A thermostable flavin-based fluorescent protein from Chloroflexus aggregans: a framework for ultra-high resolution...

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Light-Oxygen-Voltage (LOV) domains are blue-light sensing proteins found in plants, bacteria, and fungi.
• Flavin-based fluorescent proteins (FbFPs) derived from LOV domains are valuable for real-time biological process monitoring.
• Existing FbFPs offer advantages like fast folding and small size, but stability can be a limitation.

Purpose of the Study:

• To characterize a novel, thermostable flavin-based fluorescent protein, CagFbFP, derived from a thermophilic bacterium.
• To assess CagFbFP's potential as a reporter protein and for ultra-high resolution structural studies.
• To investigate the structural dynamics of key residues within the LOV domain.

Main Methods:

• Protein expression and purification of CagFbFP from Chloroflexus aggregans.
• Thermal stability assays (melting point determination).
• X-ray crystallography and small-angle scattering for structural analysis.
• Molecular dynamics simulations to study residue conformations.

Main Results:

• CagFbFP is a small (11.6 kDa), thermostable (Tm ~68 °C) dimeric protein composed of a conserved LOV core domain.
• The protein crystallizes readily and diffracts to high resolution (1.07 Å).
• Key residue Gln148 was observed in two rapidly interconverting conformations, confirmed by crystallography and simulations.

Conclusions:

• CagFbFP is a highly stable and easily crystallizable LOV-derived fluorescent protein.
• Its properties make it a promising candidate for advanced structural investigations of LOV domains.
• CagFbFP holds potential for applications as a robust fluorescent reporter in various biological systems.