Chromophorylation of a Novel Cyanobacteriochrome GAF Domain from Spirulina and Its Response to Copper Ions

Abstract

Cyanobacteriochromes (CBCRs) are phytochrome-related photoreceptor proteins in cyanobacteria and cover a wide spectral range from ultraviolet to far-red. A single GAF domain that they contain can bind bilin(s) autocatalytically via heterologous recombination and then fluoresce, with potential applications as biomarkers and biosensors. Here, we report that a novel red/green CBCR GAF domain, SPI1085g2 from Spirulina subsalsa, covalently binds both phycocyanobilin (PCB) and phycoerythrobilin (PEB). The PCB-binding GAF domain exhibited canonical red/green photoconversion with weak fluorescence emission. However, the PEB-binding GAF domain, SPI1085g2-PEB, exhibited an intense orange fluorescence (λ abs. max = 520 nm, λ fluor. max = 555 nm), with a fluorescence quantum yield close to 1.0. The fluorescence of SPI1085g2-PEB was selectively and instantaneously quenched by copper ions in a concentration-dependent manner and exhibited reversibility upon treatment with the metal chelator EDTA. This study identified a novel PEB-binding cyanobacteriochrome-based fluorescent protein with the highest quantum yield reported to date and suggests its potential as a biosensor for the rapid detection of copper ions.

Transition Metals Induce Quenching of Monomeric Near-Infrared Fluorescent Proteins

“…This technique takes advantage of the fact that zinc forms a complex with the protein-linked bilin chromophore and fluoresces in the red spectral region, but applications of this technique typically use denatured proteins. One report of a cyanobacteriochrome bound to phycoerythrobilin reported zinc-induced fluorescence for the unfolded protein but no change in fluorescence for the folded protein . There is also a mutant GFP, BFPms1, where Zn 2+ can access the chromophore through a solvent channel and bind directly, enhancing the fluorescence (Cu 2+ also binds and quenches the fluorescence) .…”

“…One report of a cyanobacteriochrome bound to phycoerythrobilin reported zinc-induced fluorescence for the unfolded protein but no change in fluorescence for the folded protein. 55 There is also a mutant GFP, BFPms1, where Zn 2+ can access the chromophore through a solvent channel and bind directly, enhancing the fluorescence (Cu 2+ also binds and quenches the fluorescence). 56 Here, we investigated how several metal ions affect the fluorescence of each miRFP (Figure 1B,C).…”

Zinc-Induced Fluorescence Turn-On in Native and Mutant Phycoerythrobilin-Binding Orange Fluorescent Proteins

“…For example, while the folded GAF-PEB protein from S. subsalsa does not undergo any changes in fluorescence in the presence of Zn 2+ , the denatured protein fluoresces. 29 To determine whether Zn 2+ may be binding directly to the bound PEB/PUB cofactor embedded within All1280g2, we compared the UV−visible absorption spectra obtained in the presence and absence of zinc (Figure 3). We expect that metal binding to the bilin cofactor would change the absorption spectrum, as observed for some metals that bind free bilins, including Zn 2+ binding to free PEB.…”

“…To overcome this obstacle, the non-photoswitching phycoerythrobilin (PEB) cofactor can be used. PEB is identical to PCB except it lacks the Δ15,16 double bond between pyrrole ring C and D, and when bound to a variety of CBCRs results in fluorescent proteins with high quantum yields and molar extinction coefficients. , These CBCR GAFs can be heterologously coexpressed in E. coli with the biosynthesis enzymes for PEB production from a heme precursor (heme oxygenase and phycoerythrobilin synthase, PebS). – Among the CBCR GAFs tested for PEB binding, All1280g2 is one of the brightest PEB-binding CBCRs (Φ F = 0.68, ε 495/546 = 49,000/24,000 M –1 cm –1 ) and shows absorption maxima that could make it suitable as an acceptor in Förster resonance energy transfer (FRET)-based platforms utilizing donor proteins that emit green fluorescence …”

Biliverdin incorporation into the cyanobacteriochrome SPI1085g3 from Spirulina

“…We have recently reported two novel PCB-binding CBCR GAF domains, SPI1085g2 and SPI1085g3, isolated from Spirulina subsalsa ( Wu et al, 2018 ; Jiang et al, 2021 ). PCB-binding SPI1085g2 exhibited typically reversible photoconversion between the green light-absorbing photoproduct and the red light-absorbing dark-adapted state but only with weak fluorescence ( Jiang et al, 2021 ), while PCB-binding SPI1085g3 exhibited unidirectional photoconversion and moderate dark reversion from the orange-absorbing photoproduct to the red-absorbing dark-adapted state with intense fluorescence ( Wu et al, 2018 ). The fluorescence could be switched off by illumination with red light, followed by a moderate recovery in the dark.…”