Fig. 1

Different designs for real time sensors to detect molecular events. Here we use GPCR activation sensors as an example for cpGFP and protein–protein interaction sensors for the other sensors. a cpGFP: cpGFP is generated from GFP by genetically linking the original N- and C-termini with a short polypeptide linker, when the original protein is broken at specific positions (typically site 144). For GPCR sensor, cpGFP is inserted at specific locations between the transmembrane helix 5 (TM5) and 6 (TM6). Conformational changes by receptor activation cause alterations in the chromophore environment and change the fluorescence intensity. b FRET: A donor fluorophore (CFP) is fused to protein A and an acceptor fluorophore (YFP) is fused to protein B. Interaction between two proteins trigger energy transfer between CFP and YFP. Thus, violet excitation triggers yellow emission (YFP). c BRET: the donor fluorophore of the FRET technique is replaced by a luciferase, an enzyme which catalyzes luciferin oxidation to oxyluciferin, producing light emission and trigger YFP emission. d ddGFP: Weak or non-fluorescent ddGFP monomers (A and B) are separately fused to different proteins. Interaction between two proteins triggers the reversible association between monomers to form a fluorescent heterodimer