R103 and R115 Affinity Mutants of ATeam ATP Biosensors.

Adenosine triphosphate (ATP) varies from nanomolar to millimolar levels across the physiological landscapes in which it serves as an energy carrier, phosphate donor, and purinergic signaling molecule. To measure these vastly different concentrations, genetically encoded sensors with different affinities are needed to match the particular ATP range and application. To this end, we mutagenized two key arginine residues in the ATP-binding domain of the ATeam family of sensors to explore how charge neutralization and charge reversal affect ATP affinity. As a result, we generated an extended family of affinity mutants with apparent dissociation constants ranging from sub-micromolar to millimolar. We then carried out live-cell imaging to demonstrate the utility of different affinity mutants in detecting mild versus extreme metabolic inhibition. Overall, these sensors add to the toolbox for understanding ATP dynamics in and around cells.