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Photosynthetic organisms have developed various light-harvesting systems to adapt to their environments 1 . Phycobilisomes are large light-harvesting protein complexes found in cyanobacteria and red algae 2 – 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 pacifica 5 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. The cryo-electron microscopy structure of a phycobilisome from the red alga Porphyridium purpureum reveals how aromatic interactions between the linker proteins and the chromophores drive a unidirectional transfer of energy.

Phycobiliproteins (PBPs), captivating water-soluble proteins found in cyanobacteria, red algae, and cryptophytes, continue to fascinate researchers and industries due to their unique properties and multifaceted applications. These proteins consist of chromophores called phycobilins (PBs), covalently linked to specific protein subunits. Major phycobiliproteins include phycocyanin (PC), allophycocyanin (APC), and phycoerythrin (PE), each distinguished by distinct absorption and emission spectra. Beyond their colorful properties, PBs exhibit a broad spectrum of biological activities, including antibacterial, antifungal, antiviral, and antidiabetic effects, making them valuable for pharmaceutical, biotechnological, and medical purposes. The extraction and purification methods for PBs have been optimized to enhance their bioavailability and stability, opening new avenues for industrial production. For this review, a comprehensive literature search was conducted using scientific databases such as PubMed, Scopus, and Web of Science, prioritizing peer-reviewed articles published between 2000 and 2025, with an emphasis on recent advances from the last five years, using keywords such as “phycobiliproteins”, “phycobilins”, “bioactivities”, “therapeutic applications”, and “industrial use”. Studies were selected based on their relevance to the biological, technological, and pharmacological applications of PBPs and PBs. This review explores the diverse applications of PBs in therapeutic, diagnostic, and environmental fields, highlighting their potential as natural alternatives in the treatment of various diseases. The future perspectives for PBs focus on their incorporation into innovative drug delivery systems, biocompatible materials, and functional foods, presenting exciting opportunities for advancing human health and well-being.

Phycocyanobilin (PCB) is a blue pigment with antioxidant, anti-inflammatory, and anticancer properties. It is used in the medical and cosmetic industries. In this study, a high-expression plasmid, pET-30a-PCB, was constructed for expression of PCB in Escherichia coli BL21(DE3). The PCB was analyzed using UV–visible absorption spectrum, MALDI-TOF-MS, and fluorescence spectra. The stability and half-life of PCB in different serum were determined. The yield of PCB was optimized through single-factor and orthogonal experiments. The optimal expression conditions were determined as a lactose concentration of 5 mmol/L, an induction time of 8 h, an induction temperature of 27 °C, and an induction duration of 22 h. PCB yield of 6.5 mg/L was achieved and subsequently purified using nickel-affinity chromatography. The purified PCB was quantified indirectly using Hist-tag ELISA detection, and the concentration was 11.66 μg/L. In the range of 0–33 μg/mL, the total antioxidant capacity and reducing the capacity of PCB were stronger than Vitamin E (Ve), with 1,1-diphenyl-2-picrylhydrazil (DPPH) scavenging reaching up to 87.07%, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate) free radical (ABTS) scavenging up to 100%, hydroxyl radicals (·OH) scavenging up to 64.19%, hydrogen peroxide (H 2 O 2 ) scavenging up to 78.75%, This study provides theoretical evidence for PCB as a potent antioxidant.

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole-binding photoreceptors in cyanobacteria that absorb visible and near-ultraviolet light. CBCRs are divided into two types based on the type of chromophore they contain: phycocyanobilin (PCB) or phycoviolobilin (PVB). PCB-binding CBCRs reversibly photoconvert at relatively long wavelengths, i.e., the blue-to-red region, whereas PVB-binding CBCRs reversibly photoconvert at shorter wavelengths, i.e., the near-ultraviolet to green region. Notably, prior to this report, CBCRs containing biliverdin (BV), which absorbs at longer wavelengths than do PCB and PVB, have not been found. Herein, we report that the typical red/green CBCR AM1_1557 from the chlorophyll d –bearing cyanobacterium Acaryochloris marina can bind BV almost comparable to PCB. This BV-bound holoprotein reversibly photoconverts between a far red light–absorbing form (Pfr, λmax = 697 nm) and an orange light–absorbing form (Po, λmax = 622 nm). At room temperature, Pfr fluoresces with a maximum at 730 nm. These spectral features are red-shifted by 48~77 nm compared with those of the PCB-bound domain. Because the absorbance of chlorophyll d is red-shifted compared with that of chlorophyll a , the BV-bound AM1_1557 may be a physiologically relevant feature of A. marina and is potentially useful as an optogenetic switch and/or fluorescence imager.

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.

Phycobilisomes are highly organized pigment–protein antenna complexes found in the photosynthetic apparatus of cyanobacteria and rhodophyta that harvest solar energy and transport it to the reaction center. A detailed bottom-up model of pigment organization and energy transfer in phycobilisomes is essential to understanding photosynthesis in these organisms and informing rational design of artificial light-harvesting systems. In particular, heterogeneous photophysical behaviors of these proteins, which cannot be predicted de novo, may play an essential role in rapid light adaptation and photoprotection. Furthermore, the delicate architecture of these pigment–protein scaffolds sensitizes them to external perturbations, for example, surface attachment, which can be avoided by study in free solution or in vivo. Here, we present single-molecule characterization of C-phycocyanin (C-PC), a three-pigment biliprotein that self-assembles to form the midantenna rods of cyanobacterial phycobilisomes. Using the Anti-Brownian Electrokinetic (ABEL) trap to counteract Brownian motion of single particles in real time, we directly monitor the changing photophysical states of individual C-PC monomers from Spirulina platensis in free solution by simultaneous readout of their brightness, fluorescence anisotropy, fluorescence lifetime, and emission spectra. These include single-chromophore emission states for each of the three covalently bound phycocyanobilins, providing direct measurements of the spectra and photophysics of these chemically identical molecules in their native protein environment. We further show that a simple Förster resonant energy transfer (FRET) network model accurately predicts the observed photophysical states of C-PC and suggests highly variable quenching behavior of one of the chromophores, which should inform future studies of higher-order complexes.

Arthrospira platensis has been utilized as a food source since ancient times due to its rich nutrient profile. In recent years, its popularity as a dietary supplement has soared, especially due to the presence of a water-soluble phycobiliprotein, C-phycocyanin C (C-PC), which is abundant and notable for its fluorescent properties. C-PC contains the chromophore phycocyanobilin B (PCB-B), a tetrapyrrole molecule, that is why it plays a dual role as a food colorant and as nutraceutical. However, comprehensive studies have mostly evaluated C-PC’s broader health-promoting properties, particularly its antioxidative and anti-inflammatory effects, which are linked to its ability to contrast oxidative stress and related pathological conditions. That is why this review explores recent advancements in optimizing culture conditions to enhance C-PC and PCB-B production, with a particular emphasis on novel extraction and purification techniques that increase yield and bioactivity. This focus on efficient production methods is crucial for expanding the commercial and therapeutic applications of C-PC, contributing to its growing relevance in the food and pharmaceutical industries

Land plant phytochromes perceive red and far-red light to control growth and development, using the linear tetrapyrrole (bilin) chromophore phytochromobilin (PΦB). Phytochromes from streptophyte algae, sister species to land plants, instead use phycocyanobilin (PCB). PCB and PΦB are synthesized by different ferredoxin-dependent bilin reductases (FDBRs): PΦB is synthesized by HY2, whereas PCB is synthesized by PcyA. The pathway for PCB biosynthesis in streptophyte algae is unknown. We used phylogenetic analysis and heterologous reconstitution of bilin biosynthesis to investigate bilin biosynthesis in streptophyte algae. Phylogenetic results suggest that PcyA is present in chlorophytes and prasinophytes but absent in streptophytes. A system reconstituting bilin biosynthesis in Escherichia coli was modified to utilize HY2 from the streptophyte alga Klebsormidium flaccidum (KflaHY2). The resulting bilin was incorporated into model cyanobacterial photoreceptors and into phytochrome from the early-diverging streptophyte alga Mesostigma viride (MvirPHY1). All photoreceptors tested incorporate PCB rather than PΦB, indicating that KflaHY2 is sufficient for PCB synthesis without any other algal protein. MvirPHY1 exhibits a red–far-red photocycle similar to those seen in other streptophyte algal phytochromes. These results demonstrate that streptophyte algae use HY2 to synthesize PCB, consistent with the hypothesis that PΦB synthesis arose late in HY2 evolution.

Phycocyanobilin (PCB) binds with high affinity (2.2 x 106 M-1 at 25°C) to human serum albumin (HSA) at sites located in IB and IIA subdomains. The aim of this study was to examine effects of PCB binding on protein conformation and stability. Using 300 ns molecular dynamics (MD) simulations, UV-VIS spectrophotometry, CD, FT-IR, spectrofluorimetry, thermal denaturation and susceptibility to trypsin digestion, we studied the effects of PCB binding on the stability and rigidity of HSA, as well as the conformational changes in PCB itself upon binding to the protein. MD simulation results demonstrated that HSA with PCB bound at any of the two sites showed greater rigidity and lower overall and individual domain flexibility compared to free HSA. Experimental data demonstrated an increase in the α-helical content of the protein and thermal and proteolytic stability upon ligand binding. PCB bound to HSA undergoes a conformational change to a more elongated conformation in the binding pockets of HSA. PCB binding to HSA stabilizes the structure of this flexible transport protein, making it more thermostable and resistant to proteolysis. The results from this work explain at molecular level, conformational changes and stabilization of HSA structure upon ligand binding. The resultant increased thermal and proteolytic stability of HSA may provide greater longevity to HSA in plasma.

Fluorescent proteins (FP) are used to study various biological processes. Recently, a series of near-infrared (NIR) FPs based on bacterial phytochromes was developed. Finding ways to improve NIR FPs is becoming progressively important. By applying rational design and molecular evolution we have engineered R. palustris bacterial phytochrome into a single-domain NIR FP of 19.6 kDa, termed GAF-FP, which is 2-fold and 1.4-fold smaller than bacterial phytochrome-based NIR FPs and GFP-like proteins, respectively. Engineering of GAF-FP involved a substitution of 15% of its amino acids and a deletion of the knot structure. GAF-FP covalently binds two tetrapyrrole chromophores, biliverdin (BV) and phycocyanobilin (PCB). With the BV chromophore GAF-FP absorbs at 635 nm and fluoresces at 670 nm. With the PCB chromophore GAF-FP becomes blue-shifted and absorbs at 625 nm and fluoresces at 657 nm. The GAF-FP structure has a high tolerance to small peptide insertions. The small size of GAF-FP and its additional absorbance band in the violet range has allowed for designing a chimeric protein with Renilla luciferase. The chimera exhibits efficient non-radiative energy transfer from luciferase to GAF-FP, resulting in NIR bioluminescence. This study opens the way for engineering of small NIR FPs and NIR luciferases from bacterial phytochromes.