Fluorogen-activating proteins: beyond classical fluorescent proteins

Xu, Shengnan; Hu, Hai-Yu

The FAP-based fluorescence detection and quantification approach also provides a platform for high-throughput screening of receptor proteins 29 . The most successful application is the discovery of novel cystic fibrosis transmembrane conductance regulator (CFTR) F508del correctors, using FAP tagging method in the trafficking studies of CFTR [43] [44] [45] . In addition, FAPs have an enormous potential for use in flow cytometry cell surface-based assays because fluorescence can be limited to proteins that are or have recently been resident on the surface membrane. Wu et al. 46 developed a platform combining FAP technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform allows drug discovery for trafficking receptors such as GPCRs, and has been validated using the β2-AR system. They later expanded the hybrid system to a new type of biosensor, which provides the opportunity to study multiple trafficking proteins in the same cell 47 . Recently, Jarvik et al. 48 developed a novel approach based on FAPs and tethered fluorogen for visualizing regions of close apposition between the surfaces of living cells, which has the potential to provide a real-time readout of the proximity status of the membranes of the two cells. More recently, we and Brönstrup group first applied MG/FAP to study translocation efficiencies of molecular scaffolds designed to transport cargos in bacteria, which provided a general method for investigating the translocation capability of compounds across the membrane of bacterial cells (Fig. 10) 49 .