ATP and FRET – A cautionary note, confirmed?

6 03 2007

In the February issue of Nature Biotechnology, a correspondence piece noted some very surprising findings regarding the sensitivity of genetically-encoded fluorescence resonance energy transfer (FRET) indicators to adenosine triphosphate.

During the development of CFP-YFP based FRET reporters for adenosine nucleotides, Willemse et. al. discovered that all of their FRET constructs, including putatively non-responsive controls had a significant response to millimolar levels of ATP. Increasing levels of ATP appeared to quench the acceptor chromophore. The effect appeared specific to ATP, as 10mM ADP had no effect and 10mM GTP had a very small one. They also tested whether there was a direct effect on either the CFP or YFP and found none. Only constructs that underwent FRET showed ATP responsiveness. The authors suggested that the effect was due to “a direct quenching of the energy-transfer step coupled to energy-induced charge displacement in the phosphate groups.”

One must be very cautious in interpreting anomalous results in FRET imaging. Genetically-encoded FRET reporters have been used for over 10 years, and their specificity has been validated by dozens of high-quality imaging labs. Furthermore, a large variety of potential confounds, including pH shifts, osmotic effects, non-linear photo-bleaching, protein precipitation and secondary effectors, must be carefully accounted for when doing FRET experiments. However, fluorescent imaging in cell biology is a rapidly developing field, and the discovery of surprising photophysics and photochemical effects is not uncommon. Therefore, I took a rather skeptical, but open-minded approach to the published results and interpretation. I needed to see for myself.

Effect of ATP on GluSnFR emission

I performed the following experiment. 5ul of 100uM pure, soluble GluSnFR (a custom genetically-encoded FRET reporter for glutamate) was diluted into 3mL of HBSS containing 0, 3.3 or 10mM ATP. pH was balanced to exactly 7.35 in each solution. The emission spectra of the solutions were then measured on a SPEX spectrophotometer with excitation set to 420nm, selective to CFP excitation. To my surprise, there was a strong effect of increasing ATP concentration on the emission spectra of the FRET construct. YFP/CFP changed from 2.12 @ 0mM ATP to 1.87 @ 3.3mM to 1.51 @10mM. There was a suprisingly small increase in the donor emission @ 474nm, perhaps reduced due to slight variations in GluSnFR concentration. Although I did not perform several possible control experiments, this data does corroborate the findings of the authors.

The authors interpretation of a quenching of the energy-transfer step seems to be a novel mechanism not supported by my understanding of FRET theory.  If the mechanism were siphoning off FRET to a quenched acceptor (ATP), then there should be a direct effect on CFP upon ATP addition.  In the supplemental figures, the authors show there is not. My expectation for mechanism would be a direct effect of ATP on the conformation of the sensor that does not depend on the linker. The presence of CFP and YFP is common between all the sensors tested.  Perhaps ATP reduces the tendency of CFP and YFP to dimerize, hence the time-averaged FRET ratios are reduced. Does this effect persist after subsitution of other variants of CFP and YFP? Certainly, more technical work needs to be done to nail down the mechanism of the effect.

Why is this finding important to the field of neuroscience? Average ATP concentrations in a neuron fluctuate between approximately 0.8 and 1.4mM, although some neurons may reach as high as 5mM. Slow changes of these levels are not likely to have a significant impact on the readout of intracellular FRET reporters in neurons. However, the authors make the point that subcellular compartments of neurons may have ATP levels as high as 20mM which undergo more dynamic fluctuations. If this is the case, and if the generality of the quenching effect holds up to future scrutiny, then FRET reporters targeted to these compartments may have their readout significantly confounded by changes in ATP levels. Development of ATP-specific sensors genetically targeted to these compartments of potentially high ATP flux will provide essential insight to the impact these findings will have on the general field of cellular FRET imaging.



One response

13 06 2010
Etta Talib

Another Good wordpress post, I will be sure to bookmark this post in my Newsvine account. Have a awesome day.

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