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

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. Structural analyses guide the rational design of mutant fluorogen-activating proteins with enhanced fluorescence.

Automated summary

This study describes the rational design of enhanced FAST mutants optimized for the N871b fluorogen. Through structural analysis, NMR relaxation analysis, and computer simulations, the study identifies mobile regions in the complex and suggests mutations that could stabilize both the protein and the ligand. The experimental results show that the mutants exhibit higher brightness and provide up to 35% enhancement for several other fluorogens of similar structure, both in vitro and in vivo.