Chemical Synthesis Of Photocaged Proteins For Elucidating The Dynamic Signal Transduction Of Lymphocytes

Chemical protein synthesis is an effective tool to generate proteins that are otherwise difficult to obtain by the biological synthesis.It enables us to effect any desired change in the covalent structure of a protein for biochemical and biomedical studies.For example,chemical protein synthesis provides convenient access to site-specifically post-translationally modified（e.g.methylation,acetylation,phosphorylation and ubiquitination）proteins,protein probes sensitive to different physical stimuli（e.g.light and magnetic field）,and protein drugs.Specifically,photocaged protein probes,due to their high spatiotemporal control of protein activities,are especially suitable for the studies of dynamic cellular processes,such as cell motility and signal transduction.However,new systems are still required to further improve the efficiency of chemical protein synthesis,and to extend the application of chemically synthesized proteins in biochemical studies.In this thesis,the author will describe the newly developed highly efficient method for protein chemical synthesis,and the utility of photocaged protein probes to investigate the dynamic signal transduction of lymphocytes.An important recent advance for protein chemical synthesis is the development of one-pot ligation methods,which can significantly improve the yield and decrease the time cost during the ligation of multiple segments.Although one-pot three-segment ligation has been well studied,a robust method for one-pot more-segment（such as four segments）ligation remains undeveloped.The author developed a robust and efficient one-pot four-segment condensation method based on the newly characterized pH-sensitive N-terminal Cys protecting group-trifluoroacetamidomethyl（Tfacm）group.The practicality of this new one-pot four-segment ligation method was demonstrated in the synthesis of a plant protein Crambin,and a human chemokine,hCCL21.The one-pot method based on Tfacm represents a powerful tool to synthesize bigger and more complex proteins for biomedical studies.In addition to the method development,the author also developed well-defined systems to investigate the mechanism of chemotaxis,and the dynamic signal transduction in the initiation of lymphocyte activation.The author developed the first two-photon-activatable h CCL5,namely hCCL5**,through an efficient one-pot total chemical synthesis strategy.Combination of focused（<0.1μm2）720nm two-photon irradiation and confocal live-imaging approach,the author demonstrated at single-cell level that PI3 K was not responsible for perceiving the direction,but was nonetheless required for persistent migration over an extended period of time for CD8+ T cells.By intravital imaging,the author successfully manipulated artificial T cell positioning,for the first time,both in cutaneous tissues and lymph nodes by two-photon activation of hCCL5**.Besides,the author designed,synthesized,and selected a photocaged protein antigen(HEL-K₉₆NPE),which effectively disrupted a picomolar affinity（20 pM）antibody（HyHEL-10）-antigen（HEL）interaction with a large interface（1800?2）.Combination of total internal reflection fluorescent microscopy（TIRFM）and photoactivatable HEL-K96 NPE,the author investigated the dynamic signal transduction in the initiation of B cell activation,for example,the formation of BCR microclusters,and single cell calcium oscillation signaling.These systems highlight the potential of photoactivatable proteins for understanding and potential therapeutic manipulation of lymphocytes.