Recently, Brain Windows mentioned the report A genetically encoded fluorescent reporter of ATP:ADP ratio. We invited Dr. Jim Berg, the lead author of the study to provide additional background to our readers. Below, Jim provides a fascinating look at rationale behind sensor development. I really like that they came at this problem with a biological question in mind, something I would recommend before anyone start the development of a genetically encoded indicator.
A pixel-by-pixel ratio of the 490 nm excitation image by the 430 nm excitation image from two cultured HEK293 cells expressing Perceval during control conditions (left) and after 40 min of metabolic inhibition with 5 mM 2-deoxyglucose (right)
Here’s a little insight into why we decided to develop a fluorescent sensor for cellular energy, and how Perceval evolved. One of the primary research interests of the Yellen lab is the interaction between diet and epilepsy. The ketogenic diet, a high fat, low carbohydrate regimen, is remarkably effective at reducing seizure number. We are investigating how the transition in brain metabolism from glucose to a mixture of glucose and ketone bodies (the metabolically active byproduct of fat metabolism) could lead to a change in neuronal excitability. Previously, we described how acute application of ketone bodies reduces the excitability of substantia nigra neurons, an effect that relies on the opening of ATP-sensitive potassium (KATP) channels. Our hypothesis is that the inhibition of glycolysis by ketone body metabolism leads to a reduction in sub-membrane ATP, resulting in an opening of KATP channels and a decrease in neuronal excitability. This relies on the controversial idea that sub-membrane ATP is provided by glycolysis (possibly by glycolytic enzymes tethered to the membrane), and that the diffusion of ATP is restricted between the submembrane space and bulk cytoplasm, and concept known as “compartmentation”. To fully test this hypothesis, we required an optical sensor for ATP levels.
When planning these experiments, our first thought was to use Luciferase to detect different subcellular ATP levels. For a number of reasons, primarily Luciferase’s weak signal, we decided that a fluorescent sensor for ATP would be much more useful for our application. Our initial approach was a FRET-based design, with CFP and YFP tethered to a bacterial periplasmic binding protein that dimerized upon ATP application. Although these sensors gave some encouraging results, we never got the change in signal that would be required for cellular assays. We then adopted the ‘circularly permuted fluorescent protein (cpFP) approach that had previously produced sensors for calcium (pericam) and hydrogen peroxide (HyPer). We inserted the yellow fluorescent protein cpmVenus into the loop of the bacterial ATP binding protein, GlnK1 (involved in the regulation of ammonia transport) and found that application of small amounts of ATP to the purified sensor led to a substantial change in the excitation spectrum of the sensor. The affinity of the sensor for ATP was extremely high, orders of magnitude more sensitive than would be appropriate for cellular assays. We also found that our sensor responded to ADP application, only with a much smaller fluorescence change. It was then that we determined that these two perceived negatives (too high affinity and ADP binding) would lead to a sensor that reports the ratio of ATP to ADP. In a bit of good fortune, our design for an ATP sensor had in fact given us a sensor for the more valuable ATP:ADP ratio. After tinkering with our sensor by semirandom mutagenesis of the GlnK1 portion of the protein, we expressed the improved sensor, which we named Perceval (for permuted reporter of cellular energy value) into cultured cells and monitored a change in fluorescence with metabolic inhibition.
Right now, we are excited to use Perceval to investigate neuronal/glial metabolism in mammals. We may target subcellular ATP by either tethering Perceval to a membrane protein, or by using TIRF microscopy. In addition, we are continuing to design improved versions of Perceval, as well as sensors for other metabolic intermediates. We also hope that these sensors will be useful in applications beyond neuronal metabolism, from studies of cancer cells to bacterial metabolism.
Brain Windows 
NIce to hear the backstory of something we published. I’m usually more likely to hear this side of story for papers published elsewhere that we cover in a research highlight.