A genetically encoded fluorescent reporter of ATP:ADP ratio

Nat Methods. 2009 Feb;6(2):161-6. doi: 10.1038/nmeth.1288. Epub 2009 Jan 4.


We constructed a fluorescent sensor of adenylate nucleotides by combining a circularly permuted variant of GFP with a bacterial regulatory protein, GlnK1, from Methanococcus jannaschii. The sensor's affinity for Mg-ATP was <100 nM, as seen for other members of the bacterial PII regulator family, a surprisingly high affinity given that normal intracellular ATP concentration is in the millimolar range. ADP bound the same site of the sensor as Mg-ATP, competing with it, but produced a smaller change in fluorescence. At physiological ATP and ADP concentrations, the binding site is saturated, but competition between the two substrates causes the sensor to behave as a nearly ideal reporter of the ATP:ADP concentration ratio. This principle for sensing the ratio of two analytes by competition at a high-affinity site probably underlies the normal functioning of PII regulatory proteins. The engineered sensor, Perceval, can be used to monitor the ATP:ADP ratio during live-cell imaging.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine Diphosphate / analysis*
  • Adenosine Triphosphate / analysis*
  • Archaeal Proteins / chemistry*
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Binding Sites
  • Biosensing Techniques / methods
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Fluorescent Dyes / chemistry*
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / genetics
  • Microscopy, Fluorescence
  • Polymerase Chain Reaction


  • Archaeal Proteins
  • Bacterial Proteins
  • DNA, Bacterial
  • Fluorescent Dyes
  • Luminescent Proteins
  • yellow fluorescent protein, Bacteria
  • Adenosine Diphosphate
  • Adenosine Triphosphate