Modifications Constructions And Functional Research For Phosphoarginine Related Peptides And Protiens

Protein arginine phosphorylation(pArg)is a novel post-translational modification.In gram-positive bacteria,pArg proteins are involved in almost all aspects of physiological processes,such as regulating gene transcription and controlling protein turnover.While pArg proteins mainly participate in physiological processes that related to nucleic acid in Jurkat cells of human acute T-cell leukemia,for example,RNA splicing and nucleic acid binding.Therefore,pArg is of significance in orgnisms.Recently,although some progress has been made in the study of pArg,its own chemical properties are still the decisive factor of the progress.Acid instability and thermal sensitivity of P-N bond in pArg make it difficult to obtain pArg samples,which results in the difficulty in producing of pArg antibody and impedes the functional research of pArg proteins.Thus,in this thesis,preparation of samples containing stable pArg analogues and functional research of pArg-related peptides and proteins are simultaneously proceeded.The research results are as follows:(1)Thiol of Cys and dehydrogenation alanine(Dha)conjugate with stable pArg analogues(pAIE,pBIE,pCIE,pTIE)through exchange reaction of disulfide bond,Michael addition reaction and radical addition reaction,which for the first time siteselectively introduce four kinds of pArg analogues on the polypeptide and CtsR protein.The conjugation strategies provides the research basis for the development of sequence dependent pArg-antibody and monoclonal antibody,even for the study of pArg protein function;additionally,the site-selective introduction of ?-thiolysine achieves the bidirectional introduction.(2)N-terminal acetylated polypeptide Ac-KRGGGGYIKIIKV(PR2)can bind to 7re-dsDNA that is the seven-base sequence repeat unit in clpC operon with the same magnitude 107 M-1 of binding constant to the binding of CtsR and DNA,it shows that PR2 serves as a potential inhibitor to hold back the interactions between CtsR and DNA.Futhermore,pArg regulates the interactions of PR2 with 7re-dsDNA,it indicates that pArg may play important role in regulating gene transcription.(3)Protein arginine phosphatase YwlE negatively regulates the level of pArg,and its phosphatase activity is closely related to cell signalling transduction.In vitro,the activity of YwlE is discriminatively impacted by metal ions,which are natively in Bacillus subtilis.The results lay a foundation for the study of the relationship between metal ions and YwlE in Bacillus subtilis.Moreover,the study on the effect of oxyanions on the activity of YwlE can provide ideas for the development of its inhibitors.Thereby,the thesis has established a reliable basis for exploring the significant role of pArg in cell signaling pathway and expanded the understanding of pArg.