Interfacial Functional Terminals Enhance The Heterogeneous Nucleation Of Lysozyme Crystals

Protein crystallization is widely used in drug design,purification,and delivery.The global market for biopharmaceuticals is recently growing rapidly.Since the crystallization effect of protein is constrained by many factors,and the study of the crystallization process is not thorough enough,so obtaining high-quality protein crystals still faces many technical difficultiesIn this article,the crystallizing conditions of proteinase K,lysozyme and papain were systematically screened and optimized.Based on experimental results,lysozyme was selected as the target protein.Taking into account the amino acid properties and chemical bond composition,we prepared a series of functional surfaces including S-PhAc,S-biPh,andS-PhCF₃withacetamidobenzene,biphenylor trifluoromethylbenzene as functional terminals and then we determined the effect of functional surfaces on lysozyme crystallization process.In hanging drop method,the obtained lysozyme crystals have the largest modal size of about 270?m on S-PhAc within 24 h and the concentration of lysozyme was 15mg/mL.As for batch method,the length of lysozyme crystals grown on S-PhAc was150²75?m,whereas on the control coverslip,the crystals appear to be somewhat aggregates with small size.The crystals obtained by batch method were characterized by Fourier transform infrared spectroscopy and X-ray powder diffraction.It is demonstrated that the functional surfaces do not cause the significant change of lysozyme structure during the crystallization process.The value of N^heteroetero decreases in the order of???illustrating that the heterogeneous nucleation is enhanced greatly.The in situ liquid-cell AFM images reflect the fact that the particle aggregation rate is greatly associated with the functional terminals at the beginning of crystallization,i.e.,the fast rate is achieved as 0.088±0.004 nm/s on S-PhAc,much higher than that on commercial siliconized square cover slides?0.025±0.005 nm/s?.When lysozyme crystals reach a certain size,functional terminals have no significant influence on the rate of theirs growth.Combining the crystallization results with the molecular modeling,it is indicated that the stronger the interactions between lysozyme loop residues and the functional terminals,the more easily the heterogeneous nucleation occurs.It is suggested that designing functional terminals targeting the feature loop residues of proteins would be a promising route to modulate protein crystallization at the early stage.