DEVELOPMENT OF A RESEARCH TOOL FOR PHOSPHOHISTIDINE

Abstract

Post-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.