Assembly of CpcL-phycobilisomes.

CpcL-phycobilisomes (CpcL-PBSs) are a reduced type of phycobilisome (PBS) found in several cyanobacteria. They lack the traditional PBS terminal energy emitters, but still show the characteristic red-shifted fluorescence at ~670 nm. We established a method of assembling in vitro a rod-membrane linker protein, CpcL, with phycocyanin, generating complexes with the red-shifted spectral features of CpcL-PBSs. The red-shift arises from the interaction of a conserved key glutamine, Q57 of CpcL in Synechocystis sp. PCC 6803, with a single phycocyanobilin chromophore of trimeric phycocyanin at one of the three ß82-sites. This chromophore is the terminal energy acceptor of CpcL-PBSs and donor to the photosystem(s). This mechanism also operates in PBSs from Acaryochloris marina MBIC11017. We then generated multichromic complexes harvesting light over nearly the complete visible range via the replacement of phycocyanobilin chromophores at sites α84 and ß153 of phycocyanins by phycoerythrobilin and/or phycourobilin. The results demonstrate the rational design of biliprotein-based light-harvesting elements by engineering CpcL and phycocyanins, which broadens the light-harvesting range and accordingly improves the light-harvesting capacity and may be potentially applied in solar energy harvesting.

Infrared Signatures of Phycobilins within the Phycocyanin 645 Complex.

Aquatic photosynthetic organisms evolved to use a variety of light frequencies to perform photosynthesis. Phycobiliprotein phycocyanin 645 (PC645) is a light-harvesting complex in cryptophyte algae able to transfer the absorbed green solar light to other antennas with over 99% efficiency. The infrared signatures of the phycobilin pigments embedded in PC645 are difficult to access and could provide useful information to understand the mechanism behind the high efficiency of energy transfer in PC645. We use visible-pump IR-probe and two-dimensional electronic vibrational spectroscopy to study the dynamical evolution and assign the fingerprint mid-infrared signatures to each pigment in PC645. Here, we report the pigment-specific vibrational markers that enable us to track the spatial flow of excitation energy between the phycobilin pigment pairs. We speculate that two high-frequency modes (1588 and 1596 cm-1) are involved in the vibronic coupling leading to fast (

The Cyanobacterial "Nutraceutical" Phycocyanobilin Inhibits Cysteine Protease Legumain.

The blue biliprotein phycocyanin, produced by photo-autotrophic cyanobacteria including spirulina (Arthrospira) and marketed as a natural food supplement or "nutraceutical," is reported to have anti-inflammatory, antioxidant, immunomodulatory, and anticancer activity. These diverse biological activities have been specifically attributed to the phycocyanin chromophore, phycocyanobilin (PCB). However, the mechanism of action of PCB and the molecular targets responsible for the beneficial properties of PCB are not well understood. We have developed a procedure to rapidly cleave the PCB pigment from phycocyanin by ethanolysis and then characterized it as an electrophilic natural product that interacts covalently with thiol nucleophiles but lacks any appreciable cytotoxicity or antibacterial activity against common pathogens and gut microbes. We then designed alkyne-bearing PCB probes for use in chemical proteomics target deconvolution studies. Target identification and validation revealed the cysteine protease legumain (also known as asparaginyl endopeptidase, AEP) to be a target of PCB. Inhibition of this target may account for PCB's diverse reported biological activities.

Identification of significant residues for intermediate accumulation in phycocyanobilin synthesis.

Phycocyanobilin, the primary pigment of both light perception and light-harvesting in cyanobacteria, is synthesized from biliverdin IXα (BV) through intermediate 181, 182-dihydrobiliverdin (181, 182-DHBV) by a phycocyanobilin:ferredoxin oxidoreductase (PcyA). In our previous study, we discovered two PcyA homologs (AmPcyAc and AmPcyAp) derived from Acaryochloris marina MBIC 11017 (A. marina) that exceptionally uses chlorophyll d as the primary photosynthetic pigment, absorbing longer wavelength far-red light than chlorophyll a, the photosynthetic pigment found in most cyanobacteria. Biochemical characterization of the two PcyA homologs identified functional diversification of these two enzymes: AmPcyAc provides 181, 182-DHBV, and PCB to the cyanobacteriochrome (CBCR) photoreceptors, whereas, AmPcyAp specifically provides PCB to the light-harvesting phycobilisome subunit. In this study, we focused on the residues necessary for 181, 182-DHBV supply to the CBCR photoreceptors by AmPcyAc. Based on the SyPcyA structure, we concentrated on the 30 residues that constitute the substrate-binding pocket. Among them, we discovered that Leu151 and Val225 in AmPcyAc were both substituted with isoleucine. During the enzymatic reaction, the SyPcyA variant molecule, possessing V225I and L151I replacements, accumulates the 181, 182-DHBV and supplies it to a CBCR molecule derived from A. marina. It is worth noting that the substitution of Val225 with isoleucine was specifically conserved among the Acaryochloris genus. Collectively, we propose that the specific evolution of PcyA among the Acaryochloris genus may correlate with the acquisition of Chl. d synthetic ability and growth in long-wavelength far-red light environments.

Red fluorescent protein from cyanobacteriochrome chromophorylated with phycocyanobilin and biliverdin.

Cyanobacteriochromes are the extended family of phytochrome photosensors characterized in cyanobacteria. Alr1966g2C56A is a cyanobacteriochrome mutant of Alr1966g2 in Nostoc sp. PCC 7120 from freshwater. In this paper, we truncated ten residues in the N-terminus and ten residues in the C-terminus of Alr1966g2C56A and obtained truncated Alr1966g2C46A, termed as Alr1966g2C46A-tr. Alr1966g2C46A-tr binded covalently not only phycocyanobilin but also biliverdin via Cys74 of the conserved CH motif, and showed a significant improvement in binding-PCB efficiency in E. coli, compared with that of untruncated Alr1966g2C56A. We also captured a persistent red fluorescence of Alr1966g2C46A-tr-PCB or Alr1966g2C46A-tr-BV expressed in live E. coli. Thus, Alr1966g2C46A-tr was suitable for the stable red fluorescent probe as a starting material.

The impact of chromophore choice on the assembly kinetics and primary photochemistry of a red/green cyanobacteriochrome.

Cyanobacteriochromes (CBCRs) are bi-stable photoreceptor proteins with high potential for biotechnological applications. Most of these proteins utilize phycocyanobilin (PCB) as a light-sensing co-factor, which is unique to cyanobacteria, but some variants also incorporate biliverdin (BV). The latter are of particular interest for biotechnology due to the natural abundance and red-shifted absorption of BV. Here, AmI-g2 was investigated, a CBCR capable of binding both PCB and BV. The assembly kinetics and primary photochemistry of AmI-g2 with both chromophores were studied in vitro. The assembly reaction with PCB is roughly 10× faster than BV, and the formation of a non-covalent intermediate was identified as the rate-limiting step in the case of BV. This step is fast for PCB, where the formation of the covalent thioether bond between AmI-g2 and PCB becomes rate-limiting. The photochemical quantum yields of the forward and backward reactions of AmI-g2 were estimated and discussed in the context of homologous CBCRs.

Hydrogen-Bond Network Determines the Early Photoisomerization Processes of Cph1 and AnPixJ Phytochromes.

Phytochrome proteins are light receptors that play a pivotal role in regulating the life cycles of plants and microorganisms. Intriguingly, while cyanobacterial phytochrome Cph1 and cyanobacteriochrome AnPixJ use the same phycocyanobilin (PCB) chromophore to absorb light, their excited-state behavior is very different. We employ multiscale calculations to rationalize the different early photoisomerization mechanisms of PCB in Cph1 and AnPixJ. We found that their electronic S1 , T1 , and S0 potential minima exhibit distinct geometric and electronic structures due to different hydrogen bond networks with the protein environment. These specific interactions influence the S1 electronic structures along the photoisomerization paths, ultimately leading to internal conversion in Cph1 but intersystem crossing in AnPixJ. This explains why the excited-state relaxation in AnPixJ is much slower (ca. 100 ns) than in Cph1 (ca. 30 ps). Further, we predict that efficient internal conversion in AnPixJ can be achieved upon protonating the carboxylic group that interacts with PCB.

Pressurized Liquid Extraction of a Phycocyanobilin Chromophore and Its Reconstitution with a Cyanobacteriochrome Photosensor for Efficient Isotopic Labeling.

Linear tetrapyrrole compounds (bilins) are chromophores of the phytochrome and cyanobacteriochrome classes of photosensors and light-harvesting phycobiliproteins. Various spectroscopic techniques, such as resonance Raman, Fourier transform-infrared and nuclear magnetic resonance, have been used to elucidate the structures underlying their remarkable spectral diversity, in which the signals are experimentally assigned to specific structures using isotopically labeled bilin. However, current methods for isotopic labeling of bilins require specialized expertise, time-consuming procedures and/or expensive reagents. To address these shortcomings, we established a method for pressurized liquid extraction of phycocyanobilin (PCB) from the phycobiliprotein powder Lina Blue and also the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). PCB was efficiently cleaved in ethanol with three extractions (5 min each) under nitrogen at 125�C and 100 bars. A prewash at 75�C was effective for removing cellular pigments of Synechocystis without PCB cleavage. Liquid chromatography and mass spectrometry suggested that PCB was cleaved in the C3-E (majority) and C3-Z (partial) configurations. 15N- and 13C/15N-labeled PCBs were prepared from Synechocystis cells grown with NaH13CO3 and/or Na15NO3, the concentrations of which were optimized based on cell growth and pigmentation. Extracted PCB was reconstituted with a recombinant apoprotein of the cyanobacteriochrome-class photosensor RcaE. Yield of the photoactive holoprotein was improved by optimization of the expression conditions and cell disruption in the presence of Tween 20. Our method can be applied for the isotopic labeling of other PCB-binding proteins and for the commercial production of non-labeled PCB for food, cosmetic and medical applications.

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

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 (λabs.max = 520 nm, λfluor.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.

Carbon Atoms Speaking Out: How the Geometric Sensitivity of 13C Chemical Shifts Leads to Understanding the Colour Tuning of Phycocyanobilin in Cph1 and AnPixJ.

We present a combined quantum mechanics/molecular mechanics (QM/MM) molecular dynamics-statistical approach for the interpretation of nuclear magnetic resonance (NMR) chemical shift patterns in phycocyanobilin (PCB). These were originally associated with colour tuning upon photoproduct formation in red/green-absorbing cyanobacteriochrome AnPixJg2 and red/far-red-absorbing phytochrome Cph1Δ2. We pursue an indirect approach without computation of the absorption frequencies since the molecular geometry of cofactor and protein are not accurately known. Instead, we resort to a heuristic determination of the conjugation length in PCB through the experimental NMR chemical shift patterns, supported by quantum chemical calculations. We have found a characteristic correlation pattern of 13C chemical shifts to specific bond orders within the π-conjugated system, which rests on the relative position of carbon atoms with respect to electron-withdrawing groups and the polarisation of covalent bonds. We propose the inversion of this regioselective relationship using multivariate statistics and to apply it to the known experimental NMR chemical shifts in order to predict changes in the bond alternation pattern. Therefrom the extent of electronic conjugation, and eventually the change in absorption frequency, can be derived. In the process, the consultation of explicit mesomeric formulae plays an important role to qualitatively account for possible conjugation scenarios of the chromophore. While we are able to consistently associate the NMR chemical shifts with hypsochromic and bathochromic shifts in the Pg and Pfr, our approach represents an alternative method to increase the explanatory power of NMR spectroscopic data in proteins.

Revisiting high-resolution crystal structure of Phormidium rubidum phycocyanin.

The crystal structure of phycocyanin (pr-PC) isolated from Phormidium rubidum A09DM (P. rubidum) is described at a resolution of 1.17 Å. Electron density maps derived from crystallographic data showed many clear differences in amino acid sequences when compared with the previously obtained gene-derived sequences. The differences were found in 57 positions (30 in α-subunit and 27 in ß-subunit of pr-PC), in which all residues except one (ß145Arg) are not interacting with the three phycocyanobilin chromophores. Highly purified pr-PC was then sequenced by mass spectrometry (MS) using LC-MS/MS. The MS data were analyzed using two independent proteomic search engines. As a result of this analysis, complete agreement between the polypeptide sequences and the electron density maps was obtained. We attribute the difference to multiple genes in the bacterium encoding the phycocyanin apoproteins and that the gene sequencing sequenced the wrong ones. We are not implying that protein sequencing by mass spectrometry is more accurate than that of gene sequencing. The final 1.17 Å structure of pr-PC allows the chromophore interactions with the protein to be described with high accuracy.

Spectroscopic and molecular docking studies on the interaction of phycocyanobilin with peptide moieties of C-phycocyanin.

The binding of C-phycocyanin (CPC), a light harvesting pigment with phycocyanobilin (PCB), a chromophore is instrumental for the coloration and bioactivity. In this study, structure-mediated color changes of CPC from Spirulina platensis during various enzymatic hydrolysis was investigated based on UV-visible, circular dichroism, infra-red, fluorescence, mass spectrometry, and molecular docking. CPC was hydrolyzed using 7.09 U/mg protein of each enzyme at their optimal hydrolytic conditions for 3 h as follows: papain (pH 6.6, 60 °C), dispase (pH 6.6, 50 °C), and trypsin (pH 7.8, 37 °C). The degree of hydrolysis was in the order of papain (28.4%) > dispase (20.8%) > trypsin (7.3%). The sequence of color degradation rate and total color difference (ΔE) are dispase (82.9% and 40.37), papain (72.4% and 24.70), and trypsin (58.7% and 25.43). The hydrolyzed peptides were of diverse sequence length ranging from 8 to 9 residues (papain), 7-12 residues (dispase), and 9-63 residues (trypsin). Molecular docking studies showed that key amino acid residues in the peptides interacting with chromophore. Amino acid residues such as Arg86, Asp87, Tyr97, Asp152, Phe164, Ala167, and Val171 are crucial in hydrogen bonding interaction. These results indicate that the color properties of CPC might associate with chromopeptide sequences and their non-covalent interactions.

Structural basis of energy transfer in Porphyridium purpureum phycobilisome.

Photosynthetic organisms have developed various light-harvesting systems to adapt to their environments1. Phycobilisomes are large light-harvesting protein complexes found in cyanobacteria and red algae2-4, although how the energies of the chromophores within these complexes are modulated by their environment is unclear. Here we report the cryo-electron microscopy structure of a 14.7-megadalton phycobilisome with a hemiellipsoidal shape from the red alga Porphyridium purpureum. Within this complex we determine the structures of 706 protein subunits, including 528 phycoerythrin, 72 phycocyanin, 46 allophycocyanin and 60 linker proteins. In addition, 1,598 chromophores are resolved comprising 1,430 phycoerythrobilin, 48 phycourobilin and 120 phycocyanobilin molecules. The markedly improved resolution of our structure compared with that of the phycobilisome of Griffithsia pacifica5 enabled us to build an accurate atomic model of the P. purpureum phycobilisome system. The model reveals how the linker proteins affect the microenvironment of the chromophores, and suggests that interactions of the aromatic amino acids of the linker proteins with the chromophores may be a key factor in fine-tuning the energy states of the chromophores to ensure the efficient unidirectional transfer of energy.

Structural elements regulating the photochromicity in a cyanobacteriochrome.

The three-dimensional (3D) crystal structures of the GAF3 domain of cyanobacteriochrome Slr1393 (Synechocystis PCC6803) carrying a phycocyanobilin chromophore could be solved in both 15-Z dark-adapted state, Pr, λmax = 649 nm, and 15-E photoproduct, Pg, λmax = 536 nm (resolution, 1.6 and 1.86 Å, respectively). The structural data allowed identifying the large spectral shift of the Pr-to-Pg conversion as resulting from an out-of-plane rotation of the chromophore's peripheral rings and an outward movement of a short helix formed from a formerly unstructured loop. In addition, a third structure (2.1-Å resolution) starting from the photoproduct crystals allowed identification of elements that regulate the absorption maxima. In this peculiar form, generated during X-ray exposition, protein and chromophore conformation still resemble the photoproduct state, except for the D-ring already in 15-Z configuration and tilted out of plane akin the dark state. Due to its formation from the photoproduct, it might be considered an early conformational change initiating the parental state-recovering photocycle. The high quality and the distinct features of the three forms allowed for applying quantum-chemical calculations in the framework of multiscale modeling to rationalize the absorption maxima changes. A systematic analysis of the PCB chromophore in the presence and absence of the protein environment showed that the direct electrostatic effect is negligible on the spectral tuning. However, the protein forces the outer pyrrole rings of the chromophore to deviate from coplanarity, which is identified as the dominating factor for the color regulation.

Photoreversible interconversion of a phytochrome photosensory module in the crystalline state.

A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in Thermosynechococcus elongatus assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on Fobs - Fobs difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion.

Red, Orange, Green: Light- and Temperature-Dependent Color Tuning in a Cyanobacteriochrome.

Cyanobacteriochromes (CBCRs) are photoreceptor proteins that photoconvert between two parent states and thereby regulate various biological processes. An intriguing property is their variable ultraviolet-visible (UV-vis) absorption that covers the entire spectral range from the far-red to the near-UV region and thus makes CBCRs promising candidates for optogenetic applications. Here, we have studied Slr1393, a CBCR that photoswitches between red- and green-absorbing states (Pr and Pg, respectively). Using UV-vis absorption, fluorescence, and resonance Raman (RR) spectroscopy, a further orange-absorbing state O600 that is in thermal equilibrium with Pr was identified. The different absorption properties of the three states were attributed to the different lengths of the conjugated π-electron system of the phycocyanobilin chromophore. In agreement with available crystal structures and supported by quantum mechanics/molecular mechanics (QM/MM) calculations, the most extended conjugation holds for Pr whereas it is substantially reduced in Pg. Here, the two outer pyrrole rings D and A are twisted out of the plane defined by inner pyrrole rings B and C. For the O600 state, the comparison of the experimental RR spectra with QM/MM-calculated spectra indicates a partially distorted ZZZssa geometry in which ring A is twisted while ring D and the adjacent methine bridge display essentially the same geometry as Pr. The quantitative analysis of temperature-dependent spectra yields an enthalpy barrier of ∼30 kJ/mol for the transition from Pr to O600. This reaction is associated with the movement of a conserved tryptophan residue from the chromophore binding pocket to a solvent-exposed position.

Near-Infrared Markers based on Bacterial Phytochromes with Phycocyanobilin as a Chromophore.

Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IXα (BV) cofactor as a chromophore are increasingly used in cell biology and biomedicine, since their absorption and fluorescence spectra lie within the so-called optical "transparency window" of biological tissues. However, the quantum yield of BV fluorescence in these biomarkers does not exceed 0.145. The task of generating biomarkers with a higher fluorescence quantum yield remains relevant. To address the problem, we proposed the use of phycocyanobilin (PCB) as a chromophore of biomarkers derived from bacterial phytochromes. In this work, we characterized the complexes of iRFP713 evolved from RpBphP2 and its mutant variants with different location of cysteine residues capable of covalent tetrapyrrole attachment with the PCB cofactor. All analyzed proteins assembled with PCB were shown to have a higher fluorescence quantum yield than the proteins assembled with BV. The iRFP713/V256C and iRFP713/C15S/V256C assembled with PCB have a particularly high quantum yield of 0.5 and 0.45, which exceeds the quantum yield of all currently available near-infrared biomarkers. Moreover, PCB has 4 times greater affinity for iRFP713/V256C and iRFP713/C15S/V256C proteins compared to BV. These data establish iRFP713/V256C and iRFP713/C15S/V256C assembled with the PCB chromophore as promising biomarkers for application in vivo. The analysis of the spectral properties of the tested biomarkers allowed for suggesting that the high-fluorescence quantum yield of the PCB chromophore can be attributed to the lower mobility of the D-ring of PCB compared to BV.

Quantum dynamics of vibration-assisted excitation energy transfer in phycobiliprotein light-harvesting complex.

Phycobiliprotein is a light-harvesting complex containing linear tetrapyrrole bilin pigments that are responsible for absorption and funneling the sun's energy in cryptophytes algae. In particular, the protein structure determines relative positions and orientations of the pigments and thus controls energy transfer pathways. The present research reveals the impact of molecular vibrations (in the 850-2700 cm-1 region) on excitation energy transfer in phycobiliprotein. The analysis of the excitation energy transfer pathways indicates a possibility of the coherent mechanism of energy transfer (delocalization) in central dihydrobiliverdin pigments and incoherent vibration-assisted energy transfer to peripheral phycocyanobilin pigments at a sub-picosecond time scale. A computational approach that enables modeling the dynamics of the excitation energy transfer with the quantum master equation formalism employing Huang-Rhys factors to describe electronic-vibrational coupling has been developed. The computational methodology has been implemented in PyFREC software.

Antioxidant activity of proteins extracted from red alga dulse harvested in Japan.

In this study, we investigated antioxidant activity of proteins from the red alga dulse (Palmaria sp.) harvested in Hokkaido, Japan. The dulse proteins that contain phycoerythrin (PE) as the main component showed a high radical scavenging activity. To clarify the key constituent of antioxidant activity in dulse proteins, we prepared recombinant dulse PE ß-subunit (rPEß) (apoprotein) and chromophores from the dulse proteins. As a result, the rPEß showed lower radical scavenging activity than that of dulse proteins. On the other hand, the dulse chromophores composed mainly of phycoerythrobilin (PEB) indicated extremely higher radical scavenging activity (90.4% ± 0.1%) than that of dulse proteins (17.9% ± 0.1%) on ABTS assay. In addition, on cell viability assay using human neuroblastoma SH-SY5Y cells, the dulse chromophores showed extracellular and intracellular cytoprotective effects against H2 O2 -induced cell damage. From these data, we concluded that the dulse proteins have antioxidant ability and the activity principally derives from the chromophores. PRACTICAL APPLICATION: Dulse is an abundant and underused resource, which contains a lot of proteins, especially phycoerythrin. We here demonstrated that the practically prepared dulse proteins possessed antioxidant activity and clarified that chromophores from the dulse proteins were the key components. Therefore, the dulse proteins have a potential for functional material.

Very Bright Phycoerythrobilin Chromophore for Fluorescence Biolabeling.

Biliproteins have extended the spectral range of fluorescent proteins into the far-red (FR) and near-infrared (NIR) regions. These FR and NIR fluorescent proteins are suitable for the bioimaging of mammalian tissues and are indispensable for multiplex labeling. Their application, however, presents considerable challenges in increasing their brightness, while maintaining emission in FR regions and oligomerization of monomers. Two fluorescent biliprotein triads, termed BDFP1.2/1.6:3.3:1.2/1.6, are reported. In mammalian cells, these triads not only have extremely high brightness in the FR region, but also have monomeric oligomerization. The BDFP1.2 and BDFP1.6 domains covalently bind to biliverdin, which is accessible in most cells. The BDFP3.3 domain noncovalently binds phycoerythrobilin that is added externally. A new method of replacing phycoerythrobilin with proteolytically digested BDFP3.3 facilitates this labeling. BDFP3.3 has a very high fluorescence quantum yield of 66 %, with maximal absorbance at λ=608 nm and fluorescence at λ=619 nm. In BDFP1.2/1.6:3.3:1.2/1.6, the excitation energy that is absorbed in the red region by phycoerythrobilin in the BDFP3.3 domain is transferred to biliverdin in the two BDFP1.2 or BDFP1.6 domains and fluoresces at λ≈670 nm. The combination of BDFP3.3 and BDFP1.2/1.6:3.3:1.2/1.6 can realize dual-color labeling. Labeling various proteins by fusion to these new fluorescent biliproteins is demonstrated in prokaryotic and mammalian cells.