Abstract
Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. Subsequent analysis of purified monomers of the used proteins showed that ATP has a direct effect on the fluorescence lifetime properties of CFP. Since the fluorescence lifetime analysis of CFP is rather complicated by the existence of different lifetimes, we tested a variant of CFP, i.e. Cerulean, as a monomer and in our FRET constructs. Surprisingly, this CFP variant shows no ATP concentration dependent changes in the fluorescence lifetime. The most important difference between CFP and Cerulean is a histidine residue at position 148. Indeed, changing this histidine in CFP into an aspartic acid results in identical fluorescence properties as observed for the Cerulean fluorescent based FRET sensor. We therefore conclude that the changes in fluorescence lifetime of CFP are affected specifically by possible electrostatic interactions of the negative charge of ATP with the positively charged histidine at position 148. Clearly, further physicochemical characterization is needed to explain the sensitivity of CFP fluorescence properties to changes in environmental (i.e. ATP concentrations) conditions.
Highlights
In a recent paper [1] we described that ATP induces changes in YFP/CFP fluorescence intensities in YFP/CFP based Fluorescence Resonance Energy Transfer (FRET) sensors
When we subsequently analyzed a range of constructs encoding sensors composed of different donor and acceptor fluorescent proteins [15] we found that the effect of ATP on the fluorescence intensity ratio’s was only observed in constructs composed of CFP-YFP based versions
We postulated that ATP could have an effect on FRET signals in CFP-YFP based sensors by quenching of the energy transfer between the two fluorophores
Summary
In a recent paper [1] we described that ATP induces changes in YFP/CFP fluorescence intensities in YFP/CFP based Fluorescence Resonance Energy Transfer (FRET) sensors. We presented this finding as a cautionary note, we pointed out that this ability can possibly serve as basis for development of a new range of genetically encoded biosensors for monitoring ATP concentrations. Tools to monitor dynamic changes in the intracellular distribution of ATP at near physiological concentrations in living cells are urgently needed. The development of Perceval, a new reporter for ATP:ADP ratios in cells was described [7]. As the authors of this work point out, this sensor has its limitations and a ratiometric sensor based on FRET would still be welcome
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