Figure 3.

Schematics of the designs of fluorescent probes to follow small molecule second messengers, protein kinase activities and membrane phospholipids. Throughout, Black arrows indicates direction of energy transfer (A) The original design of the “cameleons”, containing calmodulin (CaM) and the M13 peptide of MLCK placed between CFP and YFP. Ca²⁺ binding makes the peptide bind to CaM evoking the conformational change detected by a FRET change. This design served as template for many subsequently generated probes. (B) cAMP detection based on Epac. Here the truncated regulatory domain of Epac (Reg) binds a small peptide sequence (red) in a cAMP-dependent manner, which, in turn, relieves the inhibition by an inhibitory domain (In) of the (catalytically inactivated) guanine-nucleotide exchange domain (GEF) ⁶⁷. This rearrangement can be detected as a FRET change when the two fluorescent proteins are placed at the two ends of the reporter. (C) Ins(1,4,5)P₃ binding to the ligand binding domain of the InsP₃ receptor [that consists of helical (H) and β-trefoil (B) domains] induces a conformational change that can be read as a FRET decrease between the two fluorophores attached at each end of the domain. (D) Protein kinase activity reporters use a consensus peptide sequence (green) specific for the particular kinase and a relatively low specificity and -affinity phosphopeptide recognizing domain (yellow) paired in the form of a FRET sensor. Phosphorylation causes binding of the phosphopeptide to the binding domain inducing a conformational transition that is detected as a FRET change. More specific and higher affinity phosphopeptide binding modules tend to protect the peptide from dephosphorylation making the probes less sensitive to detect the termination of the event. (E) Changes in membrane lipids can be monitored by specific lipid binding modules fused to GFP. Because the regulatory lipids are in higher abundance than proteins, such simple probes can detect lipid changes by changing their distribution between the membranes and the cytosol. The PH domain of PLCδ1 can monitor the amounts of PtdIns(4,5)P₂ in the membrane, while the C1a domain of PKC (or some other proteins) will detect the formation of DAG. For example, during PLC activation, the PLCδ1-PH domain falls off the membrane as the amount of PtdIns(4,5)P₂ decreases and concomitantly, the PKC-C1 domain will be recruited to the membrane from the cytosol as DAG is produced. Such probes have been developed to follow changes in a number of different phosphoinositides. See text for original citations.