Rational Design of a Split Flavin-Based Fluorescent Reporter.

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Protein-fragment complementation assays (PCAs) are vital for studying protein-protein interactions.
• Existing split fluorescent proteins have limitations, including aerobic condition requirements, irreversible assembly, or the need for exogenous chromophores.
• Flavin-based fluorescent proteins (FbFPs) derived from light-oxygen-voltage (LOV) domains show promise as alternatives to traditional fluorescent proteins.

Purpose of the Study:

• To develop a novel split fluorescent reporter system based on a LOV domain.
• To enable protein-protein interaction studies under anaerobic conditions without exogenous fluorophores.
• To create a versatile tool for optogenetics and further development of fluorescent reporters.

Main Methods:

• Engineered CagFbFP, a thermostable FbFP from *Chloroflexus aggregans*, into a split reporter system.
• Identified and utilized specific loops (Aβ-Bβ, Eα-Fα, Hβ-Iβ) for splitting the protein.
• Fused split CagFbFP pairs to interacting proteins and assessed fluorescence in vivo in *E. coli* and human cells.

Main Results:

• Demonstrated successful reassembly and fluorescence of split CagFbFP in vivo in both bacterial and human cells.
• Observed low background fluorescence, indicating a high signal-to-noise ratio.
• Validated the system's utility for probing protein-protein interactions under anaerobic conditions.

Conclusions:

• CagFbFP serves as an effective split fluorescent reporter for protein-protein interactions.
• The developed system overcomes limitations of existing reporters, enabling anaerobic studies without external dyes.
• This work provides a foundation for advanced LOV and PAS domain-based fluorescent tools and optogenetics.