Discovery of Red-Shifting Mutations in Firefly Luciferase Using High-Throughput Biochemistry.

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Photinus pyralis luciferase (FLuc) is crucial for bioluminescence imaging.
• Native FLuc emission is absorbed by biomolecules, limiting deep-tissue imaging resolution.
• Red-shifted luciferases are needed for improved imaging.
• A predictive model of protein structure and emission color is lacking.

Purpose of the Study:

• To systematically identify red-shifting mutations in FLuc using deep mutational scanning.
• To contribute to understanding the mechanism of emission color determination in FLuc.
• To facilitate the engineering of improved bioluminescence imaging probes.

Main Methods:

• Deep mutational scanning was applied to 20 functionally important amino acid positions in FLuc.
• Mutations were assessed for their effect on emission color, specifically red-shifting.
• Characterization of key mutations (N229T, T352M) was performed.

Main Results:

• Dozens of red-shifting mutations were identified, with most being novel.
• Mutations N229T and T352M resulted in unimodal emission with photons predominantly >600 nm.
• The identified mutations provide biochemical evidence for a multiple-emitter mechanism and the role of water networks.

Conclusions:

• High-throughput screening successfully identified numerous red-shifting FLuc mutations.
• These findings support a multiple-emitter mechanism and highlight the role of the binding pocket's water network.
• The generated dataset aids mechanistic understanding and engineering of FLuc for enhanced deep-tissue imaging.