Fluorogen-activating proteins: beyond classical fluorescent proteins

Xu, Shengnan; Hu, Hai-Yu

The FAP technology is a fluorogenic tagging approach that utilizes molecular recognition to directly activate the fluorescence of otherwise nonfluorescent small-molecule dyes (fluorogens). Selected FAPs bind TO and MG with nanomolar affinity and increase their respective green and red fluorescence by as much as thousands of fold. The fluorescence enhancement results from FAPs constraining the rapid rotation around a single bond within the chromophore (Fig. 2) 6 . The non-covalent interactions between the fluorogens and FAPs are like those of ligands and their receptors, mainly including van der Waals forces, π-effects and hydrogen bonds. Molecular recognition capabilities are largely determined by these loops of FAPs, termed complementarity determining regions (CDRs), which undergo somatic hypermutation during the immune response to generate specific high affinity binding to the antigen (Fig. 3) 20, 21 . FAPs represent a new class of fluorogen-based reporters, which provide a fluorescent tool for imaging fusion protein's location and abundance in time and space. FAP-fluorogen imaging system offers a number of distinct advantages in bio-applications: 1) unbound dye remains nonfluorescent in solution, allowing for the simple addition of dyes to the cellular media without any need for fixation or washout, a property that will enable imaging in more complicated tissue environments and live-cell imaging; 2) fluorogen binding to most FAPs occurs within seconds of addition, and can be carried out in a near physiological buffer or medium of choice. The interaction between the fluorogen and FAP is highly specific, with some FAP clones exhibiting subnanomolar affinity; 3) since FAPs are small in size (o30 kDa), they are easy to genetically engineer. The FAP technology thus allows specific fluorescent labeling of fusion proteins of interest in both living or chemically fixed cells; 4) the possibility offered to completely control the concentration of fluorogens paves the way for on-demand applications wherein fluorescence is desired only at a specific time or at a given density as exemplified with the FAPs; 5) fluorescence visualization can be spatially controlled by the appropriate choice of the membrane permeable and impermeable fluorogens, enabling one to selectively observe FAP fusion proteins inside cells, on the cell surface, or within trafficking vesicles; 6) variations of the fluorogens have been shown to produce a variety of distinct spectral and sensing properties for a given FAP, which is very useful in a variety of multicolor experiments. To sum up, the FAP-fluorogen system is a versatile, effective fluorogenic labeling strategy.