation results also showed that the amino acid residues in simulation system 1 fluctuated significantly in the range of 0.05 to 0.5 nm, indicating that the TmAFP structure underwent a large conformational change during the simulation process (see Figure 6 In contrast, the RMSF values in simulation system 2 fluctuated within a narrow range of 0.05 to 0.15 nm, indicating minimal conformational changes in TmAFP during the simulation. Therefore, it can be preliminarily concluded that the inhibitory mechanisms of IBS and non-IBS systems differ significantly. IBS systems may experience greater perturbations, whereas non-IBS systems may not.

[0057] 2.2 Methane hydrate growth characteristics

[0058] The different growth processes of methane hydrates by IBS and non-IBS in the presence of TmAFP were studied by calculating the cage numbers of methane hydrates. For comparison, the cage numbers in simulation systems 3 and 4 were also calculated. The unit cell of methane hydrate sI contains 6 large 5 12 6 2 Cage and 2 small 5 12 Cage (Duenas, DC; Dunn-Rankin, D.; Chien, YCMethaneHydrate Structure IDissociation Process and Free Surface Analysis. Energy Fuels 2024, 38, 7862–7872.). GRADE is used to identify the hydrate structure of clathrate (Mahmoudinobar, F.; Dias, CLGRADE: A Code to Determine Clathrate Hydrate Structures. Comput Phys Commun 2019, 244, 385–391.). 12 Cage and Big 5 12 6 2 The number of cages is calculated as Figure 7 As shown. It can be seen that the small 5 12 Cage and Big 5 12 6 2 The number of cages increases gradually from 0 to 150 ns. After that, the growth rate of simulation system 1 and simulation system 2 increases very slowly, indicating that the system gradually reaches a stable state. Both IBS and non-IBS have the ability to inhibit the growth of methane hydrate. It should be noted that the small 5 12 Cage and Big 5 12 6 2 The number of cages is less than that of the other three simulation systems. Similarly, the small 5 12 Cage and Big 5 12 6 2 The same statistics were performed on the number of cages (see Figure 8 The results showed that there was no difference in the inhibitory ability between IBS and non-IBS on non-(001) planes, which is consistent with the P A2D2 The statistical results of the values are highly consistent. The results show that when the IBS faces the (001) surface of methane hydrate, it has a strong perturbation effect on water molecules, making it difficult for water molecules to form regular cages.

[0059] In order to further study the growth process of methane hydrate in different simulation systems, the four-body order parameter (F4) (e.g. Figure 9As shown in Figure 2 ). F4 can characterize the state of water molecules, that is, whether they are in a liquid or hydrated state (Choudhary, N.; Hande, V.R.; Roy, S.; Chakrabarty, S.; Kumar, R. Effect of Sodium Dodecyl Sulfate Surfactant on Methane Hydrate Formation: A Molecular Dynamics Study. J Phys Chem B 2018, 122, 6536–6542.). The slope of the curve represents the growth rate of the hydrate. The F4 of the hydrate phase is 0.7, and the F4 of liquid water is 0. The results show that the slope of the curve of simulation system 1 is smaller than that of the other three simulation systems, that is, the methane hydrate growth rate of simulation system 1 is the lowest. At 150 ns, the F4 value is only 0.11. Since the F4 of the entire simulation system is calculated, rather than the portion between the methane hydrate surface and the IBS / non-IBS, the value is slightly smaller. When the simulation time is 50 ns, the F4 value of the other three molecular combinations is 0.11. Correspondingly, the same statistical analysis was performed on the F4 values of four different simulation systems on non-(001) planes (see Figure 10 The results show no difference in the inhibitory ability of IBS and non-IBS on non-(001) planes, which is highly consistent with other previously obtained statistical results. Furthermore, it can be seen that the presence of TmAFP and the IBS facing the (001) plane of methane hydrate do affect the formation kinetics of methane hydrate.

[0060] 2.3 TmAFP's inhibitory mechanism for IBS and non-IBS

[0061] In order to reveal the mechanism by which IBS and non-IBS inhibit the growth of methane hydrate, we first analyzed the hydrogen bonds formed between amino acids on IBS and surrounding water molecules in simulation system 1 and between amino acids on non-IBS and surrounding water molecules in simulation system 2. The results are as follows: Figure 11 As shown in Figure 1, the number of hydrogen bonds formed between amino acids on the IBS and surrounding water molecules in Simulation System 1 is significantly lower than that formed between amino acids on the non-IBS and surrounding water molecules in Simulation System 2. This result indicates that the non-IBS in Simulation System 2 more readily binds to methane hydrate, further enhancing the likelihood of amino acid residues adsorbing on methane hydrate. The simulation trajectories (see Movies 1 and 2) also clearly demonstrate that Simulation System 2 inhibits methane hydrate growth by adsorbing specific amino acids on the non-IBS onto the surface, while Simulation System 1 inhibits methane hydrate growth by perturbing amino acids on the IBS.

[0062] In order to further reveal the difference between the mechanisms of IBS and non-IBS in inhibiting the growth of methane hydrate, the structure of non-IBS adsorbed on methane hydrate was analyzed, such as Figure 12 As shown in Figure 2, non-IBS can be stably adsorbed on the surface of methane hydrate, and the three key residues are approximately located above the empty half cage adjacent to the hydrate surface. Figure 12 As can be seen in Figure 3, residues HIS33, PHE59, and TYR71 can reach the half-cage of the newly formed methane hydrate. This forms a stable adsorption structure, effectively inhibiting the growth of methane hydrate. This is consistent with the literature reporting that AFP inhibits methane hydrate growth through an adsorption-inhibition mechanism (Maddah, M.; Maddah, M.; Peyvandi, K. The Influence of a Type III Antifreeze Protein and Its Mutants on Methane Hydrate Adsorption-inhibition: A Molecular Dynamics Simulation Study. Phys Chem Chem Phys 2019, 21, 21836–21846.). Furthermore, the simulated trajectory of the IBS toward the methane hydrate was also studied. Unlike non-IBS, the IBS inhibits methane hydrate growth primarily through perturbation rather than direct adsorption. In summary, the amino acids on non-IBS inhibit the growth of methane hydrate by binding to methane hydrate, while IBS mainly relies on perturbation to inhibit the growth of methane hydrate.

[0063] In order to further explore the adsorption stability of key adsorption residues in the non-IBS model, the key residues HIS33, PHE59 and TYR71 were The analysis of dihedral angles shows the following results: Figure 13 As shown. Dihedral angle It is formed by the methyl group, nitrogen, carbon and other atoms contained in the residue structure, which can reflect the stability of the binding between the residue and the half cage. Figure 13 AC shows that three key residues It remained stable during the 300ns simulation, indicating that the corresponding dihedral angle changes were relatively stable, that is, the methyl groups on these three key adsorption residues had good stability as semi-caged guest molecules.

[0064] 2.4 Reasons why IBS cannot be adsorbed

[0065] To investigate the reasons why the residues on the IBS cannot serve as adsorption sites, we studied the degree of matching between the spacing between adjacent hydrate cages and the methyl spacing of THR residues and calculated the methyl spacing of THR residues on the well-aligned IBS surface on the β-sheet. The specific THR residues we selected are distributed as follows: Figure 14 As shown in A. From the statistics of the distances between adjacent sI methane hydrate cages, it can be seen (Li, S.; Lv, R.; Yan, Z.; Huang, F.; Zhang, X.; Chen, GJ; Yue, T. Design of Alanine-rich Short Peptides as a Green Alternative of Gas Hydrate Inhibitors: Dual Methyl Group Docking for Efficient Adsorption on the Surface of GasHydrates. ACS Sustain Chem Eng 2020, 8, 4256–4266.), two 5 12 The distance between the small cages is 0.764-0.816nm, and the two 5 12 6 2 The distance between the large cages is 0.742-0.990nm, 5 12 6 2 Big cage with 5 12 The distance between the small cages is 0.753-0.903nm. Figure 14Comparing the data in B with the distances between adjacent cages of methane hydrate, it was found that the distances between THR29 and THR53 and THR27 and THR51 residues meet the interval value of the distance between adjacent cages, while THR29 and THR41 and THR27 and THR39 do not meet the spacing requirements between the two small cages. In addition, these residues on the β-sheet are difficult to be affected by disulfide and hydrogen bonds to produce deformation (Chen, C.; Zhang, Y.; Sun, J.; Liu, Y.; Qin, Y.; Ling, Z.; Liu, W.; Li, W. The Roles of Functional Groups of Antifreeze Protein in Inhibition of Hydrate Growth. Fuel 2022, 327, 125060.). Analysis of residues on the IBS that cannot be adsorbed further supports the conclusion that the TxxxAxxxAxx motif shared by AFPs is not involved in clathrate binding (Huard, DJE; Johnson, AM; Fan, Z.; Kenney, LG; Xu, M.; Drori, R.; Gumbart, JC; Dai, S.; Lieberman, RL; Glass, JB. Molecular Basis for Inhibition of Methane Clathrate Growth by a Deep Subsurface Bacterial Protein. PNAS Nexus 2023, 2, 268–276.). To bind to methane hydrate, all adjacent THR residues must meet the required distance between adjacent cages. Taken together, the above analysis indicates that the inability of THR residues to enter the half-cage is a key reason why the binding site residues on the IBS cannot serve as adsorption sites.

[0066] The dihedral angles formed by THR residues and nitrogen and carbon atoms were further analyzed based on simulation system 1 and simulation system 2. and in The curve trend shows that the methyl group is stable as a half-cage guest molecule. The curve trend shows the stability of hydrogen bonds between hydroxyl groups and surrounding water molecules. Figure 15 As shown. Whether it is THR4 located on the α helix or THR27 and THR29 on the β fold, The fluctuations with the simulation time are large, indicating that the methyl group has not stably entered the methane hydrate cage. Although the fluctuation is small, it can be seen from the simulation trajectory (Movie1) that the amino acids on the IBS do not bind to methane hydrates, but inhibit the growth of methane hydrates by perturbing them. For comparison, we counted the THR4, THR27 and THR29 in simulation system 2. and As the simulation time changes, Figure 16 The results also showed that the strength of hydrogen bonds formed between the hydroxyl groups of the amino acids on IBS and the surrounding water molecules and the stability of the methyl groups as semi-caged guest molecules were reduced. These results confirmed that IBS exerted its influence through a perturbation mechanism, thereby inhibiting the formation of methane hydrate.

[0067] 2.5 Design strategies for efficient methane hydrate inhibitors

[0068] Design of adsorption-type targets. Using HIS33, PHE59, and TYR71 as templates, small molecules (such as polyphenol derivatives, aromatic amides, and benzamide derivatives) containing a pyridine ring (simulating the imidazole group of HIS), a benzene ring (simulating the hydrophobic group of PHE), and a phenolic hydroxyl group (simulating the phenolic hydroxyl group of TYR) as key characteristic structures are designed. At the same time, hydrophobic groups (such as aromatic rings and methyl groups) and hydrogen bond donors / acceptors are included. The methane hydrate cage spacing (0.742-0.990nm), the spatial distribution of adsorbed residues, and the number of hydrogen bonds (such as an average of 3-5 hydrogen bonds formed when non-IBS binds to the surface) are used as geometric constraints for molecular design to simulate the interaction between non-IBS residues and the half-cage of the methane hydrate surface.

[0069] Design of perturbation targets. The ice-binding surface perturbs the mechanism that inhibits the growth of methane hydrates. Molecules with rigid polycyclic structures (such as adamantane derivatives such as adamantane alcohol and naphthalene derivatives such as naphthol) are designed to disrupt the orderly arrangement of water molecules through steric hindrance.

[0070] The basic principles, main features and advantages of the present invention are shown and described above. Without departing from the spirit and scope of the present invention, the present invention may also be subject to various changes and improvements, which fall within the scope of the present invention to be protected.

Claims

1. A method for designing a small molecule methane hydrate inhibitor based on the antifreeze protein inhibition mechanism, characterized in that The design process of adsorption-type targets is as follows: using HIS33, PHE59 and TYR71 as templates, small molecules containing pyridine rings, benzene rings and phenolic hydroxyl groups as key characteristic structures are designed, while also containing hydrophobic groups and hydrogen bond donors / acceptors. At the same time, the methane hydrate cage spacing of 0.742-0.990 nm, the spatial distribution of adsorption residues and the number of hydrogen bonds are used as geometric constraints for molecular design.

2. The method for designing a methane hydrate small molecule inhibitor based on the antifreeze protein inhibition mechanism according to claim 1, characterized in that: The pyridine ring is used to simulate the imidazole group of HIS, the benzene ring is used to simulate the hydrophobic group of PHE, and the phenolic hydroxyl group is used to simulate the phenolic hydroxyl group of TYR; the hydrophobic group is an aromatic ring or a methyl group; the specific design requirements for the spatial distribution of the adsorption residues and the number of hydrogen bonds are that an average of 3-5 hydrogen bonds are formed when the non-IBS binds to the surface.

3. A method for designing a small molecule methane hydrate inhibitor based on the antifreeze protein inhibition mechanism, characterized in that The design process of the perturbation target is as follows: the ice-binding surface inhibits the growth of methane hydrate by perturbing the mechanism, and molecules with rigid multi-ring structures are designed to interfere with the orderly arrangement of water molecules through steric hindrance.

4. The method for designing a methane hydrate small molecule inhibitor based on the antifreeze protein inhibition mechanism according to claim 3, characterized in that: The molecule having a rigid polycyclic structure is specifically an adamantane derivative or a naphthalene derivative.

5. The method for designing a methane hydrate small molecule inhibitor based on the antifreeze protein inhibition mechanism according to claim 4, characterized in that: The adamantane derivative is specifically adamantane alcohol, and the naphthalene derivative is specifically naphthol.

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