The use of fluorescent reporter proteins for the identification, localization, and quantification of target gene and protein expression in cells and tissues has revolutionized the field of microscopic imaging. Fluorescent reporter systems have permitted researchers to elucidate important cellular processes in the context of living cells and tissues, enabling deeper understanding into the dynamics of cellular function. However, while useful, fluorescent reporter systems are not without their limitations. Many reporter proteins lack thermostability and denature with prolonged exposure to laser irradiation. Additionally reporter proteins tend to be large, with the increased genetic load and bulky structure at times limiting the expression and function of the gene/protein of interest.

In a recent study, researchers generated a small extremely thermostable fluorescent reporter protein, CagFbFP. This flavin-based fluorescent protein is derived from the light, oxygen, or voltage sensing (LOV) domain of a soluble histidine kinase belonging to Chloroflexus aggregans, a thermophilic bacterium. LOV domains are highly conserved in plants, bacteria and fungi, and are known for their ability to bind flavins and act as reversible photo switches in response to UV/blue light. In fact, optimization of LOV domains led to the engineering of a fluorescent protein iLOV, which was found to be a superior reporter of plant virus infection than GFP. At just 11.6 kDa, CagFbFP is the smallest member of the family of flavin-based fluorescent proteins (FbFPs). In addition to being functional in anaerobic conditions, with a melting point of 68°C, CagFbFP is very thermostable. CagFbFP also possesses a fluorescence brightness that is higher than most comparable FbFP members, and researchers anticipate that there is potential to further enhance CagFbFP function.

Through structural characterization and molecular dynamics, the research team found that CagFbFP possessed an unusual glutamine residue, Gln148. This residue was predicted to interconvert between a "buried" and "exposed" conformations, setting it apart from other homologs such as iLOV, where the homologous Gln residue (Gln489) remains buried. While more work needs to be conducted to elucidate the mechanistic differences in signaling, this research certainly highlights the capability for CagFbFP to be used as a reporter for gene and protein expression in vivo.

Nazarenko, et al. Aedes aegypti Thermostable flavin-based fluorescent protein from Chloroflexus aggregans: a framework for ultra-high resolution structural studies. Photochemical & Photobiological Sciences (2019). doi:10.1039/c9pp00067d

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