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Department of ChemistryPost-translational modification of protein is an important phenomenon in biology. Protein phosphorylation is one of post-translational modification and is responsible for signal transduction system and many kinds of cellular metabolisms. Especially, our research focused on histidine phosphorylation. Chapter 1 introduces the characteristics of histidine phosphorylation and the related research so far. Histidine phosphorylation is N-phosphorylation, which is the phosphorylation on N atom of imidazole to form P-N bond and has acid labile property. Histidine phosphorylation was observed in many biological events. The most famous histidine phosphorylation is two-component system (TCS), which is the fundamental signal transduction system in bacteria. Also, it was found in eukaryotes such as nucleoside diphosphate kinase (NDPK), protein phosphohistidine phosphatase 1 (PHPT1), histone H4 and so on. However, because of its acid-labile chemical instability, there were insufficient tools for studying histidine phosphorylation and histidine phosphorylation was rarely explored. Currently available tools for phosphohistidine are radiolabeling analysis and antibody detection, which are not suitable for continuous enzyme assays. So we try to develop new tool for phosphohistidine, continuous and available for kinetic assay. Chapter 2 describes our main research subject and progress. We devised Sox-based fluorescence sensor for phosphohistidine, which was basically a short peptide having kinase recognition domain, phosphorylation site (histidine) and Sox fluorophore. When histidine was phosphorylated, histidine phosphate group and Sox fluorophore chelated with magnesium cation and increased fluorescence by chelation-enhanced fluorescence (CHEF) effect, while no fluorescence when histidine was not phosphorylated. We designed simple peptide sensors, and then they were chemically phosphorylated and evaluated for their feasibility as phosphohistidine sensors. In chapter 3, we briefly describe the protein labeling strategy using isotope-labeled desthiobiotin tags by APEX system. Especially, we synthesized a series of isotope-labeled desthiobiotinyl tyramine tags through enzymatic decarboxylation and acid coupling reaction.ope

Specific Fluorescent Probe for Protein Histidine Phosphatase Activity

Protein histidine phosphorylation plays a vital role in cell signaling and metabolic processes, and phosphohistidine (pHis) phosphatases such as protein histidine phosphatase 1 (PHPT1) and LHPP have been linked to cancer and diabetes, making them novel drug targets and biomarkers. Unlike the case for other classes of phosphatases, further studies of PHPT1 and other pHis phosphatases have been hampered by the lack of specific activity assays in complex biological mixtures. Previous methods relying on radiolabeling are hazardous and technically laborious, and small-molecule phosphatase probes are not selective toward pHis phosphatases. To address these issues, we herein report a fluorescent probe based on chelation-enhanced fluorescence (CHEF) to continuously measure the pHis phosphatase activity of PHPT1. Our probe exhibited excellent sensitivity and specificity toward PHPT1, enabling the first specific measurement of PHPT1 activity in cell lysates. Using this probe, we also obtained more physiologically relevant kinetic parameters of PHPT1, overcoming the limitations of previously used methods