Aggregation Behavior And Crystallization Of Lysozyme Under The Modulation Of Small Organic Molecules

The abnormal aggregation and crystallization behavior of proteins in the human body are closely related to the occurrence of certain diseases.Exploring the interaction between small organic molecules with different structural characteristics and protein molecules,and their influence on the aggregation and crystallization behavior of protein molecules,which is important for in-depth understanding of protein-related behaviors and have guiding significance for the prevention and treatment of diseases.In this paper,Hen egg white lysozyme(HEWL)was selected as the model protein to study the effects of several organic small molecules,such as fluorinated pesticides,chiral drugs,neurotransmitter-dopamine,on the aggregation and crystallization properties of HEWL,the mechanism of protein conformation regulation,aggregation and crystal nucleation influenced by organic molecules was investigated.The research results have certain guiding significance for revealing protein structure and corresponding biological functions.The main research work in the paper includes:Firstly,the effects of two fluorinated pesticides,chlorfenapyr(CHL)and picoxystrobin(PIC),on the aggregation and crystallization of HEWL were investigated.The study found that at low concentrations(30 μM)of CHL or PIC,the nucleation of HEWL crystals were promoted,while at high concentrations(100 μM)of CHL or PIC,both crystalline phase and amorphous precipitate were produced in the drops;The aggregation rates of protein in solution were promoted to varying degrees by CHL and PIC,the difference is closely related to the binding mode of the two pesticides and HEWL as well as the changes in HEWL conformation.Molecular simulations suggested that HEWL and CHL are dominated by non-polar interactions,resulting in a larger hydrophobic surface area for protein molecules,while HEWL and PIC form more stable hydrogen bonds.These results suggest that small amounts of pesticides can provide interactions strong enough to influence the aggregate and nucleation of protein,which would affect the competing formation of protein crystals against the amorphous fiber-like precipitate.Secondly,the effects of chiral drug enantiomers,S-ibuprofen(SIBP)and Ribuprofen(RIBP)on the aggregation and crystallization of HEWL were explored.The study found that low concentration(30 μM)of SIBP or RIBP favored the nucleation of HEWL,while the nucleation number of lysozyme crystals decreased significantly at high concentration(100 μM)of SIBP or RIBP,but was still higher than the control group.SIBP and RIBP increased the aggregation rate of proteins in solution to different degrees,especially in the presence of SIBP,the aggregation rate of proteins was faster.Molecular simulation found that SIBP and RIBP had different effects on the conformation of lysozyme in solution,and the binding of the chiral enantiomer of ibuprofen to HEWL was stereoselective,which caused different effects on protein aggregation and crystallization.Finally,the effects of dopamine(DA)on the crystallization behavior of HEWL was investigated.The study found that DA increased the solubility of HEWL and promoted the nucleation of HEWL crystals in solution.The shape of HEWL crystals changed from bulk to rod after DA addition.The growth rate of(101)plane decreased from 3.9 nm/s to 3.1 nm/s while the growth rate(110)plane decreased significantly from 4.1 nm/s to 1.8 nm/s with the increase of DA content.Molecular dynamics simulations showed that the hydrogen bonding and hydrophobic interactions between DA and HEWL were mainly concentrated in the pocket region of protein HEWL,which regulated the assembly behavior of HEWL,resulting in anisotropic growth of HEWL crystals.These results suggest that small organic molecules in vivo are an important factor in modulating protein crystal morphology by adjusting the conformational structure of protein molecules,and affect the biological function of proteins.