FMN-binding fluorescent protein

Upon illumination with blue light, LOV-domains undergo a photocyle, during which a covalent bond is formed between a conserved cysteine-residue and the FMN.

[3][4][5] In order to make better use of the fluorescence properties of these proteins, the natural photocycle of these LOV-domains was abolished by exchanging the conserved cysteine against an alanine on a genetic level.

The difference to free FMN is even more significant in the case of the photostabaility, the proteins resistance to bleach out during prolonged and intense irradiation with blue light.

Based on the bleaching-halftime (the times it takes to reduce the initial fluorescence intensity to 50% upon illumination) the genetically engineered variant phiLOV2.1[2] is approximately 40x as stable as free FMN.

Since all GFP derivatives and homologues require molecular oxygen for the maturation of their chromophore, these fluorescent proteins are of limited use under anaerobic or hypoxic conditions.

[13] Since FbFPs bind FMN as chromophore, which is synthesized independently of molecular oxygen, their fluorescence signal does not differ between aerobic and anaerobic conditions.

[6] Due to their extraordinary long average fluorescence lifetime of up to 5.7 ns they are also very well suited for the use as donor domains in FRET systems in conjunction with e.g. YFP (see photophysical properties).

A fusion of EcFbFP and YFP was e.g. used to develop the first genetically encoded fluorescence biosensor for oxygen (FluBO)[15] The main disadvantage compared to GFP variants is their lower brightness (the product of ε and Φ).