Molecular Dynamics Simulation Study Of The Effect Of PH And Ca²⁺ On The Structure And Dimerization Of αs1-Casein

Casein micelles,as a novel drug-loaded material,can self-assemble with insoluble macromolecular drugs to form micelle structures.This structure can increase drug solubility,protect drugs from enzymatic degradation and improves the bioavailability of drugs,also has the functions of targeting and controlled release.It is important to find out the structure of casein and the molecular mechanism of micelle formation for the further design of better casein micelle-related drug-loaded materials.However,the structure of casein and casein micelles and the molecular mechanism of micelle formation are remain unclear.Moreover,αs1-casein that the core component of the micelles structure is flexible,and the experimental method is difficult to determine the three-dimensional structure of casein.At the same time,external conditions such as pH and ionic strength will affect the formation of aggregates,which makes the molecular mechanism of casein micelle formation unknown.Compared with traditional experimental methods,the structure of casein and the molecular mechanism of micelle formation are discussed by means of homology modeling,molecular docking and molecular dynamics simulation.The detailed research includes the following three aspects.In the first part,αs1-casein monomer and dimer structure was constructed and optimized.Homologous modeling technique was used to predict the three-dimensional structure of the most importantαs1-casein monomer in the casein micelle structure.The dimer structure ofαs1-casein was constructed by protein-protein docking method.Then the obtained structure was optimized by molecular dynamics simulation.The obtainedαs1-casein monomers were evaluated by ramachandran plot.The structure ofαs1-casein dimers was determined by binding free energy calculation and protein binding interface analysis.This work solves the problem that the experimental method is difficult to obtain the spatial structure ofαs1-casein and provides a structural basis for further study of the effects of environmental factors on the structure ofαs1-casein and the initial micelle formation process.In the second part,the effect of pH and Ca²⁺on the monomer structure ofαs1-casein was studied by molecular dynamics simulation.We performed 300 ns molecular dynamics simulations of twoαs1-casein monomers under pH=6 and 7conditions and four systems incorporating gradient Ca²⁺concentrations,respectively.At pH=6,the spatial inversion of the residue 143-160 region resulted in the increase of structural flexibility.And it turns out pH=6:00,The residue 143-160 region is spatially flipped,resulting in increased structural flexibility.h8 n（δ）protonation,leading to the disappearance of hydrogen bonds and van der Waals interactions with the q108.To investigate the mechanism of pH and Ca²⁺on the structure ofαs1-casein,300 ns of molecular dynamics simulations were performed on twoαs1-casein monomers under the conditions of pH=6 and pH=7 and four systems adding gradient Ca²⁺concentrations,respectively.The results shown that the region of residue143-160 was flipped in space at pH=6,which resulted in the increase of structural flexibility.H8 resulted in the disappearance of hydrogen bonds and van der waals interactions with Q108 due to N（δ）protonation.Moreover,new interactions are formed with L11,increasing the distance between L11 and E14、V15.Meanwhile,it promotes the formation of new interactions between H121（HIP）and residues such as E4、V15,which increases theβ-sheet conversion to turn,structural flexibility.The interaction between S（P）46 and E69、S（P）67 and E110、S（P）68 and E117 disappears for the system added with Ca²⁺due to the chelation of serine phosphate,which increases the structural flexibility of the protein,changes the surface structure of casein and forms a spatial structure conducive to complexing more Ca²⁺,which explains the principle of casein delivery of minerals.The results of this work explain the mechanism of pH=6 and Ca²⁺onαs1-casein monomer structure and provide a theoretical basis for further exploring pH=6 and Ca²⁺on casein aggregation.In the third part,the molecular mechanism of the effect of pH and Ca²⁺on the dimerization process ofαs1-casein was studied based on molecular dynamics simulation.Through the simulation calculation and analysis of 500 ns of the four research systems,it is found that under the acidic conditions of pH=6,H8（HIP）directly increases the interaction with B_E47,B_S48,B_T49,B_Q52 and B_Y166,and H128（HIP）indirectly increases the interaction between H128-E148 of A chain and R22-A26 of B chain.Thus,it is beneficial to the stability ofαs1-casein dimer structure.The addition of Ca²⁺transforms the structure of residues around phosphoserine from helix andβstructures to coil and turn structures,forming conformations that favor dimer formation.And thus,based on the originally spontaneously formed interaction,indirectly increased the interaction of H4 with LD157,A_L142 with B_Y154,A_A143 with B_D157,A_E148 with B_R151,A_L149 with B_Y154,A_S191 with B_Y144,B_E148,A_T194 with B_S178 and A_T195 with B_S180 in the binding interface,so that the solvent accessible surface area was reduced,and theαs1-casein dimer was more stable.This work explains the molecular mechanism of the formation ofαs1-casein dimers from the molecular level and provides a reference for the study of casein micelle structure and aggregation mechanism.In summary,this paper constructs a three-dimensional structure ofαs1-casein monomers and dimers,and explains the molecular mechanism of the effects of pH and Ca²⁺on the structure ofαs1-casein monomers and the dimerization from the molecular level.The results provide a reference for the further study of casein micelle structure and micelle formation,and provide valuable theoretical guidance for the design of casein micelle related drug loading materials with excellent performance.