Genetically Encoded Unnatural Amino Acid For Probing The Biological Function Of Protein Post-translational Modification

Proteins post-translational modifications(PTMs)are ubiquitous in all living organisms and can participate in various biological processes in vivo,regulating protein structure,function,stability,cell localization and interaction.At present,more than 400 different modification types have been identified,including phosphorylation,acetylation,lipidation,methylation,ubiquitination,and glycosylation.On account of the dynamic property of proteins and the technical limitations for acquiring homogeneous proteins,the investigation of the exact biological function of specific post-translational modifications remains extremely challenging.Unnatural amino acids carrying natural modifications or their analogues can be site-specifically incorporated into the proteins of bacteria,yeast,mammalian cells or whole animals aided by genetic code expansion strategy,so that homogeneous proteins with site-selective modifications can be obtained for the analysis of physical and chemical properties.In this thesis,we genetically incorporated several electron-rich Trp derivatives in a site-specific manner into the tryptophan position of the histone reader,and we observed that the binding affinity of the reader with 6-methoxy-Trp to histone H3K4me3 increased by more than eightfold.This finding helps us to better understand the chemical mechanism of cation-π interactions,and paved the way for the design of other Trp derivatives that could further improve the binding affinity of this noncovalent interactions.Moreover,in combination with the design of multivalent readers,we further improved the binding affinity of engineered PHD domains to H3K4me3 to single-digit binding affinity for efficient detection and imaging of H3K4me3 signals.In addition,we developed a strategy for computational-aided virtual screening of lipid mimics,which then could be site-specifically incorporated into protein in E.coli and mammalian cells by genetic code expansion strategy.Next,we demonstrated that these lipid mimics could be used to enhance the binding affinity of the target protein to human serum albumin,which could be used to extend the half-life of the disease therapeutic protein in living mice.Moreover,these length-tunable lipid analogues can be incorporated into specific sites of various functional proteins to mimic natural lipidation modifications and help them anchor to membranes for localization and signal transduction.In conclusion,our strategy has enabled the precise synthesis of lipid-modified proteins in all living systems and the ability to perform gain-of-function studies of protein lipidation on hundreds of substrate proteins under different physiological and pathological conditions,which lays the foundation for the development of therapeutic drug candidates.