Abstract
In recent years, scientists discover that proteins and metabolites are involved in many diseases. The concentration of proteins and metabolites often influence patient’s conditions. Therefore, methods which can detect concentration of proteins and metabolites are important in biology and medicine. Here we introduce two approach for the detection of proteins and metabolites, fluorescence sensor protein and fluorescence probe. Fluorescent sensor proteins offer the possibility to study the concentration of key metabolites in living cells. The approaches currently used to generate such fluorescent sensor proteins lack generality, as these sensors rely on a conformational change of a protein upon ligand binding to generate sinal. Here we devalope a strategy to overcome this limitation. We use a self-labeling protein tag to link with hCA to form SNAP-hCA. This recombinant protein was labeled with defferent synthetic moleculer containing BG group and fluorophore. In our fluorescent sensor protein, the metabolite of interest competes with labeling ligand. The fluorescence decrease, as the metabolite displaces the intramolecular ligand and makes the cyclic system open. The defference of readout can also be fitted with dose-response equation to obtain IC50. Furthermore, the modular design of our sensors provides a cyclic system that can change ligand binding protein or self-labeling protein. This adventage can let us detect defferent metabolite by aimply changing ligand binding protein in further approaches. In the second part, fluorescence probe containing a fluorophore and a ligand is designed to detact protein. Since the ligand part of fluorescence probe can specificly inhibit target protein, our probe can show a high specificity for target protein identification. Our fluorophore in this part not only has polarity sensitivity but also viscosity sensitivity. When the ligand part of our probe recognizes the binding site of target protein, the fluorophore will be closer to the binding pocket. Subsequently the fluorescent turn on, because of the restricted environment.