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Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, are a resource for photosynthetic, photonic and fluorescence labeling elements. They cover an exceptionally broad spectral range, but the complex superstructure and assembly have been an obstacle. By replacing in Synechocystis sp. PCC 6803 the biliverdin reductases, we studied the role of chromophores in the assembly of the phycobilisome core. Introduction of the green-absorbing phycoerythrobilin instead of the red-absorbing phycocyanobilin inhibited aggregation. A novel, trimeric allophycocyanin (Dic-APC) was obtained. In the small (110 kDa) unit, the two chromophores, phycoerythrobilin and phytochromobilin, cover a wide spectral range (550 to 660 nm). Due to efficient energy transfer, it provides an efficient artificial light-harvesting element. Dic-APC was generated in vitro by using the contained core-linker, L

The phycobilisomes (PBSs) of cyanobacteria and red-algae are unique megadaltons light-harvesting protein-pigment complexes that utilize bilin derivatives for light absorption and energy transfer. Recently, the high-resolution molecular structures of red-algal PBSs revealed how the multi-domain core-membrane linker (LCM ) specifically organizes the allophycocyanin subunits in the PBS's core. But, the topology of LCM in these structures was different than that suggested for cyanobacterial PBSs based on lower-resolution structures. Particularly, the model for cyanobacteria assumed that the Arm2 domain of LCM connects the two basal allophycocyanin cylinders, whereas the red-algal PBS structures revealed that Arm2 is partly buried in the core of one basal cylinder and connects it to the top cylinder. Here, we show by biochemical analysis of mutations in the apcE gene that encodes LCM , that the cyanobacterial and red-algal LCM topologies are actually the same. We found that removing the top cylinder linker domain in LCM splits the PBS core longitudinally into two separate basal cylinders. Deleting either all or part of the helix-loop-helix domain at the N-terminal end of Arm2, disassembled the basal cylinders and resulted in degradation of the part containing the terminal emitter, ApcD. Deleting the following 30 amino-acids loop severely affected the assembly of the basal cylinders, but further deletion of the amino-acids at the C-terminal half of Arm2 had only minor effects on this assembly. Altogether, the biochemical data are consistent with the red-algal LCM topology, suggesting that the PBS cores in cyanobacteria and red-algae assemble in the same way.

Far-red and near-infrared fluorescent proteins can be used as fluorescence biomarkers in the region of maximal transmission of most tissues and facilitate multiplexing. Recently, we reported the generation and properties of far-red and near-infrared fluorescent phycobiliproteins, termed BeiDou Fluorescent Proteins (BDFPs), which can covalently bind the more readily accessible biliverdin. Far-red BDFPs maximally fluoresce at ∼670 nm, while near-infrared BDFPs fluoresce at ∼710 nm. In this work, we molecularly evolved BDFPs as follows: (a) mutations L58Q, S68R and M81K of BDFPs, which can maximally enhance the effective brightness in vivo by 350 %; (b) minimization and monomerization of far-red BDFPs 2.1, 2.2, 2.3, and near-infrared BDFPs 2.4, 2.5 and 2.6. These newly developed BDFPs are remarkably brighter than the formerly reported far-red and near-infrared fluorescent proteins. Their advantages are demonstrated by biolabeling in mammalian cells using super-resolution microscopy.

Phytochrome photoreceptors operate via photoisomerization of a bound bilin chromophore. Their typical architecture consists of GAF, PAS and PHY domains. Knotless phytochromes lack the PAS domain, while retaining photoconversion abilities, with some being able to photoconvert with just the GAF domain. Therefore, we investigated the ultrafast photoisomerization of the Pr state of a knotless phytochrome to reveal the effect of the PHY domain and its “tongue” region on the transduction of the light signal. We show that the PHY domain does not affect the initial conformational dynamics of the chromophore. However, it significantly accelerates the consecutively induced reorganizational dynamics of the protein, necessary for the progression of the photoisomerization. Consequently, the PHY domain keeps the bilin and its binding pocket in a more reactive conformation, which decreases the extent of protein reorganization required for the chromophore isomerization. Thereby, less energy is lost along nonproductive reaction pathways, resulting in increased efficiency., More reactive, more efficient: The influence of the PHY domain on the photoconversion of a knotless phytochrome was investigated. The interaction of its “tongue” region with the GAF domain keeps the bilin chromophore and the binding pocket in a more reactive conformation. This results in accelerated reorganization of the protein environment necessary to accommodate the photoisomerization of the chromophore and, in effect, in an increased overall quantum efficiency.

Phytochromes regulate central responses of plants and microorganisms such as shade avoidance and photosystem synthesis. Canonical phytochromes comprise a photosensory module of three domains. The C-terminal phytochrome-specific (PHY) domain interacts via a tongue element with the bilin chromophore in the central GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain. The bilin isomerizes upon illumination with red light, transforming the receptor from the Pr state to the Pfr state. The "knotless" phytochrome All2699 from the cyanobacterium Nostoc sp. PCC7120 comprises three GAF domains as a sensory module and a histidine kinase as an effector. GAF1 and GAF3 both bind a bilin, and GAF2 contains a tongue-like element. We studied the response of All2699, GAF1-GAF2, and GAF1 to red light by Fourier transform infrared difference spectroscopy, including a 13C-labeled protein moiety for assignment. In GAF1-GAF2, a refolding of the tongue from β-sheet to α-helix and an upshift of the ring D carbonyl stretch from 1700 to 1712 cm-1 were observed. Therefore, GAF1-GAF2 is regarded as the smallest model system available to study the tongue response and interaction with the chromophore. Replacement of an arginine in the tongue with proline (R387P) did not affect the unfolding of the β-sheet to Pfr but strongly impaired α-helix formation. In contrast, the Y55H mutation close to bilin ring D did not interfere with conversion to Pfr. Strikingly, the presence of GAF3 in the full-length All2699 diminished the response of the tongue and generated the signal pattern found for GAF1 alone. These results point to a regulatory or integrative role of GAF3 in All2699 that is absent in canonical phytochromes.

Phycobilisomes are large light-harvesting complexes attached to the stromal side of thylakoids in cyanobacteria and red algae. They can be remodeled or degraded in response to changing light and nutritional status. Both the core and the peripheral rods of phycobilisomes contain biliproteins. During biliprotein biosynthesis, open-chain tetrapyrrole chromophores are attached covalently to the apoproteins by dedicated lyases. Another set of non-bleaching (Nb) proteins has been implicated in phycobilisome degradation, among them NblA and NblB. We report in vitro experiments with lyases, biliproteins and NblA/B which imply that the situation is more complex than currently discussed: lyases can also detach the chromophores and NblA and NblB can modulate lyase-catalyzed binding and detachment of chromophores in a complex fashion. We show: (i) NblA and NblB can interfere with chromophorylation as well as chromophore detachment of phycobiliprotein, they are generally inhibitors but in some cases enhance the reaction; (ii) NblA and NblB promote dissociation of whole phycobilisomes, cores and, in particular, allophycocyanin trimers; (iii) while NblA and NblB do not interact with each other, both interact with lyases, apo- and holo-biliproteins; (iv) they promote synergistically the lyase-catalyzed chromophorylation of the β-subunit of the major rod component, CPC; and (v) they modulate lyase-catalyzed and lyase-independent chromophore transfers among biliproteins, with the core protein, ApcF, the rod protein, CpcA, and sensory biliproteins (phytochromes, cyanobacteriochromes) acting as potential traps. The results indicate that NblA/B can cooperate with lyases in remodeling the phycobilisomes to balance the metabolic requirements of acclimating their light-harvesting capacity without straining the overall metabolic economy of the cell.

Methylobacteria are facultative methylotrophic phytosymbionts of great industrial and agronomical interest, and they are considered as opportunistic pathogens posing a health threat to humans. So far only a few reports mention photoreceptor coding sequences in Methylobacteria genomes, but no investigation at the molecular level has been performed yet. We here present comprehensive in silico research into potential photoreceptors in this bacterial phylum and report the photophysical and photochemical characterisation of two representatives of the most widespread photoreceptor classes, a blue-light sensing LOV (light, oxygen, voltage) protein and a red/far red light sensing BphP (biliverdin-binding bacterial phytochrome) from M. radiotolerans JCM 2831. Overall, both proteins undergo the expected light-triggered reactions, but peculiar features were also identified. The LOV protein Mr4511 has an extremely long photocycle and lacks a tryptophan conserved in ca. 75% of LOV domains. Mutation I37V accelerates the photocycle by one order of magnitude, while the Q112W change underscores the ability of tryptophan in this position to perform efficient energy transfer to the flavin chromophore. Time-resolved photoacoustic experiments showed that Mr4511 has a higher triplet quantum yield than other LOV domains and that the formation of the photoproduct results in a volume expansion, in sharp contrast to other LOV proteins. Mr4511 was found to be astonishingly resistant to denaturation by urea, still showing light-triggered reactions after incubation in urea for more than 20 h. The phytochrome MrBphP1 exhibits the so far most red-shifted absorption maxima for its Pr- and Pfr forms (λmax = 707 nm and 764 nm for the Pr and Pfr forms). The light-driven conversions in both directions occur with relatively high quantum yields of 0.2. Transient ns absorption spectroscopy (μs-ms time range) identifies the decay of the instantaneously formed lumi-intermediate, followed by only one additional intermediate before the formation of the respective final photoproducts for Pr-to-Pfr or Pfr-to-Pr photoconversion, in contrast to other BphPs. The relatively simple photoconversion patterns suggest the absence of the shunt pathways reported for other bacterial phytochromes.

SUMMARYVertebrate embryogenesis is a remarkably dynamic process during which numerous cell types of different lineages generate, change, or disappear within a short period of time. A major challenge in understanding this process is the lack of topographical transcriptomic information that can help correlate microenvironmental cues within the hierarchy of cell fate decisions. Here, we employed Stereo-seq, a high-definition spatially resolved transcriptomic technology, to dissect the spatiotemporal dynamics of gene expression and regulatory networks in the developing zebrafish embryos. We profiled 91 embryo sections covering six critical time points during the first 24 hours of development, obtaining a total of 139,391 spots at cellular size (∼100 μm2) with spatial coordinates. Meanwhile, we identified spatial modules and co-varying genes for specific tissue organizations. By performing the integrative analysis of the Stereo-seq and scRNA-seq data from each time point, we reconstructed the spatially resolved developmental trajectories of cell fate transitions and molecular changes during zebrafish embryogenesis. We further investigated the spatial distribution of ligand-receptor pairs for major signaling pathways and identified novel interactions that potentially crosstalk with the Notch signaling pathway during zebrafish development. Our study constitutes a fundamental reference for further studies aiming to understand vertebrate development.

Naturally-occurring orange carotenoid protein (OCP) is synthesized in cyanobacteria and red algae for photoprotection. Holo-OCP can be produced with three plasmids in E. coli, which needs two inducers (arabinose and isopropyl β-D-thiogalactoside) to initiate two processes: one for generation of carotenoid and the other for generation of apo-OCP, so takes about two days. Afterwards, a two-plasmid method using two plasmids in E. coli is established, in which E. coli cells are induced only by isopropyl β-D-thiogalactoside, so can yield different holo-OCPs from several cyanobacteria within three days. In this work, we optimized the two-plasmid method as follows: (1) re-organization of the two plasmids, letting carotenoid-generating gene, crtW, be arranged together with apo-OCP-generating gene, ocp, in a single plasmid, which causes that both carotenoid and apo-protein were properly produced, (2) modification of several amino acids at the N-terminus of apo-OCP, in this way increasing the yield and purity of holo-OCP. After these optimizations, we can generate much more amount of holo-OCP within shorter time of only 16 h, and pure holo-OCP be conveniently prepared after routine purification. Comparing with the reported data, the general yield of holo-OCP is increased by ∼10-fold under similar conditions. The high quality of the prepared holo-OCPs is verified by fluorescence quenching of the phycobilisomes.

Microorganisms living in animals can function as drug delivery systems or as detectors for some diseases. Here, we developed a biosensor constructed by the deletion of hemF and harboring ho1, chuA, and bdfp1.6 in Escherichia coli. HemF is an enzyme involved in heme synthesis in E. coli. ChuA and HO1 can transfer extracellular heme into cells and generate biliverdin (BV). BDFP1.6 can bind BV autocatalytically, and it emits a far-red fluorescence signal at 667 nm. Therefore, we named this biosensor as the far-red light for bleeding detector (FRLBD). Our results indicated that the FRLBD was highly efficient and specific for detecting heme or blood in vitro. Moreover, the FRLBD could be used to detect bleeding in the zebrafish induced by aspirin, and a convolutional neural network was an appropriate model to identify the fluorescence features in the images.

Cyanobacteria sense and respond to various colors of light employing a large number of bilin-based phytochrome-like photoreceptors. All2699 from Nostoc 7120 has three consecutive GAF domains with GAF1 and GAF3 binding a phycocyanobilin chromophore. GAF1, even when expressed independently, can be photoconverted between red-absorbing Pr and far-red-absorbing Pfr states, while the nonphotosensory GAF2 domain is structurally and functionally homologous to the PHY domains in canonical and Cph2-like phytochromes. Here, we characterize possible bilin chromophore conformers using solid-state NMR spectroscopy on the two lyophilized All2699 samples (GAF1-only and GAF1-PHY constructs). On the basis of complete 1H, 13C, and 15N assignments for the chromophore obtained on the two Pr lyophilizates, multiple static conformations of the chromophore in both cases are identified. Moreover, most atoms of the chromophore in the bidomain sample show only subtle changes in the mean chemical shifts relative to those in frozen solution (FS), indicating an optimized interaction of the GAF2 domain with the GAF1-bound chromophore. Our results confirm the conservation of key chromophore-protein interactions and the photoreversibility in both All2699 lyophilizates, offering the possibility to investigate conformational distributions of the heterogeneous chromophore and its functional consequences in phytochromes and other bilin-dependent photoreceptors intractable by the solid-state NMR technique as FSs.

Phytochromes are a diverse family of bilin-binding photoreceptors that regulate a wide range of physiological processes. Their photochemical properties make them attractive for applications in optogenetics and superresolution microscopy. Phytochromes undergo reversible photoconversion triggered by the Z ⇄ E photoisomerization about the double bond in the bilin chromophore. However, it is not fully understood at the molecular level how the protein framework facilitates the complex photoisomerization dynamics. We have studied a single-domain bilin-binding photoreceptor All2699g1 (Nostoc sp. PCC 7120) that exhibits photoconversion between the red light-absorbing (P(r)) and far red-absorbing (P(fr)) states just like canonical phytochromes. We present the crystal structure and examine the photoisomerization mechanism of the P(r) form as well as the formation of the primary photoproduct Lumi-R using time-resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations. We show that the unusually long excited state lifetime (broad lifetime distribution centered at ∼300 picoseconds) is due to the interactions between the isomerizing pyrrole ring D and an adjacent conserved Tyr142. The decay kinetics shows a strongly distributed character which is imposed by the nonexponential protein dynamics. Our findings offer a mechanistic insight into how the quantum efficiency of the bilin photoisomerization is tuned by the protein environment, thereby providing a structural framework for engineering bilin-based optical agents for imaging and optogenetics applications.

Proceedings of the National Academy of Sciences of the United States of America 117(5), 2432 - 2440 (2020). doi:10.1073/pnas.1910208117, Phytochromes and related photoreceptors distinguish themselves for their long-wavelength absorption and large spectral shift between parental state and photoproduct. Both features are not well understood, partly due to lack of high-resolution structural data and insufficient support from quantum-chemical calculations. The red–green switching cyanobacteriochrome Slr1393g3 shows an absorption shift larger than 110 nm. Both parental state and photoproduct could be crystallized with high resolution, together with a “hybrid” form carrying the chromophore in parental state geometry, whereas the protein remained in the photoproduct conformation. The crystal structures reveal how chromophore and protein mutually regulate their conformational changes, yielding the observed spectral shift. Quantum-chemical calculations, based on these structures, provide a deeper understanding of the spectral tuning mechanisms., Published by National Acad. of Sciences, Washington, DC

Cyanobacteriochromes (CBCRs) are photochromic proteins in cyanobacteria that act as photosensors. CBCRs bind bilins as chromophores and sense nearly the entire visible spectrum of light, but the regulation of the chromophorylation of CBCRs is unknown. Slr1393 from Synechocystis sp. PCC 6803 is a CBCR containing three consecutive GAF (cGMP phosphodiesterase, adenylyl cyclase, and FhlA protein) domains, of which only the third one (Slr1393g3) can be phycocyanobilin-chromophorylated. The protein Slr2111 from Synechocystis sp. PCC 6803 includes a cystathionine β-synthase (CBS) domain pair of an as yet unknown function at its N terminus. CBS domains are often characterized as sensors of cellular energy status by binding nucleotides. In this work, we demonstrate that Slr2111 strongly interacts with Slr1393 in vivo and in vitro, which generates a complex in a 1:1 molar ratio. This tight interaction inhibits the chromophorylation of Slr1393g3, even if the chromophore is present. Instead, the complex stability and thereby the chromophorylation of Slr1393 are regulated by the binding of nucleotides (ATP, ADP, AMP) to the CBS domains of Slr2111 with varying affinities. It is demonstrated that residues Asp-53 and Arg-97 of Slr2111 are involved in nucleotide binding. While ATP binds to Slr2111, the association between the two proteins gets weaker and chromophorylation of Slr1393 are enabled. In contrast, AMP binding to Slr2111 leads to a stronger association, thereby inhibiting the chromophorylation. It is concluded that Slr2111 acts as a sensor of the cellular energy status that regulates the chromophorylation of Slr1393 and thereby its function as a light-driven histidine kinase.

Cyanobacteriochromes (CBCRs) are biliprotein photoreceptors that only exist in cyanobacteria and have a broad spectral response range from ultra-violet to far-red. The red/green-type CBCRs can show red/green reversible photoconversion via a covalently bound phycocyanobilin (PCB). In recent years, several CBCRs binding with not only PCB but also biliverdin (BV) have been discovered, which raises the possibility of CBCRs being applied as optogenetic tools. Through molecular modification, we hope to engineer BV-binding CBCRs responsive to the near-infrared spectral region (650–900 nm), of which the red/green type of CBCRs are suitable resources for experimentation. Here, we use Slr1393g3 (the third GAF domain of a red/green photoswitching CBCR from Synechocystis sp. PCC 6803) as a template to perform such molecular evolution using both random mutagenesis and site-directed mutagenesis. After several rounds of random mutagenesis, we obtained several BV-binding variants of Slr1393g3. These BV-binding variants have a maximal absorbance at ̃690 nm and a fluorescence at ̃720 nm. Additionally, some of them have remarkable photochromicity between a far-red light-absorbing state and a red light-absorbing state. Based on the primary amino acid sequence and structural models, the Phe474 surrounding ring D of BV is thought as a crucial site for chromophore selectivity.

Cyanobacterial phycobilisomes funnel the harvested light energy to the reaction centers via two terminal emitters, allophycocyanin B and the core-membrane linker. ApcD is the α-subunit of allophycocyanin B responsible for its red-shifted absorbance (λmax 665 nm). Far-red photo-acclimated cyanobacteria contain certain allophycocyanins that show even further red-shifted absorbances (λmax > 700 nm). We studied the chromophorylation of the three far-red induced ApcD subunits ApcD2, ApcD3 and ApcD4 from Chroococcidiopsis thermalis sp. PCC7203 during the expression in E. coli. The complex behavior emphasizes that a variety of factors contribute to the spectral red-shift. Only ApcD2 bound phycocyanobilin covalently at the canonical position C81, while ApcD3 and ApcD4 gave only traces of stable products. The product of ApcD2 was, however, heterogeneous. The major fraction had a broad absorption around 560 nm and double-peaked fluorescence at 615 and 670 nm. A minor fraction was similar to the product of conventional ApcD, with maximal absorbance around 610 nm and fluorescence around 640 nm. The heterogeneity was lost in C65 and C132 variants; in these variants only the conventional product was formed. With ApcD4, a red-shifted product carrying non-covalently bound phycocyanobilin could be detected in the supernatant after cell lysis. While this chromophore was lost during purification, it could be stabilized by co-assembly with a far-red light-induced β-subunit, ApcB3.

Kai-Hong Zhao,1 Can Zhang,2 Yue Bai,1 Yan Li,1 Xun Kang,1 Jian-Xin Chen,1 Kun Yao,3 Tao Jiang,4 Xiao-Song Zhong,2 Wen-Bin Li1 1Department of Glioma, 2Clinical Center of Gene And Cell Engineering, Beijing Shijitan Hospital, 3Department of Neurosurgery, Beijing Tiantan Hospital, 4Department of Pathology, Sanbo Brain Hospital, Capital Medical University, Beijing, People’s Republic of China Abstract: Leptomeningeal metastasis (LM) of high-grade glioma is a highly lethal disease requiring new effective therapeutic measures. For both de novo or relapsed glioma with LM, intrathecal cytarabine chemotherapy is not frequently used for first-line and relapse protocols. We encountered a clinical case demonstrating effective application of cytarabine in high-grade glioma with LM, prompting us to explore the effects of cytarabine on malignant glioma and molecular mechanisms of such effects through in vivo and in vitro experiments. The U87 cell line was selected to represent human glioma for studies. Cell viability was measured by MTT assay, plate colony formation assay, and trypan-blue dye exclusion test. Apoptosis was assessed by flow cytometry. Protein expression levels were detected by Western blot assay and immunohistochemistry. mRNA expression was examined by quantitative real-time reverse transcription polymerase chain reaction. Cytarabine inhibited tumor growth during the in vivo experiment. The present study confirmed that cytarabine inhibits proliferation and promotes apoptosis of U87 cells, and molecular analysis of this effect showed that cytarabine significantly reduces expression of phosphatidylinositol 3-kinase/serine/threonine kinase also known as the protein kinaseB/mechanistic target of rapamycin (PI3K/Akt/mTOR) pathway, Ki-67, BCL2, and 4-1BB, and upregulates Bax and cleaved caspase-3. Our findings indicated that intrathecal administration of cytarabine manifests potential in prophylaxis and treatment of malignant glioma with LM. Effective medications for high-grade glioma with LM should contain cytarabine. Keywords: leptomeningeal metastases, malignant glioma, cytarabine, PI3K/Akt/mTOR

Photoprotection is essential for efficient photosynthesis. Cyanobacteria have evolved a unique photoprotective mechanism mediated by a water-soluble carotenoid-based photoreceptor known as orange carotenoid protein (OCP). OCP undergoes large conformational changes in response to intense blue light, and the photoactivated OCP facilitates dissipation of excess energy via direct interaction with allophycocyanins at the phycobilisome core. However, the structural events leading up to the OCP photoactivation remain elusive at the molecular level. Here we present direct observations of light-induced structural changes in OCP captured by dynamic crystallography. Difference electron densities between the dark and illuminated states reveal widespread and concerted atomic motions that lead to altered protein-pigment interactions, displacement of secondary structures, and domain separation. Based on these crystallographic observations together with site-directed mutagenesis, we propose a molecular mechanism for OCP light perception, in which the photochemical property of a conjugated carbonyl group is exploited. We hypothesize that the OCP photoactivation starts with keto-enol tautomerization of the essential 4-keto group in the carotenoid, which disrupts the strong hydrogen bonds between the bent chromophore and the protein moiety. Subsequent structural changes trapped in the crystal lattice offer a high-resolution glimpse of the initial molecular events as OCP begins to transition from the orange-absorbing state to the active red-absorbing state.

The last decade has seen development and application of a large number of novel fluorescence-based techniques that have revolutionized fluorescence microscopy in life sciences. Preferred tags for such applications are genetically encoded fluorescent proteins (FP), mostly derivatives of the green fluorescent protein (GFP). Combinations of FPs with wavelength-separated absorption/fluorescence properties serve as excellent tools for molecular interaction studies, for example, protein-protein complexes or enzyme-substrate interactions, based on the FRET phenomenon (Förster resonance energy transfer). However, alternatives are requested for experimental conditions where FP proteins or FP couples are not or less efficiently applicable. We here report as a "proof of principle" a specially designed, non-naturally occurring protein (LG1) carrying a combination of a flavin-binding LOV- and a photochromic bilin-binding GAF domain and demonstrate a FRET process between both chromophores.

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.

Due to the low absorbance in the far-red (FR) and near-infrared (NIR) “optical window”, NIR fluorescent proteins (FPs) are powerful tools for deep imaging. Here, we report three new, highly bright NIR FPs termed BDFP1.8, BDFP1.8:1.8 (tandem BDFP1.8) and BDFP1.9, which evolved from a previously reported FR FP, BDFP1.6: a derivative of ApcF2 from Chroococcidiopsis thermalis sp. PCC7203. ApcF2 binds phycocyanobilin (PCB) non-covalently, while BDFPs, the derivatives of ApcF2, can bind biliverdin (BV) covalently. We identified that dimeric BDFP1.8 and monomeric BDFP1.8:1.8 have a 2.4-and 4.4-fold higher effective brightness, respectively, than iRFP720, which has the highest effective brightness among the reported NIR FPs. Monomeric DBFP1.9 (17 kDa) has one of the smallest masses among highly bright FPs in the FR and NIR regions. Enhancing the affinity between the apo-proteins and the BV chromophore is an effective method to improve the effective brightness of biliprotein FPs. Moreover, BDFP1.8 and 1.9 exhibit higher stability to temperature, pH and light than iRFP720. Finally, the highly bright NIR BDFP1.8 together with FR BDFP1.6 could effectively biolabel cells in dual colors.

Cyanobacterial light-harvesting complexes, phycobilisomes, can undergo extensive remodeling under varying light conditions. Acclimation to far-red light involves not only generation of red-shifted chlorophylls in the photosystems, but also induction of additional copies of core biliproteins that have been related to red-shifted components of the phycobilisome (Gan et al., Life 5, 4, 2015). We are studying the molecular basis for these acclimations in Chroococcidiopsis thermalis sp. PCC7203. Five far-red induced allophycocyanin subunits (ApcA2, ApcA3, ApcB2, ApcB3 and ApcF2) were expressed in Escherichia coli, together with S-type chromophore-protein lyases and in situ generated chromophore, phycocyanobilin. Only one subunit, ApcF2, shows an unusual red-shift (λAmax~675nm, λFmax~698nm): it binds the chromophore non-covalently, thereby preserving its full conjugation length. This mechanism operates also in two Cys-variants of the induced subunits of bulky APC. All other wild-type subunits bind phycocyanobilin covalently to the conventional Cys-81 under catalysis of the lyase, CpcS1. Although three of them also show binding to additional cysteines, all absorb and fluoresce similar to conventional APC subunits (λAmax~610nm, λFmax~640nm). Another origin of red-shifted complexes was identified, however, when different wild-type α- and β-subunits of the far-red induced bulky APC were combined in a combinatorial fashion. Strongly red-shifted complexes (λFmax≤722nm) were formed when the α-subunit, PCB-ApcA2, and the β-subunit, PCB-ApcB2, were generated together in E. coli. This extreme aggregation-induced red-shift of ~90nm of covalently bound chromophores is reminiscent, but much larger, than the ~30nm observed with conventional APC.

Phycobiliproteins that bind bilins are organized as light-harvesting complexes, phycobilisomes, in cyanobacteria and red algae. The harvested light energy is funneled to reaction centers via two energy traps, allophycocyanin B and the core-membrane linker, ApcE1 (conventional ApcE). The covalently bound phycocyanobilin (PCB) of ApcE1 absorbs near 660 nm and fluoresces near 675 nm. In cyanobacteria capable of near infrared photoacclimation, such as Synechococcus sp. PCC7335, there exist even further spectrally red shifted components absorbing >700 nm and fluorescing >710 nm. We expressed the chromophore domain of the extra core-membrane linker from Synechococcus sp. PCC7335, ApcE2, in E. coli together with enzymes generating the chromophore, PCB. The resulting chromoproteins, PCB-ApcE2(1-273) and the more truncated PCB-ApcE2(24-245), absorb at 700 nm and fluoresce at 714 nm. The red shift of ~40 nm compared with canonical ApcE1 results from non-covalent binding of the chromophore by which its full conjugation length including the Δ3,3(1) double bond is preserved. The extreme spectral red-shift could not be ascribed to exciton coupling: dimeric PCB-ApcE2(1-273) and monomeric-ApcE2(24-245) absorbed and fluoresced similarly. Chromophorylation of ApcE2 with phycoerythrobilin- or phytochromobilin resulted in similar red shifts (absorption at 615 and 711 nm, fluorescence at 628 or 726 nm, respectively), compared to the covalently bound chromophores. The self-assembled non-covalent chromophorylation demonstrates a novel access to red and near-infrared emitting fluorophores. Brightly fluorescent biomarking was exemplified in E. coli by single-plasmid transformation.

The genome of the cyanobacterium Nostoc sp. PCC 7120 encodes a large number of putative bacteriophytochrome and cyanobacteriochrome photoreceptors that, due to their long-wavelength absorption and fluorescence emission, might serve as fluorescent tags in intracellular investigations. We show that the PAS-GAF domain of the bacteriophytochrome, AphB, binds biliverdin covalently and exhibits, besides its reversible photochemistry, a moderate fluorescence in the near infrared (NIR) spectral region. It was selected for further increasing the brightness while retaining the NIR fluorescence. In the first step, amino acids assumed to improve fluorescence were selectively mutated. The resulting variants were then subjected to several rounds of random mutagenesis and screened for enhanced fluorescence in the NIR. The brightness of optimized PAS-GAF variants increased more than threefold compared to that of wt AphB(1-321), with only insignificant spectral shifts (Amax around 695 nm, and Fmax around 720 nm). In general, the brightness increases with decreasing wavelengths, which allows for a selection of the fluorophore depending on the optical properties of the tissue. A spectral heterogeneity was observed when residue His260, located in close proximity to the chromophore, was mutated to Tyr, emphasizing the strong effects of the environment on the electronic properties of the bound biliverdin chromophore.

Cyanobacteria are among the main contributors to global photosynthesis and show a high degree of metabolic plasticity. Synechocystis sp. PCC 6803 can grow under photoautotrophic, photomixotrophic or photoheterotrophic conditions. We have characterized a novel periplasmic protein (Slr0280) that tunes the photomixotrophic growth of Synechocystis sp. PCC 6803. Slr0280 is a multi-domain protein consisting mainly of β-sheets. Several proteins that interact with Slr0280 were identified via bacterial two-hybrid screening. Slr0280 may interact through its DUF2233 domain with partners that participate in sugar metabolism, thereby coordinating the respective regulations. When slr0280 was deleted, the mutant grew more slowly than wild-type in the presence of glucose, which is ascribed to the down-regulation of glycolysis, glycogen catabolism, oxidative pentose phosphate pathway, Calvin cycle and glucose utilization. A positive regulation of Slr0280 on these sugar catabolic enzymes was confirmed by transcript (qPCR) analyses. Based on these findings, we proposed a speculative model that Slr0280 plays a coordinating regulatory role in sugar metabolism.

Photosynthesis relies on energy transfer from light-harvesting complexes to reaction centers. Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, attach to the membrane via the multidomain core-membrane linker, L(CM). The chromophore domain of L(CM) forms a bottleneck for funneling the harvested energy either productively to reaction centers or, in case of light overload, to quenchers like orange carotenoid protein (OCP) that prevent photodamage. The crystal structure of the solubly modified chromophore domain from Nostoc sp. PCC7120 was resolved at 2.2 Å. Although its protein fold is similar to the protein folds of phycobiliproteins, the phycocyanobilin (PCB) chromophore adopts ZZZssa geometry, which is unknown among phycobiliproteins but characteristic for sensory photoreceptors (phytochromes and cyanobacteriochromes). However, chromophore photoisomerization is inhibited in L(CM) by tight packing. The ZZZssa geometry of the chromophore and π-π stacking with a neighboring Trp account for the functionally relevant extreme spectral red shift of L(CM). Exciton coupling is excluded by the large distance between two PCBs in a homodimer and by preservation of the spectral features in monomers. The structure also indicates a distinct flexibility that could be involved in quenching. The conclusions from the crystal structure are supported by femtosecond transient absorption spectra in solution.

Phycobiliproteins are constituents of phycobilisomes that can harvest orange, red, and far-red light for photosynthesis in cyanobacteria and red algae. Phycobiliproteins in the phycobilisome cores, such as allophycocyanins, absorb far-red light to funnel energy to the reaction centers. Therefore, allophycocyanin subunits have been engineered as far-red fluorescent proteins, such as BDFP1.6. However, most current fluorescent probes have small Stokes shifts, which limit their applications in multicolor bioimaging. mCherry is an excellent fluorescent protein that has maximal emittance in the red spectral range and a high fluorescence quantum yield, and thus, can be used as a donor for energy transfer to a far-red acceptor, such as BDFP1.6, by FRET. In this study, mCherry was fused with BDFP1.6, which resulted in a highly bright far-red fluorescent protein, BDFP2.0, with a large Stokes shift (≈79 nm). The excitation energy was absorbed maximally at 587 nm by mCherry and transferred to BDFP1.6 efficiently; thus emitting strong far-red fluorescence maximally at 666 nm. The effective brightness of BDFP2.0 in mammalian cells was 4.2-fold higher than that of iRFP670, which has been reported as the brightest far-red fluorescent protein. The large Stokes shift of BDFP2.0 facilitates multicolor bioimaging. Therefore, BDFP2.0 not only biolabels mammalian cells, including human cells, but also biolabels various intracellular components in dual-color imaging.

Biliproteins have extended the spectral range of fluorescent proteins into the near-infrared region (NIR, 700–770 nm) of maximal transmission of most tissues and are also favorable for multiplex labeling. Their application, however, presents considerable challenges to increase their stability under physiological conditions and, in particular, to increase their brightness while maintaining the emission in near-infrared regions: their fluorescence yield generally decreases with increasing wavelengths, and their effective brightness depends strongly on the environmental conditions. We report a fluorescent biliprotein triad, termed BDFP1.1:3.1:1.1, that combines a large red-shift (722 nm) with high brightness in mammalian cells and high stability under changing environmental conditions. It is fused from derivatives of the phycobilisome core subunits, ApcE2 and ApcF2. These two subunits are induced by far-red light (FR, 650–700 nm) in FR acclimated cyanobacteria. Two BDFP1.1 domains engineered from ApcF2 covalently bind biliverdin that is accessible in most cells. The soluble BDFP3 domain, engineered from ApcE2, binds phytochromobilin non-covalently, generating BDFP3.1. This phytochromobilin chromophore was added externally; it is readily generated by an improved synthesis in E. coli and subsequent extraction. Excitation energy absorbed in the FR by covalently bound biliverdins in the two BDFP1.1 domains is transferred via fluorescence resonance energy transfer to the non-covalently bound phytochromobilin in the BDFP3.1 domain fluorescing in the NIR around 720 nm. Labeling of a variety of proteins by fusion to the biliprotein triad is demonstrated in prokaryotic and mammalian cells, including human cell lines.

Cyanobacteriochromes (CBCRs) are photoreceptors in cyanobacteria that present a bilin chromophore-binding GAF domain as a photochromic element to control the activity of a downstream enzyme or regulator. CBCR Slr1393 from Synechocystis PCC 6803 carries three GAF domains, but only the third one binds phycocyanobilin covalently. Slr1393 shows photochromicity between red and green absorbing states and regulates a C-terminally located histidine kinase. In this work, we fused this third GAF domain to an adenylyl cyclase (AC) from Microcoleus chthonoplastes PCC7420 that in its genuine form is under blue-light control from a LOV domain. A series of RGS-AC variants were constructed with various lengths of the linkers between RGS and AC. Assays in vitro and in living Escherichia coli cells (AC-deletion mutant) demonstrated that the activity of AC was light regulated, namely, the red-light-converted form of RGSΔ14-Δ4AC (in vitro) was about three times more active than the green-light-converted form. Expression of the fusion protein RGSΔ14-Δ4AC in vivo again showed highest light regulation with at least threefold amplification of the AC function. In some experiments, even tenfold higher activity was observed, which indicated that the protein, if expressed under in vivo conditions, was part of the E. coli physiological conditions and thereby subjected to more complex and variable regulation through other E. coli inherent factors.

Far-red and near-infrared emitting chromophores extend applications of fluorescent proteins to regions of maximal transmission of most tissues, but present considerable engineering challenges. Far-red adapting cyanobacteria generate a novel set of biliproteins. One of them, ApcF2, from a thermophilic cyanobacterium was subjected to structure-guided, site-directed random and specific mutagenesis, and was screened for bright far-red emission. We report the generation of chromoproteins, termed BDFPs, that are small, bind auto-catalytically the ubiquitous biliverdin as chromophore, express well, and retain their fluorescence in mammalian cells and in the nematode, C. elegans. They are, moreover, photostable and tolerate high temperature, low pH and chemical denaturation. Homo-bichromophoric tandems of these proteins improve labeling, while hetero-bichromophoric systems with large Stokes shifts are suitable for applications like FRET, multi-channel or super-resolution microscopy. The BDFPs compare favorably to other biliproteins and provide a novel, extremely versatile labeling tool-box.

The initial light-induced processes of the photochromic, phycocyanobilin-binding GAF domain of Slr1393 from Synechocystis sp. PCC6803 have been studied by ultrafast transient absorption spectroscopy. We use lifetime density analysis as a model-independent method for the evolution of the experimental data, which gives a comprehensive overview of the excitation wavelength dependence of the photoconversion kinetics. The method is particularly suitable for this highly complex and not purely exponential kinetics. In contrast to previously studied cyanobacteriochromes (CBCRs), here both the red- and the green-absorbing forms show significantly slower reaction dynamics, which proceed also via ground state intermediates. The photoconversion of the green-absorbing form is faster than that of the red state, which allowed a clear detection of the primary photoproduct Lumi-G. Strong coherent oscillations of the recorded transient absorption due to wavepacket motion on the excited state potential energy surface were observed and analyzed for both (red and green) forms of Slr1393g3. The vibrational modes responsible for the coherent oscillations could play a role in the dynamics of the initially heterogeneous excited state (ES) population and direct the system towards the minima on the potential energy surface that determine the ES decay pathway. Furthermore, the coherent oscillations appear to be a common feature of bilin-based photoreceptors and thus deserve further attention. The investigated CBCR exhibits an extraordinary high level of heterogeneity due to the remarkable flexibility of the phycocyanobilin and the protein binding pocket. These properties should allow spectrally tuned response to the light stimuli and thus have significant biological implications.

The brightly fluorescent phycobiliproteins are light-harvesting pigments in cyanobacteria and certain algae, covering almost the entire visible spectrum. Another type of biliproteins, the phytochrome family, comprises regulatory photoswitches in plants, fungi and many bacteria; their spectra cover an even wider range extending into the near-infrared and near-ultraviolet. In both types, the chromophores can be tuned by modulating chromophore-protein interactions, including the transition between fluorescence and photoswitching. These properties render biliproteins useful for bioimaging. Applications require not only the introduction of genes coding for apoproteins, but also genes coding for biosynthesis of the chromophores. Strategies for tuning and heterologous biosynthesis will be discussed.

AbbreviationsASUasymmetric unitBV, PCB, PVB (bilin compounds serving as chromophores)biliverdin Ixα, phycocyanobilin, phycoviolobilinCAPSON-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acidCBCRcyanobacteriochromeGAF (protein domain)cGMP-specific phosphodiesterases adenylyl cyclases and FhlAIMACimmobilized metal-affinity chromatographyMRmolecular replacementPAS (protein domain)Per-Arnt-SimPHY (protein domain)phytochrome-specificPfr, Pg, Prfar red-, green-, and red-absorbing states of phytochromes and CBCRsSummaryThe GAF3 domain of cyanobacteriochrome Slr1393 (Synechocystis PCC6803) with an in vivo assembled phycocyanobilin (PCB) chromophore has been crystallized in parental state (1.8 Å) and photoproduct state (1.86 Å), identified by 15-Z and 15-E chromophore configuration. Comparison of both structures for the same protein allows precise determination of structural changes after photo-activation. The chromophore photoisomerization causes an outward movement and partial helix formation of a formerly unstructured loop. A tryptophan residue located in this loop, in π-π stacking distance to PCB in the dark state, moves away by 14 Å opening the binding cleft for the entry of water molecules. Also the in vitro assembled protein (chromophore addition to apo-protein) has been crystallized (1.6 Å resolution). Most importantly, an intermediate structure was solved (2.1 Å) with the protein in photoproduct conformation and the chromophore already isomerized into the parental 15-Z configuration, thereby giving insight into chromophore-initiated conformational protein changes.Impact StatementThis manuscript presents crystal structures of a photochromic protein in both states, before (1.6 Å) and after (1.9 Å) the light induced photochemical event with sufficient resolution to allow detailed description of conformational changes of chromophore and protein. The light driven reaction, double bond photoisomerization of a covalently bound bilin chromophore is presented here for the first time. Our results allow determining the impact of the chromophore photochemistry on the protein conformation. In addition, we succeeded in trapping an intermediate carrying the chromophore already in isomerized state with the protein still in unchanged conformation. Absorption spectra of this intermediate clearly demonstrate a color change, thus allowing conclusion that the absorption of phytochromes is predominantly determined by the chromophore conformation alone with only moderate effect of the surrounding protein.Authors’ ContributionsXX, KHZ, and WG designed the experiment. XX generated the protein. AH performed crystallization trials, collected the X-ray diffraction data and solved the structure. All authors contributed in preparing the manuscript.

Biliproteins have extended the spectral range of fluorescent proteins into the region of maximal transmission of most tissues and are favorable for multiplexing, but their application presents considerable challenges. Their fluorescence derives from open-chain tetrapyrrole chromophores which often require the introduction of dedicated reductases and lyases. In addition, their fluorescence yield generally decreases with increasing wavelengths and depends strongly on the state of the binding protein. We report fluorescent biliproteins, termed BDFPs, that are derived from the phycobilisome core subunit, ApcF2: this subunit is induced in the thermophilic cyanobacterium, Chroococcidiopsis thermalis, by far-red light and binds phycocyanobilin non-covalently. The BDFPs obtained by molecular evolution of ApcF2 bind the more readily accessible biliverdin covalently while retaining the red-shifted fluorescence in the near-infrared spectral region (~710nm). They are small monomers (~15kDa) and not only show excellent photostability, but are also thermostable up to 80°C, tolerate acid down to pH2 and high concentrations of denaturants. The result indicates far-red adapting cyanobacteria as a useful source for designing extremely red-shifted fluorescent markers. In vivo performance of BDFPs as biomarkers in conventional and super-resolution microscopy, alone or fused to target proteins, is exemplified in several mammalian cells, including, human cell lines, in the nematode, Caenorhabditis elegans and, at low pH, in Lactobacillus lactis.

Time-resolved photoacoustics (PA) is uniquely able to explore the energy landscape of photoactive proteins and concomitantly detects light-induced volumetric changes (ΔV) accompanying the formation and decay of transient species in a time window between ca. 20 ns and 5 μs. Here, we report PA measurements on diverse photochromic bilin-binding photoreceptors of prokaryotic origin: (1) the chromophore-binding GAF3 domain of the red (R)/green (G) switching cyanobacteriochrome 1393 (Slr1393g3) from Synechocystis; (2) the red/far red (R/FR) Synechocystis Cph1 phytochrome; (3) full-length and truncated constructs of Xanthomonas campestris bacteriophytochrome (XccBphP), absorbing up to the NIR spectral region. In almost all cases, photoisomerization results in a large fraction of energy dissipated as heat (up to 90%) on the sub-ns scale, reflecting the low photoisomerization quantum yield (

Proceedings of the National Academy of Sciences of the United States of America 114(50), 13170 - 13175 (2017). doi:10.1073/pnas.1715495114, The light-harvesting phycobilisome in cyanobacteria and red algae requires the lyase-catalyzed chromophorylation of phycobiliproteins. There are three functionally distinct lyase families known. The heterodimeric E/F type is specific for attaching bilins covalently to α-subunits of phycocyanins and phycoerythrins. Unlike other lyases, the lyase also has chromophore-detaching activity. A subclass of the E/F-type lyases is, furthermore, capable of chemically modifying the chromophore. Although these enzymes were characterized >25 y ago, their structures remained unknown. We determined the crystal structure of the heterodimer of CpcE/F from Nostoc sp. PCC7120 at 1.89-Å resolution. Both subunits are twisted, crescent-shaped α-solenoid structures. CpcE has 15 and CpcF 10 helices. The inner (concave) layer of CpcE (helices h2, 4, 6, 8, 10, 12, and 14) and the outer (convex) layer of CpcF (h16, 18, 20, 22, and 24) form a cavity into which the phycocyanobilin chromophore can be modeled. This location of the chromophore is supported by mutations at the interface between the subunits and within the cavity. The structure of a structurally related, isomerizing lyase, PecE/F, that converts phycocyanobilin into phycoviolobilin, was modeled using the CpcE/F structure as template. A H$_{87}$C$_{88}$ motif critical for the isomerase activity of PecE/F is located at the loop between h20 and h21, supporting the proposal that the nucleophilic addition of Cys-88 to C10 of phycocyanobilin induces the isomerization of phycocyanobilin into phycoviolobilin. Also, the structure of NblB, involved in phycobilisome degradation could be modeled using CpcE as template. Combined with CpcF, NblB shows a low chromophore-detaching activity., Published by National Acad. of Sciences, Washington, DC

Cyanobacteriochromes (CBCRs) are chromophorylated proteins that acting as sensory photoreceptors in cyanobacteria. Based on the bioinformatics of All4261 in Nostoc sp. PCC7120, All4261 is a CBCR apoprotein composed of GAF domains in the N-terminal region. Via polymerase chain reaction with specific primers, All4261 was amplified with genome DNA of Nostoc sp. PCC7120 as template and then subcloned into the expression vector pET30(a+). To survey the fluorescence spectral properties, All4261 was coexpressed with the plasmid that catalyzes phycocyanobilin (PCB) biosynthesis, pACYC-ho1-pcyA, in E.coli BL21. Fluorescence emission spectra and excitation spectra showed that chromophorylated cells containing All4261-PCB had a fluorescence emission peak at 645 nm and a fluorescence excitation peak at 550 nm, but no reversible photoconversion. In order to identify the binding site of PCB in All4261, we obtained three variants All4261(C296L), All4261(C328A), and All4261(C339L), via sitedirected mutagenesis. The binding site was identified as C339 based on the lack of PCB binding of All4261(C339L).

Cyanobacteriochromes are a structurally and spectrally highly diverse class of phytochrome-related photosensory biliproteins. They contain one or more GAF domains that bind phycocyanobilin (PCB) autocatalytically; some of these proteins are also capable of further modifying PCB to phycoviolobilin or rubins. We tested the chromophorylation with the non-photochromic phycoerythrobilin (PEB) of 16 cyanobacteriochrome GAFs from Nostoc sp. PCC 7120, of Slr1393 from Synechocystis sp. PCC 6803, and of Tlr0911 from Thermosynechococcus elongatus BP-1. Nine GAFs could be autocatalytically chromophorylated in vivo/in E. coli with PEB, resulting in highly fluorescent biliproteins with brightness comparable to that of fluorescent proteins like GFP. In several GAFs, PEB was concomitantly converted to phycourobilin (PUB) during binding. This not only shifted the spectra, but also increased the Stokes shift. The chromophorylated GAFs could be oligomerized further by attaching a GCN4 leucine zipper domain, thereby enhancing the absorbance and fluorescence of the complexes. The presence of both PEB and PUB makes these oligomeric GAF-"bundles" interesting models for energy transfer akin to the antenna complexes found in cyanobacterial phycobilisomes. The thermal and photochemical stability and their strong brightness make these constructs promising orange fluorescent biomarkers.

Phycobiliproteins are brightly-fluorescent light-harvesting pigments for photosynthesis in cyanobacteria and red algae. They are also of interest as fluorescent biomarkers, but their heterologous generation in vivo has previously required multiple transformations. We report here a modular approach that requires only two DNA segments. The first codes for the apo-protein. The second codes for fusions capable of chromophore biosynthesis and its covalent attachment to the apo-protein; it contains the genes of heme oxygenase, a bilin reductase, and a chromophore lyase. Phycobiliproteins containing phycoerythrobilin (λ(fluor) ~ 560 nm), phycourobilin (λ(fluor) ~ 500 nm), phycocyanobilin (λ(fluor) ~ 630 nm) or phycoviolobilin (λ(fluor) ~ 580 nm) were obtained in high yield in E. coli. This approach facilitates chromophorylation studies of phycobiliproteins, as well as their use for fluorescence labeling based on their high fluorescence.

The phycocyanin lyase CpcT1 (encoded by gene all5339) and lyase CpcS1 (encoded by gene alr0617) are capable of catalyzing the phycocyanobilin (PCB) covalently bound to the different sites of phycocyanin's and phycoerythrocyanin's β subunits, respectively. Lyase CpcS1, whose catalytic mechanism had been researched clearly, participates in the covalent coupling of phycobilin and apoprotein in the form of chaperone, and its important amino acids have been confirmed. In order to identify the functional amino acid residues of CpcT1, chemical modification was conducted to arginine, histidine, tryptophan, lysine and amino acid carboxyl of CpcT1. The results indicated that the catalytic activity of the CpcT1 was changed. After the modification of arginine, tryptophan and histidine, site-directed mutations were performed to those highly conserved amino acids which were selected by means of homologous comparison. The mutated lyase, apoprotein and the enzymes that synthesize the phycobilins were recombined in Escherichia coli (E. coli) and in vitro, yielding chromoproteins, which were detected by fluorescence and UV absorption spectrometry. The spectra were compared with that of the chromoprotein catalyzed by wild type lyase CpcT1, achieving relative specific activities of the various mutants. Meanwhile, the mutants were expressed in E. coli, and then circular dichroism structure of near-UV region was determined. The results demonstrated that H33F, W175S, R97A, C137S and C116S influence the catalytic activity of CpcT1. Being different from wild CpcT1, a great deal of α helix was involved in the structure of circular dichroism of R97A and W13S. CpcT1 or its mutants and the enzymes that synthesize the phycobilins, were reconstituted in E. coli and detected by spectra to check the bounding of lyases and PCB. The results of spectra and SDS-PAGE confirm that CpcT1 and its mutants cannot bind phycobilin, differing from the catalytic mechanism of CpcS1.