Chemical Synthesis And Structural Analysis Of Di-SUMO And Their Crosslinking Analysis With Binding Proteins

Proteins play an important physiological role in life.Post-translational modifications(acylation,phosphorylation,ubiquitination,etc.)make proteins more complex and versatile.In order to conduct biophysical research on it,it is necessary to obtain a sufficient quantity of homogeneous samples.The methods of biological extraction and recombinant expression have some limitations in obtaining some functional proteins,especially post-translational modification proteins and circular proteins.Although the development of genetic incorporation of unnatural amino acid solves some key issues,the limitations of the number of unnatural amino acids that can be embedded,the limitations of sites,and the limitations of low yields make this method also have certain problem.Protein chemical synthesis technology can design and synthesize proteins on the atomic scale and obtain some important functional proteins through effective means.After decades of development,the protein chemical synthesis has undergone qualitative changes.The solid-phase peptide synthesis technology developed by Professor Merrifield simplifies the operation steps and improves the synthesis efficiency.The native chemical ligation method developed by Professor Kent has made it possible to obtain longer and complex proteins.The development of various methods including auxiliary group and peptide hydrazides have broadened the scope of application of protein chemistry for protein synthesis.From the synthesis of the first short peptides to the synthesis of various complex post-translational modification proteins,protein chemical synthesis has become a powerful tool in chemical biology research.In this paper,we used protein chemical synthesis technology to synthesize a variety of functional proteins and conducted preliminary studies on the biochemical and biophysical properties of these proteins.This thesis consists of four chapters.The first chapter briefly introduced the development of protein chemical synthesis,including the history of early protein chemical synthesis,solid phase peptide synthesis techniques,peptide fragment ligation techniques,and protein chemical synthesis applications based on these technologies.The second chapter introduces the chemical synthesis and biochemical structure of natural Di-SUM02 protein based on peptide hydrazide and auxiliary ligation strategy.First,we introduced the synthesis of natural Di-SUM02 protein.After obtaining the Di-SUM02 protein,it was determined that the synthetic Di-SUM02 protein has similar biochemical properties as the expressed protein by comparison with the linear Di-SUM02 protein in secondary structure and protein-protein interaction.Finally,we used the quasi-racemic crystallization method to try to obtain the three-dimensional structure of Di-SUM02 with different truncated bodies,and obtained a preliminary crystal.In Chapter 3,the chemical synthesis of Di-SUM02 photocrosslinking probes and their interactions between SUMO-SIM interactions are introduced.We first chemically synthesize Di-SUM02 probes containing benzophenone photocrosslinking molecules and cross-link verification experiments with known SUMO chain-interacting proteins RNF4 and RNF111.In this process,we also conducted cross-linking verification experiments under complex systems and competitive systems.Finally,we used probes to crosslink multiple interacting proteins in HeLa cell lysates.Chapter 4 introduces the chemical synthesis of linaclotide and its structure acquisition.We first chemically synthesised L-and D-type linaclotide and obtained the crystal structure of linaclotide by racemic crystallization experiments.Based on the structural information,we learned that linaclotide forms a compact,three-?-turn spatial structure through intramolecular three pairs of disulfide bonds.