Fluorogen-activating proteins: beyond classical fluorescent proteins

Xu, Shengnan; Hu, Hai-Yu

For many of the scFvs, both V H and V L domains are essential for dye binding and fluorescence, however, the analysis of other scFvs revealed that either V H or the V L domain alone is sufficient to cause the fluorogenic dye activation 19, 22 (Table 1 ). The existence of FAPs comprised of only of V H or V L domains that activate MG, such as L5, H6 and H8 FAPs, have already been demonstrated by Szent-Gyorgyi et al. 18 . In order to discover more desirable FAPs with appropriate and superior properties, such as high quantum yield and binding affinity, scFvs-based FAPs have been reconstructed and researched. For instance, the L5* FAP is a V L domain that binds MG to activate intense fluorescence, which is a leucine-to-serine point mutant (Ser89) obtained by directed evolution of L5 21 . L5* binds to MG to form a bright fluorescent complex that improves on the quantum yield of the original L5 FAP by about 5-fold (quantum yield ¼ 0.24). Another improved version of L5, the dL5** FAP (quantum yield ¼ 0.20), is a synthetic dimer of a light chain with a disulfide forming pair of cysteines in each monomer. When expressed as a monomer putatively assembled into ternary complex with MG dye, the dL5** FAP confers tighter binding while maintaining increased brightness. The V H domain alone FAP, dH6.2 FAP, is a synthetic dimer derived from a heavy chain with the second cysteine changed to alanine in each monomer 23 . This kind of FAP was normally bright but considerably less photostable across multiple compartments, which has proven useful for super-resolution imaging 22 .