Phytochromobilin Binding and Specific Amino Acid Residues Near The Chromophore Contribute To Orange Light Perception By The Dualchrome Phytochrome Region.

A novel photoreceptor dualchrome 1 (DUC1), containing a fused structure of cryptochrome and phytochrome, was discovered in the marine green alga Pycnococcus provasolli. The DUC1 phytochrome region (PpDUC1-N) binds to the bilin (linear tetrapyrrole) chromophores, phytochromobilin (PΦB) or phycocyanobilin (PCB), and reversibly photoconverts between the orange-absorbing dark-adapted state and the far-red-absorbing photoproduct state. This contrasts with typical phytochromes, which photoconvert between the red-absorbing dark-adapted and far-red-absorbing photoproduct states. In this study, we examined the molecular mechanism of PpDUC1-N to sense orange light by identifying the chromophore species synthesized by P. provasolli and the amino acid residues within the PpDUC1-N responsible for sensing orange light in the dark-adapted state. We focused on the PcyA homolog of P. provasolli (PpPcyA). Coexpression with the photoreceptors followed by an enzymatic assay revealed that PpPcyA synthesized PCB. Next, we focused on the PpDUC1-N GAF domain responsible for chromophore binding and light sensing. Ten amino acid residues were selected as the mutagenesis target near the chromophore. Replacement of these residues with those conserved in typical phytochromes revealed that three mutations (F290Y/M304S/L353M) resulted in a 23-nm red-shift in the dark-adapted state. Finally, we combined these constructs to obtain the PΦB-binding F290Y/M304S/L353M mutant and a 38-nm red-shift was observed compared with the PCB-binding wild-type PpDUC1. The binding chromophore species and the key residues near the chromophore contribute to blue-shifted orange light sensing in the dark-adapted state of the PpDUC1-N.

Conformational change in an engineered biliverdin-binding cyanobacteriochrome during the photoconversion process.

Cyanobacteriochromes (CBCRs) derived from cyanobacteria are linear-tetrapyrrole-binding photoreceptors related to the canonical red/far-red reversible phytochrome photoreceptors. CBCRs contain chromophore-binding cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that are highly diverse in their primary sequences and are categorized into many subfamilies. Among this repertoire, the biliverdin (BV)-binding CBCR GAF domains receive considerable attention for their in vivo optogenetic and bioimaging applications because BV is a mammalian intrinsic chromophore and can absorb far-red light that penetrates deep into the mammalian body. The typical BV-binding CBCR GAF domain exhibits reversible photoconversion between far-red-absorbing dark-adapted and orange-absorbing photoproduct states. Herein, we applied various biochemical and spectral studies to identify the details of the conformational change during this photoconversion process. No oligomeric state change was observed, whereas the surface charge would change with a modification of the α-helix structures during the photoconversion process. Combinatorial analysis using partial protease digestion and mass spectrometry identified the region where the conformational change occurred. These results provide clues for the future development of optogenetic tools.

Role of hypoxanthine-guanine phosphoribosyltransferase in the metabolism of fairy chemicals in rice.

Fairy chemicals (FCs), 2-azahypoxanthine (AHX), imidazole-4-carboxamide (ICA), and 2-aza-8-oxohypoxanthine (AOH), are molecules with many diverse functions in plants. The defined biosynthetic pathway for FCs is a novel purine metabolism in which they are biosynthesized from 5-aminoimidazole-4-carboxamide. Here, we show that one of the purine salvage enzymes, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), recognizes AHX and AOH as substrates. Two novel compounds, AOH ribonucleotide and its ribonucleoside which are the derivatives of AOH, were enzymatically synthesized. The structures were determined by mass spectrometry, 1D and 2D NMR spectroscopy, and X-ray single-crystal diffraction analysis. This report demonstrates the function of HGPRT and the existence of novel purine metabolism associated with the biosynthesis of FCs in rice.

Evolution-inspired design of multicolored photoswitches from a single cyanobacteriochrome scaffold.

Cyanobacteriochromes (CBCRs) are small, bistable linear tetrapyrrole (bilin)-binding light sensors which are typically found as modular components in multidomain cyanobacterial signaling proteins. The CBCR family has been categorized into many lineages that roughly correlate with their spectral diversity, but CBCRs possessing a conserved DXCF motif are found in multiple lineages. DXCF CBCRs typically possess two conserved Cys residues: a first Cys that remains ligated to the bilin chromophore and a second Cys found in the DXCF motif. The second Cys often forms a second thioether linkage, providing a mechanism to sense blue and violet light. DXCF CBCRs have been described with blue/green, blue/orange, blue/teal, and green/teal photocycles, and the molecular basis for some of this spectral diversity has been well established. We here characterize AM1_1499g1, an atypical DXCF CBCR that lacks the second cysteine residue and exhibits an orange/green photocycle. Based on prior studies of CBCR spectral tuning, we have successfully engineered seven AM1_1499g1 variants that exhibit robust yellow/teal, green/teal, blue/teal, orange/yellow, yellow/green, green/green, and blue/green photocycles. The remarkable spectral diversity generated by modification of a single CBCR provides a good template for multiplexing synthetic photobiology systems within the same cellular context, thereby bypassing the time-consuming empirical optimization process needed for multiple probes with different protein scaffolds.

Unusual ring D fixation by three crucial residues promotes phycoviolobilin formation in the DXCF-type cyanobacteriochrome without the second Cys.

Cyanobacteriochromes are linear tetrapyrrole-binding photoreceptors produced by cyanobacteria. Their chromophore-binding GAF domains are categorized into many lineages. Among them, dual Cys-type cyanobacteriochrome GAF domains possessing not only a highly conserved 'first Cys' but also a 'second Cys' are found from multiple lineages. The first Cys stably attaches to C31 of the A-ring, while the second Cys mostly shows reversible ligation to the C10 of the chromophore. Notably, the position of the second Cys in the primary sequence is diversified, and the most abundant dual Cys-type GAF domains have a 'second Cys' within the DXCF motif, which are called DXCF GAF domains. It has been long known that the second Cys in the DXCF GAF domains not only shows the reversible ligation but also is involved in isomerization activity (reduction in C4=C5 double bond) from the initially incorporated phycocyanobilin to phycoviolobilin. However, comprehensive site-directed mutagenesis on the DXCF GAF domains, AM1_6305g1 and AM1_1499g1, revealed that the second Cys is dispensable for isomerization activity, in which three residues participate by fixing the C- and D-rings. Fixation of the chromophore on both sides of the C5 bridge is necessary, even though one side of the fixation site is far from this bridge, with the other side at C31 fixed by the first Cys.

Rational conversion of chromophore selectivity of cyanobacteriochromes to accept mammalian intrinsic biliverdin.

Because cyanobacteriochrome photoreceptors need only a single compact domain for chromophore incorporation and for absorption of visible spectra including the long-wavelength far-red region, these molecules have been paid much attention for application to bioimaging and optogenetics. Most cyanobacteriochromes, however, have a drawback to incorporate phycocyanobilin that is not available in the mammalian cells. In this study, we focused on biliverdin (BV) that is a mammalian intrinsic chromophore and absorbs the far-red region and revealed that replacement of only four residues was enough for conversion from BV-rejective cyanobacteriochromes into BV-acceptable molecules. We succeeded in determining the crystal structure of one of such engineered molecules, AnPixJg2_BV4, at 1.6 Å resolution. This structure identified unusual covalent bond linkage, which resulted in deep BV insertion into the protein pocket. The four mutated residues contributed to reducing steric hindrances derived from the deeper insertion. We introduced these residues into other domains, and one of them, NpF2164g5_BV4, produced bright near-infrared fluorescence from mammalian liver in vivo. Collectively, this study provides not only molecular basis to incorporate BV by the cyanobacteriochromes but also rational strategy to open the door for application of cyanobacteriochromes to visualization and regulation of deep mammalian tissues.

Phytochromes and Cyanobacteriochromes: Photoreceptor Molecules Incorporating a Linear Tetrapyrrole Chromophore.

In this chapter, we summarize the molecular mechanisms of the linear tetrapyrrole-binding photoreceptors, phytochromes, and cyanobacteriochromes. We especially focus on the color-tuning mechanisms and conformational changes during the photoconversion process. Furthermore, we introduce current status of development of the optogenetic tools based on these molecules. Huge repertoire of these photoreceptors with diverse spectral properties would contribute to development of multiplex optogenetic regulation. Among them, the photoreceptors incorporating the biliverdin IXα chromophore is advantageous for in vivo optogenetics because this is intrinsic in the mammalian cells, and absorbs far-red light penetrating into deep mammalian tissues.

An Engineered Biliverdin-Compatible Cyanobacteriochrome Enables a Unique Ultrafast Reversible Photoswitching Pathway.

Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward Pfr → Po transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse Po → Pfr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm-1. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances.

A photoproduct of DXCF cyanobacteriochromes without reversible Cys ligation is destabilized by rotating ring twist of the chromophore.

Cyanobacteriochrome photoreceptors (CBCRs) ligate linear tetrapyrrole chromophores via their first (canonical) Cys residue and show reversible photoconversion triggered by light-dependent Z/E isomerization of the chromophore. Among the huge repertoire of CBCRs, DXCF CBCRs contain a second Cys residue within the highly conserved Asp-Xaa-Cys-Phe (DXCF) motif. In the typical receptors, the second Cys covalently attaches to the 15Z-chromophore in the dark state and detaches from the 15E-chromophore in the photoproduct state, whereas atypical ones that lack reversible ligation activity show red-shifted absorption in the dark state due to a more extended π-conjugated system. Moreover, some DXCF CBCRs show blue-shifted absorption in the photoproduct state due to the twisted geometry of the rotating ring. During the process of rational color tuning of a certain DXCF CBCR, we unexpectedly found that twisted photoproducts of some variant molecules showed dark reversion to the dark state, which prompted us to hypothesize that the photoproduct is destabilized by the twisted geometry of the rotating ring. In this study, we comprehensively examined the photoproduct stability of the twisted and relaxed molecules derived from the same CBCR scaffolds under dark conditions. In the DXCF CBCRs lacking reversible ligation activity, the twisted photoproducts showed faster dark reversion than the relaxed ones, supporting our hypothesis. By contrast, in the DXCF CBCRs exhibiting reversible ligation activity, the twisted photoproducts showed no detectable photoconversion. Reversible Cys adduct formation thus results in drastic rearrangement of the protein-chromophore interaction in the photoproduct state, which would contribute to the previously unknown photoproduct stability.

The Cruciality of Single Amino Acid Replacement for the Spectral Tuning of Biliverdin-Binding Cyanobacteriochromes.

Cyanobacteriochromes (CBCRs), which are known as linear tetrapyrrole-binding photoreceptors, to date can only be detected from cyanobacteria. They can perceive light only in a small unit, which is categorized into various lineages in correlation with their spectral and structural characteristics. Recently, we have succeeded in identifying specific molecules, which can incorporate mammalian intrinsic biliverdin (BV), from the expanded red/green (XRG) CBCR lineage and in converting BV-rejective molecules into BV-acceptable ones with the elucidation of the structural basis. Among the BV-acceptable molecules, AM1_1870g3_BV4 shows a spectral red-shift in comparison with other molecules, while NpF2164g5_BV4 does not show photoconversion but stably shows a near-infrared (NIR) fluorescence. In this study, we found that AM1_1870g3_BV4 had a specific Tyr residue near the d-ring of the chromophore, while others had a highly conserved Leu residue. The replacement of this Tyr residue with Leu in AM1_1870g3_BV4 resulted in a blue-shift of absorption peak. In contrast, reverse replacement in NpF2164g5_BV4 resulted in a red-shift of absorption and fluorescence peaks, which applies to fluorescence bio-imaging in mammalian cells. Notably, the same Tyr/Leu-dependent color-tuning is also observed for the CBCRs belonging to the other lineage, which indicates common molecular mechanisms.

Molecular characterization of DXCF cyanobacteriochromes from the cyanobacterium Acaryochloris marina identifies a blue-light power sensor.

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole-binding photoreceptors that sense a wide range of wavelengths from ultraviolet to far-red. The primary photoreaction in these reactions is a Z/E isomerization of the double bond between rings C and D. After this isomerization, various color-tuning events establish distinct spectral properties of the CBCRs. Among the various CBCRs, the DXCF CBCR lineage is widely distributed among cyanobacteria. Because the DXCF CBCRs from the cyanobacterium Acaryochloris marina vary widely in sequence, we focused on these CBCRs in this study. We identified seven DXCF CBCRs in A. marina and analyzed them after isolation from Escherichia coli that produces phycocyanobilin, a main chromophore for the CBCRs. We found that six of these CBCRs covalently bound a chromophore and exhibited variable properties, including blue/green, blue/teal, green/teal, and blue/orange reversible photoconversions. Notably, one CBCR, AM1_1870g4, displayed unidirectional photoconversion in response to blue-light illumination, with a rapid dark reversion that was temperature-dependent. Furthermore, the photoconversion took place without Z/E isomerization. This observation indicated that AM1_1870g4 likely functions as a blue-light power sensor, whereas typical CBCRs reversibly sense two light qualities. We also found that AM1_1870g4 possesses a GDCF motif in which the Asp residue is swapped with the next Gly residue within the DXCF motif. Site-directed mutagenesis revealed that this swap is essential for the light power-sensing function of AM1_1870g4. This is the first report of a blue-light power sensor from the CBCR superfamily and of photoperception without Z/E isomerization among the bilin-based photoreceptors.

Protein Engineering of Dual-Cys Cyanobacteriochrome AM1_1186g2 for Biliverdin Incorporation and Far-Red/Blue Reversible Photoconversion.

Cyanobacteria have cyanobacteriochromes (CBCRs), which are photoreceptors that bind to a linear tetrapyrrole chromophore and sense UV-to-visible light. A recent study revealed that the dual-Cys CBCR AM1_1186g2 covalently attaches to phycocyanobilin and exhibits unique photoconversion between a Pr form (red-absorbing dark state, λmax = 641 nm) and Pb form (blue-absorbing photoproduct, λmax = 416 nm). This wavelength separation is larger than those of the other CBCRs, which is advantageous for optical tools. Nowadays, bioimaging and optogenetics technologies are powerful tools for biological research. In particular, the utilization of far-red and near-infrared light sources is required for noninvasive applications to mammals because of their high potential to penetrate into deep tissues. Biliverdin (BV) is an intrinsic chromophore and absorbs the longest wavelength among natural linear tetrapyrrole chromophores. Although the BV-binding photoreceptors are promising platforms for developing optical tools, AM1_1186g2 cannot efficiently attach BV. Herein, by rationally introducing several replacements, we developed a BV-binding AM1_1186g2 variant, KCAP_QV, that exhibited reversible photoconversion between a Pfr form (far-red-absorbing dark state, λmax = 691 nm) and Pb form (λmax = 398 nm). This wavelength separation reached 293 nm, which is the largest among the known phytochrome and CBCR photoreceptors. In conclusion, the KCAP_QV molecule developed in this study can offer an alternative platform for the development of unique optical tools.

Cyanobacteriochrome Photoreceptors Lacking the Canonical Cys Residue.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that sense near-ultraviolet to far-red light. Like the distantly related phytochromes, all CBCRs reported to date have a conserved Cys residue (the "canonical Cys" or "first Cys") that forms a thioether linkage to C31 of the linear tetrapyrrole (bilin) chromophore. Detection of ultraviolet, violet, and blue light is performed by at least three subfamilies of two-Cys CBCRs that require both the first Cys and a second Cys that forms a second covalent linkage to C10 of the bilin. In the well-characterized DXCF subfamily, the second Cys is part of a conserved Asp-Xaa-Cys-Phe motif. We here report novel CBCRs lacking the first Cys but retaining the DXCF Cys as part of a conserved Asp-Xaa-Cys-Ile-Pro (DXCIP) motif. Phylogenetic analysis demonstrates that DXCIP CBCRs are a sister to a lineage of DXCF CBCR domains from phototaxis sensors. Three such DXCIP CBCR domains (cce_4193g1, Cyan8802_2776g1, and JSC1_24240) were characterized after recombinant expression in Escherichia coli engineered to produce phycocyanobilin. All three covalently bound bilin and showed unidirectional photoconversion in response to green light. Spectra of acid-denatured proteins in the dark-adapted state do not correspond to those of known bilins. One DXCIP CBCR, cce_4193g1, exhibited very rapid dark reversion consistent with a function as a power sensor. However, Cyan8802_2776g1 exhibited slower dark reversion and would not have such a function. The full-length cce_4193 protein also possesses a DXCF CBCR GAF domain (cce_4193g2) with a covalently bound phycoviolobilin chromophore and a blue/green photocycle. Our studies indicate that CBCRs need not contain the canonical Cys residue to function as photochromic light sensors and that phototaxis proteins containing DXCIP CBCRs may potentially perceive both light quality and light intensity.

Red-shifted red/green-type cyanobacteriochrome AM1_1870g3 from the chlorophyll d-bearing cyanobacterium Acaryochloris marina.

Cyanobacteriochromes (CBCRs) are diverse photoreceptors that are found only from cyanobacteria and cover wide range of light qualities. CBCRs are divided into two types regarding the chromophore species they contain: phycocyanobilin (PCB) and phycoviolobilin. Red/green-type CBCRs are widely distributed subfamily among the PCB-binding CBCRs and photoconvert between a red-absorbing thermostable form and a green-absorbing metastable form. Our recent study discovered that a red/green-type CBCR, AM1_1557g2, from a cyanobacterium Acaryochloris marina covalently binds not only PCB but also biliverdin (BV). BV-binding AM1_1557g2 photoconverts between a far-red absorbing form and an orange-absorbing form. We report, herein, that another red/green-type CBCR, AM1_1870g3, from the cyanobacterium A. marina also bound both PCB and BV. PCB- and BV-binding ones showed red/green and far-red/orange reversible photoconversions, respectively. Unexpectedly, absorbing wavelengths are 10-20 nm red-shifted compared with those of AM1_1557g2. These red-shifted characteristics may be useful for optogenetic light switches that work in various organisms.

A new type of dual-Cys cyanobacteriochrome GAF domain found in cyanobacterium Acaryochloris marina, which has an unusual red/blue reversible photoconversion cycle.

Cyanobacteriochromes (CBCRs) form a large, spectrally diverse family of photoreceptors (linear tetrapyrrole covalently bound via a conserved cysteine) that perceive ultraviolet to red light. The underlying mechanisms are reasonably well understood with, in certain cases, reversible formation of an adduct between a second cysteine and the chromophore accounting, in part, for their spectral diversity. These CBCRs are denoted as dual-Cys CBCRs, and most such CBCRs had been shown to reversibly absorb blue and green light. Herein, we report the structural and mechanistic characterization of a new type of dual-Cys CBCR, AM1_1186, which exhibits reversible photoconversion between a red-absorbing dark state (λmax = 641 nm) and a blue-absorbing photoproduct (λmax = 416 nm). The wavelength separation of AM1_1186 photoconversion is the largest found to date for a CBCR. In addition to one well-conserved cysteine responsible for covalent incorporation of the chromophore into the apoprotein, AM1_1186 contains a second cysteine in a unique position of its photosensory domain, which would be more properly classified as a red/green CBCR according to its sequence. Carboxyamidomethylation and mutagenesis of the cysteines revealed that the second cysteine forms an adduct with the tetrapyrrole, the phycocyanobilin, that can be reversed under blue light. The proline immediately upstream of this cysteine appears to determine the rate at which the cysteinylation following photoexcitation of the dark state chromophore can occur. We propose a possible reaction scheme and color-tuning mechanism for AM1_1186 in terms of its structure and its place in a phylogenetic tree.

Red/green cyanobacteriochromes acquire isomerization from phycocyanobilin to phycoviolobilin.

Cyanobacteriochromes (CBCRs) are unique cyanobacteria-specific photoreceptors that share a distant relation with phytochromes. Most CBCRs contain conserved cysteine residues known as canonical Cys, while some CBCRs have additional cysteine residues called second Cys within the DXCF motif, leading to their classification as DXCF CBCRs. They typically undergo a process where they incorporate phycocyanobilin (PCB) and subsequently isomerize it to phycoviolobilin (PVB). Conversely, CBCRs with conserved Trp residues and without the second Cys are called extended red/green (XRG) CBCRs. Typical XRG CBCRs bind PCB without undergoing PCB-to-PVB isomerization, displaying red/green reversible photoconversion, and there are also atypical CBCRs that exhibit diverse photoconversions. We discovered novel XRG CBCRs with Cys residue instead of the conserved Trp residue. These novel XRG CBCRs exhibited the ability to isomerize PCB to PVB, displaying green/teal reversible photoconversion. Through sequence- and structure-based comparisons coupled with mutagenesis experiments, we identified three amino acid residues, including the Cys residue, crucial for facilitating PCB-to-PVB isomerization. This research expands our understanding of the diversity of XRG CBCRs, highlighting the remarkable molecular plasticity of CBCRs.

Introduction of reversible cysteine ligation ability to the biliverdin-binding cyanobacteriochrome photoreceptor.

Cyanobacteriochrome (CBCR) photoreceptors are distantly related to the canonical red/far-red reversible phytochrome photoreceptors. In the case of the CBCRs, only the GAF domain is required for chromophore incorporation and photoconversion. The GAF domains of CBCR are highly diversified into many lineages to sense various colors of light. These CBCR GAF domains are divided into two types: those possessing only the canonical Cys residue and those with both canonical and second Cys residues. The canonical Cys residue stably ligates to the chromophore in both cases. The second Cys residue mostly shows reversible adduct formation with the chromophore during photoconversion for spectral tuning. In this study, we focused on the CBCR GAF domain AnPixJg2_BV4, which possesses only the canonical Cys residue. AnPixJg2_BV4 covalently ligates to the biliverdin (BV) chromophore and shows far-red/orange reversible photoconversion. Because BV is a mammalian intrinsic chromophore, BV-binding molecules are advantageous for in vivo optogenetic and bioimaging tool development. To obtain a better developmental platform molecule, we performed site-saturation random mutagenesis and serendipitously obtained a unique variant molecule that showed far-red/blue reversible photoconversion, in which the Cys residue was introduced near the chromophore. This introduced Cys residue functioned as the second Cys residue that reversibly ligated with the chromophore. Because the position of the introduced Cys residue is distinct from the known second Cys residues, the variant molecule obtained in this study would expand our knowledge about the spectral tuning mechanism of CBCRs and contribute to tool development.

A red light-responsive photoswitch for deep tissue optogenetics.

Red light penetrates deep into mammalian tissues and has low phototoxicity, but few optogenetic tools that use red light have been developed. Here we present MagRed, a red light-activatable photoswitch that consists of a red light-absorbing bacterial phytochrome incorporating a mammalian endogenous chromophore, biliverdin and a photo-state-specific binder that we developed using Affibody library selection. Red light illumination triggers the binding of the two components of MagRed and the assembly of split-proteins fused to them. Using MagRed, we developed a red light-activatable Cre recombinase, which enables light-activatable DNA recombination deep in mammalian tissues. We also created red light-inducible transcriptional regulators based on CRISPR-Cas9 that enable an up to 378-fold activation (average, 135-fold induction) of multiple endogenous target genes. MagRed will facilitate optogenetic applications deep in mammalian organisms in a variety of biological research areas.

Functional-food constituents in the fruiting bodies of Stropharia rugosoannulata.

Nine compounds (1-9) were isolated from the fruiting bodies of Stropharia rugosoannulata. Compounds 1-5, 8, and 9 suppressed the formation of osteoclast. Compounds 2 and 5 showed anti-fungal activity, and their MIC were 250 µM and 500 µM respectively. Compounds 2-6 showed inhibitory effects on thapsigargin toxicity.

Transient electronic and vibrational signatures during reversible photoswitching of a cyanobacteriochrome photoreceptor.

Cyanobacteriochromes (CBCRs) are an emerging class of photoreceptors that are distant relatives of the phytochromes family. Unlike phytochromes, CBCRs have gained popularity in optogenetics due to their highly diverse spectral properties spanning the UV to near-IR region and only needing a single compact binding domain. AnPixJg2 is a CBCR that can reversibly photoswitch between its red-absorbing (15ZPr) and green-absorbing (15EPg) forms of the phycocyanobilin (PCB) cofactor. To reveal primary events of photoconversion, we implemented femtosecond transient absorption spectroscopy with a homemade LED box and a miniature peristaltic pump flow cell to track transient electronic responses of the photoexcited AnPixJg2 on molecular time scales. The 525 nm laser-induced Pg-to-Pr reverse conversion exhibits a ~3 ps excited-state lifetime before reaching the conical intersection (CI) and undergoing further relaxation on the 30 ps time scale to generate a long-lived Lumi-G ground state intermediate en route to Pr. The 650 nm laser-induced Pr-to-Pg forward conversion is less efficient than reverse conversion, showing a longer-lived excited state which requires two steps with ~13 and 217 ps time constants to enter the CI region. Furthermore, using a tunable ps Raman pump with broadband Raman probe on both the Stokes and anti-Stokes sides, we collected the pre-resonance ground-state femtosecond stimulated Raman spectroscopy (GS-FSRS) data with mode assignments aided by quantum calculations. Key vibrational marker bands at ~850, 1050, 1615, and 1649 cm-1 of the Pr conformer exhibit a notable blueshift to those of the Pg conformer inside AnPixJg2, reflecting the PCB chromophore terminal D (major) and A (minor) ring twist along the primary photoswitching reaction coordinate. This integrated ultrafast spectroscopy and computational platform has the potential to elucidate photochemistry and photophysics of more CBCRs and photoactive proteins in general, providing the highly desirable mechanistic insights to facilitate the rational design of functional molecular sensors and devices.