Many extracellular signals are relayed intracellularly by binding a receptor that is coupled to an intracellular heterotrimeric G protein. The stimulated G-protein coupled receptor (GPCR) activates the alpha subunit of heterotrimeric G proteins (Gα), which occurs when Gα exchanges GDP for GTP. The Gα subunit is also modulated by a proteins containing the GoLoco motif, which inhibits the exchange of GDP for GTP by the Gα subunit.
Putative inhibitors of the Gα/GoLoco interaction were screened in a FRET (Förster Resonance Energy Transfer) assay using the POLARstar® Omega. To this end, the Gαi1 subunit was labeled with CFP while the the GoLoco motif was coupled to YFP. Only in proximity of both proteins, energy transfer between the fluorophores takes place leading to a high FRET ratio.
A very good Z’ value of 0.853 in a low volume of only 60 μL shows the potential to perform the assay in 384-well format. The POLARstar® Omega proved to be a reliable and robust instrument to perform these FRET-based measurements.