Study On Coiled Coil Of Grass Carp Myosin Using Molecular Dynamics Simulation

The rod-shaped tail of myosin is a typical double-stranded super-coiled structure?coiled coil?.The structure and function of myosin coiled coil determine the solubility?or aggregation performance?of myosin in solution,the ability to form thick filaments,and the gel properties of meat proteins.At present,domestic and foreign scholars'studies on myosin coiled coil mainly focus on the biological functions of myosin coiled coil.And the molecular mechanism of self-assembly and dissociation of myosin coiled coil,and the influence mechanism of the environmental conditions on the three-dimensional structure and intermolecular forces of coiled coils and coiled coil multi-dimers?multiple coiled coils aggregation?have not been clearly investigated.Coiled coil of grass carp myosin was taken as the research object in this paper.Firstly we analyzed the coiled coil amino acid sequence and constructed a coiled coil molecular model.The stretch kinetics method was applied to explore the stacking mode of coiled coils and the dynamic process of separation of myosin from the thick filament structure.And then molecular simulation was used to investigate the impact of environmental conditions on the structure of the coiled coil and its multi-dimer and the diffusion characteristics of NaCl ions in the myofibril simulation system.The study work of this paper is helpful to understand the molecular mechanism of variation of myosin structure and function during the processing and storage of fish meat products.It was provided a new theoretical basis for the quality control of fish meat products.The main results are as follows:1 The amino acid sequence of coiled coil and its molecular structureGrass carp myosin rod S2?Myosin subfragment 2,S2?and LMM?Light meromyosin,LMM?fragments have high homology with related amino acid sequences of silver carp,carp,mandarin fish,tilapia and yellow croaker,and the similarity exceeds90%.Except for the occurrence of skips at 4 amino acid residues,the probability of the amino acid sequence of the S2 and LMM fragments forming coiled coil structure is almost 100%.Skip residues can increase the"flexibility"of the myosin rod in a certain extent.The bending freedom of the LMM segment is higher than that of the S2 segment,which is more conducive to the flexibly stacking of myosin in the myofibril.Hydrophobic amino acids distributed in the coiled coil hydrophobic core area?a,d position?account for60.4%of the total number of hydrophobic amino acids,and the surface of the coiled coil molecule?b-c,e-f-g positions?is mainly distributed with polar amino acid residues.The grass carp myosin S2 and LMM fragments have at least little difference in amino acid composition.The three-dimensional structure of grass carp myosin coiled coil constructed by the Least Squares Support Vector Machine Hybrid Modeling?LSSVM-HM?method has high reliability.Compared with other modeling methods,the coiled coil structure constructed by LSSVM-HM has achieved the best performance in the structural evaluations of Raman conformation analysis,local potential distribution of residue sidechains and Qmean6z-score validation.By the spatial comparison verification of the resolved crystal structures,the accuracy of main chain and side chain of the coiled coil structure constructed in this paper are significantly better.The coiled coil constructed in this paper has a total of 1099 residues,of which the super-helix radius of the S2 fragment is 5.17?,each coil contains 99 residues,the pitch is13.3 nm,and the crossing angle of 2 alpha-helix chains is 25.2°,and the super-helix radius of the LMM fragment is 4.89?,each coil contains 103 residues,the pitch is 14.8nm,the crossing angle of 2 alpha-helix chains is 22.4°,showing the morphological characteristics of"thick and short"for the S2 fragment and"thin and long"for the LMM fragment.The structural parameters of the alpha-helix chain of S2 and LMM fragments have no obvious difference.The residue number per alpha-helix turn is 3.53,the residue rise in the main axis is 0.14 nm and the average radius of the alpha-helix is 2.25?.2 Coiled coil stacking mode and its molecular mechanism of dissociationUse tensile dynamics to study the force between coiled coil molecules and its stacking mode,as well as the dynamic process of coiled coil dissociation from the multi-dimer under different environmental conditions.The difference in charge between the contact surfaces of coiled coil molecules is closely related to the relative rotation angle between the molecules.There exist 6 peaks of charge difference at equal intervals in the range of coiled coil rotating one revolution?0o?360o?.The electrostatic interaction is the main force for forming and stabilizing the thick filament structure.When the relative rotation angle is a multiple of 60o,the electrostatic force between coiled coils in a 2-dimer is the largest and the structure is being the most stable.At this time,the axial displacement between coiled coil molecules has about 18 amino acid residues.The stacking mode of coiled coils in the myofibril thick filament is:the coiled coil at the center position and the six nearby coiled coils arranged hexagonally are stacked parallel to each other,the stacking distance is 2 nm,and the relative rotation angle between coiled coil molecules is a multiple of 60owith a axial misalignment of about 18 residues from each coiled coil.The coiled coil molecule in the center of the 7-dimer is pulled away gradually from the 7-dimer structure along the annular channel formed by other coiled coils,which can be used to simulate the process of myosin dissociation from the myofibril thick filaments.Environmental conditions such as temperature,NaCl concentration,and concentration of H⁺or OH^-?Cab?have a significant effect on the axial tensile stress and tensile potential energy of the mobile coiled coil molecule.The tensile stress and tensile potential energy of the mobile coiled coil decrease with increasing temperature.When the NaCl concentration is 0.5M and the Cab is 5.0?T=300K?,the tensile stress(<600KJ?mol^-1?nm^-1)and tensile potential energy?0.78?10⁵ KJ/mol?reach the minimum,other NaCl concentration and Cab will make the tensile stress and tensile potential energy of the mobile coiled coil become larger.The higher temperature?280K?340K?,suitable NaCl concentration?0.5M?and Cab value?5.0?will facilitate the dissociation of myosin from the thick filament structure.3 The influence of environmental conditions on the structural parameters and solvation capacity of coiled coil and its multi-dimerThe molecular simulation method was used to study the influence of the environmental conditions,such as temperature,NaCl concentration and concentration of H⁺or OH^-,on the structural parameters and the solvation ability of coiled coil and coiled coil 7-dimer in the simulated system.As the temperature rises,the contraction degree of the coiled coil structure becomes larger,and the coiled coil 7-dimer expands significantly.The higher temperature?>320K?causes the thermal conformation change of the 7-dimer structure to be an unstable state,320K is the initial conformation change temperature of coiled coil and its 7-dimer.When the temperature is 300K?26.8??,the coiled coil and its7-dimer have the largest solvation free energy,which are in a most stable state in water.Increasing temperature weakens the hydrogen bond and ionic bond,resulting in a declining trend of the solvation of coiled coil and its 7-dimer with water molecules;As the NaCl concentration increases,the contraction degree of the coiled coil molecular structure in the aqueous solution becomes larger.The 7-dimer structure shrinks slightly under low salt conditions?<0.2 M?,and at higher salt concentrations?0.4M?0.8M?the 7-dimer structure swell significantly.At extremely high NaCl concentrations?>0.8 M?,the coiled coil and its 7-dimer structure undergo a salt-induced conformation change,and the coiled coil structure shrinks violently,while the ordered stacking mode of coiled coils in the 7-dimer structure was destroyed during the simulation process.The solvation ability of coiled coil and its 7-dimer is weaker at low salt conditions.When the NaCl concentration is 0.2 M the solvation free energy of the coiled coil and its 7-dimer molecules achieve the smallest value,the stability of coiled coil and its multi-dimer in the aqueous solution is poor and it is easy to aggregate with each other.But at a higher salt concentration,increasing the NaCl concentration can obviously increase the solvation free energy of the coiled coil and its 7-dimer molecules.Without causing salt-induced conformation change,the NaCl concentration of about 0.6M is the optimal ionic environment for presentation as monomer state of coiled coil in the solution or for dissociation of the coiled coil multi-dimer;The coiled coil and its 7-dimer structure are more stable at Cab 7.0,when the Cab is far from neutral the coiled coil and its 7-dimer will obviously shrink first and then expand.The extremely acid or alkali may break the ionic and hydrogen bonds in the protein molecule,making the protein structure irreversible conformation change.When the Cab is5.0,coiled coil and its 7-dimer have the smallest solvation free energy,Cab 5.0 is the isoelectric point?p I?of grass carp myosin coiled coil,and at this Cab coiled coil and its7-dimer structure have a largest degree shrinkage.As the Cab environment of the solution deviates from p I 5.0,the solvation free energy of the molecules gradually increases,so coiled coil and its 7-dimer are more stable in acidic or alkaline solutions.4 The characteristics of NaCl ions diffusion in myofibrilThe molecular dynamics simulation method was used to study the diffusion characteristics of NaCl ions in myofibril simulated structure.NaCl ions interact electrostatically with the charged residues of coiled coil,which is the mainly resistance for the diffusion and migration of salt ions.In the simulating experiment,the simulated structure of the myofibril has expanded,the molecular gap of coiled coils becomes larger,and the electrostatic force of salt ions with the charged residues is reduced.The double-sided flat plate model of Fick's second law can be used to well describe the diffusing distribution of NaCl in the myofibril simulated structure.There exists diffusion anisotropy of NaCl in two directions?normal and parallel to the myofibril?.The NaCl diffusion coefficients D_x?normal to myofibrils?and D_y?parallel to myofibrils?show a downward trend with the extension of the simulation time,and with the temperature rising,the values of D_xand D_yincrease.The NaCl diffusion coefficients of D_y(16.7×10^-9?120×10^-9m²/s)is greater than D_x(2.91×10^-9?88.8×10^-9 m²/s).The improved Arrhenius equation can perfectly fit the relationship of NaCl diffusion coefficients D_x or D_y with temperature and simulation time,the normal diffusion activation energy of NaCl?6.144 KJ/mol?is greater than the parallel diffusion activation energy?5.510 KJ/mol?,indicating that NaCl diffusing normal to the myofibril requires more energy than diffusing parallel to the myofibril.The diffusion coefficients and diffusion activation energy of NaCl ions calculated by the salting experiment of grass carp fillets are basically consistent with the results of the simulated diffusion experiment mentioned above.Researches show that determining the diffusion coefficients and diffusion activation energy of NaCl ions in myofibril by combination of the molecular dynamics and the salting experiment has a higher accuracy.