title: A genetically encoded fluorescent reporter of ATP:ADP ratio.
abstract: 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.
authors.affiliation: Department of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, Massachusetts 02115, USA.
authors.forename: Jim
authors.initials: J
authors.lastname: Berg
authors.forename: Yin Pun
authors.initials: YP
authors.lastname: Hung
authors.forename: Gary
authors.initials: G
authors.lastname: Yellen
mesh_terms: D000244:Adenosine Diphosphate / Q000032:analysis*; D000255:Adenosine Triphosphate / Q000032:analysis*; D019843:Archaeal Proteins / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate