Structure-based rational design of an enhanced fluorogen-activating protein for fluorogens based on GFP chromophore

Abstract

Abstract“Fluorescence-Activating and absorption-Shifting Tag” (FAST) is a well-studied fluorogen-activating protein with high brightness and low size, able to activate a wide range of fluorogens. This makes FAST a promising target for both protein and fluorogen optimization. Here, we describe the structure-based rational design of the enhanced FAST mutants, optimized for the N871b fluorogen. Using the spatial structure of the FAST/N871b complex, NMR relaxation analysis, and computer simulations, we identify the mobile regions in the complex and suggest mutations that could stabilize both the protein and the ligand. Two of our mutants appear brighter than the wild-type FAST, and these mutants provide up to 35% enhancement for several other fluorogens of similar structure, both in vitro and in vivo. Analysis of the mutants by NMR reveals that brighter mutants demonstrate the highest stability and lowest length of intermolecular H-bonds. Computer simulations provide the structural basis for such stabilization.

Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2

“…In contrast, the region is rigid in the HBR-DOM2 complex, with some slow motions detected for the residues 18–26 (H3) and 29, which can be explained by the formation of an H3 helix. A comparison of dynamics of nanoFAST/HBR-DOM2 and FAST/N871b complexes [ 12 ] also reveals few differences. Mobile “hot spots” in both complexes include the loop between the helix H5 and strand B3 (62–63, numeration according to nanoFAST) and loops between the strands B3/B4 (72–76) and B4/B5 (86–90).…”

“…However, the recently determined NMR spatial structure of FAST in complex with one of its fluorogens [ 11 ] allowed the rational design of novel FAST variants. In particular, we found optimized proteins mcFAST-Y and mcFAST-L by structure-based rational mutagenesis [ 12 ]. These proteins are 20–30% brighter than FAST in complex with the fluorogen of a particular structural group.…”

Fluorescence lifetime multiplexing with fluorogen activating protein FAST variants

“…Investigating the 3D structure of the fluorogen-activating protein FAST and the nature of its interaction with various fluorogens, we created a series of its point mutants and found that even a single amino acid substitution frequently leads to dramatic changes in the properties of resulting complexes 27 . The fluorescence lifetime of chromophores strongly depends on the environment properties 28 , 29 , thus making the fluorogen-binding pocket (the environment for fluorogen) a perfect ground for the lifetime optimization.…”

“…In the present paper, we propose a genetically encoded system for multiplexed fluorescence lifetime microscopy based on the FAST protein. After testing diverse combinations of the FAST variants, previously engineered by a structure-guided protein design 27 , and several fluorogen candidates, we identified a set of protein-fluorogen pairs exhibiting well-distinguishable fluorescence lifetimes. These chemogenetic probes, nearly identical in size, color, and other steady-state optical properties, demonstrated effective performance in the mammalian cell expression system.…”

A multi-colour fluorogenic tag and its application in Candida albicans

“…Development of a similar system for C. albicans based on iFAST would greatly advance the research of protein-protein interactions in this species. Additionally, at least 60 versions of iFAST, such as mcFAST-L, mcFAST-Y, rFAST, gFAST, frFAST and even a smaller FAST, called nanoFAST, have been developed [28,29,35,36]. The combination palette of FAST and fluorogen is further expanded through the development of novel classes of fluorogen [31].…”