# R103 and R115 Affinity Mutants of ATeam ATP Biosensors

## TL;DR

Researchers modified ATP biosensors to measure a wide range of ATP concentrations, enabling better study of cellular energy dynamics.

## Contribution

The study introduces new ATP biosensor mutants with varied affinities for ATP, expanding their utility in live-cell imaging.

## Key findings

- Mutating arginine residues altered ATP affinity, producing sensors with dissociation constants from sub-micromolar to millimolar.
- Live-cell imaging showed the sensors can detect mild and extreme metabolic inhibition effectively.
- The new biosensors enhance the ability to study ATP dynamics in diverse physiological contexts.

## Abstract

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.