Certain cyanobacteria alter their photosynthetic light absorption between green and red, a phenomenon called complementary chromatic acclimation. The acclimation is regulated by a cyanobacteriochrome-class photosensor that reversibly photoconverts between green-absorbing (Pg) and red-absorbing (Pr) states. Here, we elucidated the structural basis of the green/red photocycle. In the Pg state, the bilin chromophore adopted the extended C15-Z,anti structure within a hydrophobic pocket. Upon photoconversion to the Pr state, the bilin is isomerized to the cyclic C15-E,syn structure, forming a water channel in the pocket. The solvation/desolvation of the bilin causes changes in the protonation state and the stability of π-conjugation at the B ring, leading to a large absorption shift. These results advance our understanding of the enormous spectral diversity of the phytochrome superfamily.

Far-red cyanobacteriochromes (CBCRs) are bilin-based photosensory proteins that promise to be novel optical agents in optogenetics and deep tissue imaging. Recent structural studies of a far-red CBCR 2551g3 have revealed a unique all-Z,syn chromophore conformation in the far-red-absorbing Pfr state. Understanding the photoswitching mechanism through bilin photoisomerization is important for developing novel biomedical applications. Here, we employ femtosecond spectroscopy and site-directed mutagenesis to systematically characterize the dynamics of wild-type 2551g3 and four critical mutants in the 15Z Pfr state. We captured local relaxations in several picoseconds and isomerization dynamics in hundreds of picoseconds. Most mutants exhibited faster local relaxation, while their twisting dynamics and photoproducts depend on specific protein-chromophore interactions around the D-ring and C-ring. These results collectively reveal a unique dynamic pattern of excited-state evolution arising from a relatively rigid protein environment, thereby elucidating the molecular mechanism of Pfr-state photoisomerization in far-red CBCRs.

As plant photoreceptors, phytochromes are capable of detecting red light and far-red light, thereby governing plant growth. All2699 is a photoreceptor found in Nostoc sp. PCC7120 that specifically responds to red light and far-red light. All2699g1g2 is a truncated protein carrying the first and second GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains of All2699. In this study, we found that, upon exposure to red light, the protein underwent aggregation, resulting in the formation of protein aggregates. Conversely, under far-red light irradiation, these protein aggregates dissociated. We delved into the factors that impact the aggregation of All2699g1g2, focusing on the protein structure. Our findings showed that the GAF2 domain contains a low-complexity (LC) loop region, which plays a crucial role in mediating protein aggregation. Specifically, phenylalanine at position 239 within the LC loop region was identified as a key site for the aggregation process. Furthermore, our research revealed that various factors, including irradiation time, temperature, concentration, NaCl concentration, and pH value, can impact the aggregation of All2699g1g2. The aggregation led to variations in Pfr concentration depending on temperature, NaCl concentration, and pH value. In contrast, ΔLC did not aggregate and therefore lacked responses to these factors. Consequently, the LC loop region of All2699g1g2 extended and enhanced sensory properties.

BACKGROUND: Biliary intraepithelial neoplasia (BilIN), a noninvasive precursor of cholangiocarcinoma, can manifest malignant transformation. Since cholangiocarcinoma (CCA) may progress due to chronic inflammation in the bile ducts and gallbladder, choledochal cysts are considered a precursor to CCA. However, BilIN has rarely been reported in children, to date.
METHODS: We reviewed medical records of patients (< 18 years of age, n = 329) who underwent choledochal cyst excision at Asan Medical Center from 2008 to 2022. BilIN was diagnosed in 15 patients. Subsequent analyses were performed of the demographics, surgical procedures, clinical course, and outcomes in these patients. Subgroup analysis and multivariate logistic regression test were performed to identify factors influencing BilIN occurrence.
RESULTS: The mean age of the patients included in our study was 40.1 ± 47.6 months. In 15 patients, BilIN of various grades was diagnosed. Todani type I was prevalent in 80% of the patients. The median age at surgery was 17 months. During a mean follow-up of 63.3 ± 94.0 months, no adverse events such as stone formation in the remnant intrapancreatic common bile duct and intrahepatic duct or cholangiocarcinoma were observed, indicating a favorable outcome until now.
CONCLUSIONS: The potential progression of choledochal cysts to BilIN in children was demonstrated. These results could underscore the importance of early and comprehensive excision of choledochal cysts, including resection margins for associated lesions and more thorough postoperative surveillance in patients with or at risk of BilIN.

Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B12) can act as a light-sensing chromophore heralded a new field of B12-photobiology. Although microbial genome analysis indicates that photoactive B12-binding domains form part of more complex protein architectures, regulating a range of molecular-cellular functions in response to light, experimental evidence is lacking. Here we identify and characterise a sub-family of multi-centre photoreceptors, termed photocobilins, that use B12 and biliverdin (BV) to sense light across the visible spectrum. Crystal structures reveal close juxtaposition of the B12 and BV chromophores, an arrangement that facilitates optical coupling. Light-triggered conversion of the B12 affects quaternary structure, in turn leading to light-activation of associated enzyme domains. The apparent widespread nature of photocobilins implies involvement in light regulation of a wider array of biochemical processes, and thus expands the scope for B12 photobiology. Their characterisation provides inspiration for the design of broad-spectrum optogenetic tools and next generation bio-photocatalysts.

Phytochromes constitute a family of photosensory proteins that are utilized by various organisms to regulate several physiological processes. Phytochromes bind a bilin pigment that switches its isomeric state upon absorption of red or far-red photons, resulting in protein conformational changes that are sensed by the organism. Previously, the ultrafast dynamics in bacterial phytochrome was resolved to atomic resolution by time-resolved serial femtosecond X-ray diffraction (TR-SFX), showing extensive changes in its molecular conformation at 1 picosecond delay time. However, the large excitation fluence of mJ/mm2 used in TR-SFX questions the validity of the observed dynamics. In this work, we present an excitation-dependent ultrafast transient absorption study to test the response of a related bacterial phytochrome to excitation fluence. We observe excitation power-dependent sub-picosecond dynamics, assigned to the population of high-lying excited state Sn through resonantly enhanced two-photon absorption, followed by rapid internal conversion to the low-lying S1 state. Inspection of the long-lived spectrum under high fluence shows that in addition to the primary intermediate Lumi-R, spectroscopic signatures of solvated electrons and ionized chromophore radicals are observed. Supported by numerical modelling, we propose that under excitation fluences of tens of μJ/mm2 and higher, bacterial phytochrome partly undergoes photoionization from the Sn state in competition with internal conversion to the S1 state in 300 fs. We suggest that the extensive structural changes of related, shorter bacterial phytochrome, lacking the PHY domain, resolved from TR-SFX may have been affected by the ionized species. We propose approaches to minimize the two-photon absorption process by tuning the excitation spectrum away from the S1 absorption or using phytochromes exhibiting minimized or shifted S1 absorption.

Cyanobacteriochrome (CBCR)-derived fluorescent proteins are a class of reporters that can bind bilin cofactors and fluoresce across the ultraviolet to the near-infrared spectrum. Derived from phytochrome-related photoreceptor proteins in cyanobacteria, many of these proteins use a single small GAF domain to autocatalytically bind a bilin and fluoresce. The second GAF domain of All1280 (All1280g2) from Nostoc sp. PCC7120 is a DXCF motif-containing protein that exhibits blue-light-responsive photochemistry when bound to its native cofactor, phycocyanobilin. All1280g2 can also bind non-photoswitching phycoerythrobilin (PEB), resulting in a highly fluorescent protein. Given the small size, high quantum yield, and that unlike green fluorescent proteins, bilin-binding proteins can be used in anaerobic organisms, the orange fluorescent All1280g2-PEB protein is a promising platform for designing new genetically encoded metal ion sensors. Here, we show that All1280g2-PEB undergoes a ∼5-fold reversible zinc-induced fluorescence enhancement with a blue-shifted emission maximum (572 to 517 nm), which is not observed for a related PEB-bound GAF from Synechocystis sp. PCC6803 (Slr1393g3). Zn2+ significantly enhances All1280g2-PEB fluorescence across a biologically relevant pH range from 6.0 to 9.0, with pH-dependent dissociation constants from 1 μM to ∼20-80 nM. Site-directed mutants aiming to sterically decrease and increase access to PEB show a decreased and similar amount of zinc-induced fluorescence enhancement. Mutation of the cysteine residue within the DXCF motif to alanine abolishes the zinc-induced fluorescence enhancement. Collectively, these results support the presence of a unique fluorescence-enhancing Zn2+ binding site in All1280g2-PEB likely involving coordination to the bilin cofactor and requiring a nearby cysteine residue.

Butterflies often have conspicuously patterned wings, due to pigmentary and/or structurally wing scales that cover the wing membrane. The wing membrane of several butterfly species is also pigmentary coloured, notably by the bile pigments pterobilin, pharcobilin and sarpedobilin. The absorption spectra of the bilins have bands in the ultraviolet and red wavelength range, resulting in blue-cyan colours. Here, a survey of papilionoid and nymphalid butterflies reveals that several species with wings containing bile pigments combine them with carotenoids and other short-wavelength absorbing pigments, e.g., papiliochrome II, ommochromes and flavonoids, which creates green-coloured patterns. Various uncharacterized, long-wavelength absorbing wing pigments were encountered, particularly in heliconiines. The wings thus exhibit quite variable reflectance spectra, extending the enormous pigmentary and structural colouration richness of butterflies.

Phytochromes are biliprotein photoreceptors present in plants, algae, certain bacteria, and fungi. Land plant phytochromes use phytochromobilin (PΦB) as the bilin chromophore. Phytochromes of streptophyte algae, the clade within which land plants evolved, employ phycocyanobilin (PCB), leading to a more blue-shifted absorption spectrum. Both chromophores are synthesized by ferredoxin-dependent bilin reductases (FDBRs) starting from biliverdin IXα (BV). In cyanobacteria and chlorophyta, BV is reduced to PCB by the FDBR phycocyanobilin:ferredoxin oxidoreductase (PcyA), whereas, in land plants, BV is reduced to PФB by phytochromobilin synthase (HY2). However, phylogenetic studies suggested the absence of any ortholog of PcyA in streptophyte algae and the presence of only PФB biosynthesis-related genes (HY2). The HY2 of the streptophyte alga Klebsormidium nitens (formerly Klebsormidium flaccidum) has already indirectly been indicated to participate in PCB biosynthesis. Here, we overexpressed and purified a His6-tagged variant of K. nitens HY2 (KflaHY2) in Escherichia coli. Employing anaerobic bilin reductase activity assays and coupled phytochrome assembly assays, we confirmed the product and identified intermediates of the reaction. Site-directed mutagenesis revealed 2 aspartate residues critical for catalysis. While it was not possible to convert KflaHY2 into a PΦB-producing enzyme by simply exchanging the catalytic pair, the biochemical investigation of 2 additional members of the HY2 lineage enabled us to define 2 distinct clades, the PCB-HY2 and the PΦB-HY2 clade. Overall, our study gives insight into the evolution of the HY2 lineage of FDBRs.

The wings of the purple spotted swallowtail Graphium weiskei are marked by an unusual bright colour pattern. Spectrophotometry on G. weiskei wings demonstrated the presence of a pigment with an absorption spectrum (peak wavelength λmax=676 nm) similar to that of the bile pigment sarpedobilin in the wings of the congeneric Graphium sarpedon (λmax=672 nm). Sarpedobilin alone causes cyan-blue wing areas, but the green-coloured areas of G. sarpedon wings result from subtractive colour mixing with the carotenoid lutein. Reflectance spectra of the blue-coloured areas of G. weiskei wings indicate that sarpedobilin is mixed with the short-wavelength-absorbing papiliochrome II. An enigmatic pigment, tentatively called weiskeipigment (λmax=580 nm), enhances the saturation of the blue colour. Weiskeipigment causes a purple colour in areas where the sarpedobilin concentration is low. The wings of the related papilionid Papilio phorcas contain the bile pigment pharcobilin (λmax=604 nm), as well as another sarpedobilin (λmax=663 nm). The cyan to greenish wings of P. phorcas are due to phorcabilin and sarpedobilin mixed with papiliochrome II. A survey of known subspecies of G. weiskei as well as of congeneric Graphium species of the \'weiskei\' group shows various degrees of subtractive colour mixing of bilins and short-wavelength absorbers (carotenoids and/or papiliochromes) in their wings. This study illuminates the underestimated role of bile pigments in butterfly wing colouration.