Random Mutagenesis-Based Optimization of the Fluorogen-Activating Protein picoFast

Objective: Libraries of mutant variants of fluorogen-activating protein picoFAST were constructed and screened for improved brightness of [protein-HBR-DOM2] complex. Methods: Random mutagenesis of picoFAST by error-prone PCR was done. The screening of mutant variant was conducted. Selected picoFAST variants were sequenced and result was analyzed. New round of saturation mutagenesis was done and obtained colonies were screened. Results and Discussion: We constructed mutant libraries of picoFAST through optimized random mutagenesis protocols, achieving an average of 3.75 nucleotide substitutions per picoFAST coding sequence. Screening of approximately 1.5 × 104 bacterial colonies expressing mutant variants yielded 10 picoFAST mutants exhibiting high brightness. Structural predictions using AlphaFold indicated that substitutions localized in the N- and C-terminal β-strands modulate the fluorogen-binding pocket conformation and fluorescence activation, providing insights for rational design and targeted mutagenesis to further improve fluorogenic performance. Conclusions: We obtained and analyzed libraries of mutant forms of the fluorogen-activating protein. We have outlined promising directions for further enhancement of the smallest fluorogen-activating protein currently known. The improved picoFAST variant could be useful for visualizing tag-sensitive proteins and densely packed cellular structures.