Abstract
As an important gasotransmitter, hydrogen sulfide (H2S) plays crucial roles in cell signaling. Incorporation of p-azidophenylalanine ( pAzF) into fluorescent proteins (FPs) via genetic code expansion has been a successful strategy in developing intensity-based, genetically encoded fluorescent biosensors for H2S. To extend this strategy for ratiometric measurement which eliminates many detection uncertainties via self-calibration at two wavelengths, we modified the chromophore of a circularly permutated, superfolder green fluorescent protein (cpsGFP) with pAzF to derive cpsGFP- pAzF, which subsequently served as a Förster resonance energy transfer (FRET) acceptor to EBFP2, an enhanced blue fluorescent protein. The resultant construct, namely, hsFRET, is the first ratiometric, genetically encoded fluorescent biosensor for H2S. Both in vitro and in mammalian cells, H2S reduces the azido functional group of hsFRET to amine, leading to an increase of FRET from EBFP2 to cpsGFP. Our results collectively demonstrated that hsFRET could be used to selectively and ratiometrically monitor H2S.