Study On Glycoprotein Synthesis Optimization And Molecular Dynamics Simulation

Glycosylation is a common post-translational modification of proteins,which can impact their stability,function,and localization.Chemical synthesis is an important approach for obtaining glycoproteins,as it enables the production of glycoproteins with well-defined structures and compositions.However,current limitations in chemical synthesis technology have hindered progress in this field.This thesis systematically optimizes the chemical synthesis of glycoproteins,using the carbohydrate binding module(CBM)of cellulase as a model molecule.The CBM was chosen due to its ease and rapidity of synthesis,as well as its representation of glycoproteins in general.The aim of the study was to construct a library of glycoforms with systematically varied glycan structures,which is expected to have a significant impact on understanding the composition-properties and structure-function relationship of glycosylation.The optimization process involved selecting the appropriate resin,determining the optimal reaction temperature,utilizing special amino acids,synthesizing difficult peptides,and optimizing the composition and proportion of cleavage cocktails.These optimized conditions facilitated the high yield and large-scale synthesis of unglycosylated CBM000,achieving a target product UV peak purity of 64.44%in approximately 11 hours with the CEM Liberty Blue peptide synthesizer.Subsequently,building upon this foundation,we also optimized the synthesis of glycosylated CBM,mainly by focusing on removing protective groups from the glycans and inhibiting βelimination side reactions during the deprotection process.This work laid a solid foundation for constructing a library of CBM glycoforms.In addition to the method combining chemical synthesis and biological research,molecular dynamics simulation methods can also make important contributions to the study of the composition-properties and structure-function relationship of glycosylation.In this thesis,the molecular dynamics simulation method was optimized and used to study the binding of different CBM glycoforms to substrates like cellohexaose and cellulose.The results indicated that glycosylation can affect substrate binding by competing or coordinating with protein hydrophobic interactions through the formation of hydrogen bonds.