Backbone Cyclization of Flavin Mononucleotide-Based Fluorescent Protein Increases Fluorescence and Stability

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Flavin mononucleotide-binding proteins exhibit fluorescence but have limitations in reporter applications due to low fluorescence and thermostability.
• Existing fluorescent reporters struggle with performance under varying oxygen conditions and thermal stress.

Purpose of the Study:

• To engineer cyclic fluorescent proteins with enhanced properties for reporter applications.
• To investigate the impact of protein cyclization and linker length on fluorescence, stability, and performance in different oxygen conditions.

Main Methods:

• Incorporation of a codon-optimized fluorescent protein from Chlamydomonas reinhardtii into a redox-responsive split intein construct using two different linkers.
• Overexpression of precursor proteins in Escherichia coli SHuffle T7 strain.
• In vitro cyclization of target proteins using a reducing agent.

Main Results:

• The cyclic protein with a short linker showed enhanced fluorescence and increased thermal stability and exopeptidase resistance compared to the linear protein.
• Cyclization with a long linker (including myc-tag and protease cleavage site) resulted in slightly increased fluorescence compared to the linearized protein.
• Fluorescent E. coli cells overexpressing the cyclic reporter under oxygen-deficient conditions were brighter than those with the linear construct.

Conclusions:

• Protein cyclization significantly enhances fluorescence and stability of flavin mononucleotide-binding domain-based reporters.
• The engineered cyclic reporter demonstrates improved performance, particularly in anaerobic conditions.
• This cyclic reporter holds promise for applications in thermophilic anaerobes and other demanding environments.