Fluorescent, Protein-Based Sensors for ADP

ATP conversion to ADP is a central process in all living organisms and is catalyzed by a vast number of different enzymes. The energy generated can drive metabolic processes, directed transport, force-generation and movement as well as signal transduction and regulation. While ATPases generate ADP and free phosphate, kinases transfer the terminal phosphate of ATP to a wide variety of substrates, from metabolic intermediates to proteins, so controlling their activity. Hence, assays to monitor ADP concentrations have wide applications in biochemical and biomedical research, ranging from detailed understanding of mechanochemical coupling in motor proteins to screening for ATPase and kinase inhibitors.Fluorescent, protein-based biosensors have been reported for a number of biomolecules such as sugars, amino acids, metal ions and phosphate. This approach takes advantage of the highly specific interaction of a protein with the target molecule, which can be coupled to an optical signal by attaching fluorophores in suitable positions on the protein. Following this strategy, we have developed sensors for ADP based on fluorescently labelled mutants of the bacterial actin homologue ParM. We report two ADP sensors with distinct optical properties and ADP-binding characteristics, suitable for different types of in vitro assays. A coumarin-labeled variant binds ADP tightly and fast and can detect submicromolar concentrations of ADP. The sensor is particularly useful for mechanistic studies, where high sensitivity and high time resolution are required. The second variant is labeled with two rhodamine dyes, exploiting the stacking of rhodamines to generate a signal change. This variant has a more photostable fluorophore, higher wavelength excitation and lower ADP binding affinity, making it more suitable for high-throughput screening assays.