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Researchers developed new adenosine triphosphate (ATP) sensors with varying affinities to measure its wide concentration range. These tools help study cellular energy and signaling in different physiological conditions.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Background:

  • Adenosine triphosphate (ATP) is crucial for cellular energy, phosphate transfer, and purinergic signaling, with concentrations varying widely from nanomolar to millimolar.
  • Accurate measurement of diverse ATP levels requires genetically encoded sensors tailored to specific affinities and applications.
  • Existing sensors may not cover the full physiological range of ATP concentrations, limiting comprehensive analysis.

Purpose of the Study:

  • To engineer a new family of adenosine triphosphate (ATP) sensors with a broad spectrum of affinities.
  • To investigate the impact of specific amino acid mutations on ATP sensor affinity.
  • To validate the utility of these novel sensors in live-cell imaging for metabolic studies.

Main Methods:

  • Site-directed mutagenesis of key arginine residues within the ATP-binding domain of ATeam sensors.
  • Characterization of ATP binding affinity for engineered sensor variants, determining apparent dissociation constants (Kd).
  • Live-cell imaging experiments to assess sensor performance under conditions of metabolic stress.

Main Results:

  • Generation of an extended ATeam sensor family with affinities spanning sub-micromolar to millimolar ranges.
  • Demonstration that charge neutralization and reversal at specific arginine residues significantly alter ATP binding affinity.
  • Successful application of diverse affinity sensors to distinguish between mild and severe metabolic inhibition in living cells.

Conclusions:

  • The engineered ATP sensors provide a versatile toolkit for measuring a wide range of intracellular and extracellular ATP concentrations.
  • These new sensors enhance the ability to study ATP dynamics and cellular responses to metabolic challenges.
  • The findings offer improved methods for investigating purinergic signaling and cellular bioenergetics.