Development of a Chemoproteomic Phosphohistidine Probe and Identification of Phosphohistidine Regulated Proteins

Abstract

Protein post-translational modifications (PTMs) and small-molecule metabolites serve as fundamental mechanisms for dynamically regulating protein structure, activity, and signaling across biological systems. Among the diverse PTMs, histidine phosphorylation (pHis) is a chemically labile and historically underexplored posttranslational modification due to the intrinsic instability of its phosphoramidate bond. To overcome the limitations of conventional proteomics, a chemoproteomic platform was established using a stable pyrazole-based τ-pHis analog (pPyp-BP) conjugated to a photocrosslinker and alkyne handle for visualization and enrichment of labeled proteins. This probe enabled selective covalent labeling and enrichment of pHis-recognizing proteins under native conditions. Application to Escherichia coli lysates revealed 13 high-confidence candidate pHis acceptors, many of which participate in central carbon metabolism. Comprehensive biochemical validation demonstrated distinct regulatory behaviors among these targets. Phosphofructokinase (PfkA) was identified as a bona fide pHis-regulated enzyme. His249 phosphorylation by the phosphotransferase system, PtsI–PtsH cascade, and dephosphorylation by phosphatase SixA establish a reversible signaling axis that couples carbon source availability to glycolytic flux. In contrast, phosphoglucomutase (GlmM) and citrate synthase (GltA) showed probe reactivity but provided limited evidence for functional pHis regulation in vivo. Pyruvate kinase II (PykA) was phosphorylated at the previously unannotated His41, a residue essential for catalytic activity, although its phosphorylation appeared independent of the phosphotransferase system, indicating an alternative upstream regulator. In parallel, this thesis explored whether endogenous metabolites could drive covalent protein modification. Ascorbic acid- and melatonin-derived chemical probes were designed to mimic reactive metabolites. While melatonin- and AMK-based alkyne probes were successfully synthesized, neither melatonin nor AMK probes produced specific protein labeling in cellular or lysate experiments. Ascorbic acid-based probe synthesis was hindered by instability during key coupling steps, preventing downstream biological evaluation. These findings highlight the need for systematic interrogation of the biological conditions under which metabolite-derived covalent modifications occur, as well as the development of chemically rigorous and synthetically reliable strategies to enable metabolite-based probe synthesis. Together, this work provides an integrated chemical biology framework for investigating two complementary modes of protein regulation, reversible pHis-dependent signaling and potential metabolite-driven covalent modification. The chemoproteomic strategy developed here expands the toolkit available for studying labile PTMs and uncovers a previously uncharacterized pHis regulatory mechanism governing bacterial glycolysis. In addition, the exploratory metabolite-probe efforts outline foundational steps toward mapping covalent interactions between cellular metabolites and protein targets. This thesis underscores the value of chemical approaches in revealing hidden layers of metabolic and signaling regulation across biological systems.