Figure 1.

Schematic of FluoSTEP

To measure the signaling activities around a protein of interest (POI) expressed at endogenous levels, the POI is tagged with GFP₁₁ via CRISPR/Cas9 gene editing (component 1). In the presence of a GFP_1-10-containing FluoSTEP (component 2), GFP will then reconstitute and become fluorescent. The reconstituted GFP functions as a FRET donor, and FRET efficiency will be modulated by signaling based on the conformation of the signal-specific sensing domain contained within the probe. In this example, a FluoSTEP Indicator of cAMP Using Epac (FluoSTEP-ICUE) contains a fragment of the cAMP-binding guanine-nucleotide exchange factor Epac1 sandwiched between GFP_1-10 and RFP. The binding of cAMP to the Epac1 domain induces a conformation change that alters the distance and orientation between the fluorescent proteins, leading to a FRET change. Monitoring cAMP dynamics next to the POI allows interrogation of dynamic regulation and functional behaviors of the POI.