Here we describe the development of a single fluorescent protein (FP)-based cGMP indicator, Green cGull, based on the cGMP binding domain from mouse phosphodiesterase 5α. The dynamic range of Green cGull was enhanced to a 7.5-fold fluorescence change upon cGMP binding by optimization of the amino acid linkers between the cGMP binding domain and FP. Green cGull has excitation and emission peaks at 498 and 522 nm, respectively, and specifically responds to cGMP in a dose-dependent manner. Live cell imaging analysis revealed that addition of a nitric oxide (NO) donor induced different cGMP kinetics and was cell-type dependent. We also found that the NO donor induced an increase of intracellular cGMP, while intracellular Ca2+ exhibited a complex profile, as revealed by dual-color imaging of cGMP and Ca2+. The results suggest that Green cGull sheds new light on understanding the complex interactions between various signaling molecules by multicolor imaging and that our systematic strategy for expanding the dynamic range of single-FP-based indicators is valuable to generate indicators for molecules of interest.
Keywords: biosensors; cGMP; fluorescent protein; live cell imaging; mutant screening.