Journal
NATURE METHODS
Volume 6, Issue 2, Pages 161-166Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.1288
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Funding
- US National Institutes of Health-National Institute of Neurological Disorders and Stroke [NS029693, NS055031]
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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.
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