Identification of the Deprotonated Pyrrole Nitrogen of the Bilin-Based Photoreceptor by Raman Spectroscopy with an Advanced Computational Analysis

Osoegawa, Shinsuke; Miyoshi, Risako; Watanabe, Keita; Hirose, Yuu; Fujisawa, Tomotsumi; Ikeuchi, Masahiko; Unno, Masashi

doi:10.1021/acs.jpcb.9b00965

Cited by 31 publications

(51 citation statements)

References 37 publications

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“…The similarity in the down-filed shift of 15 (Figs. 3 and S8), which is consistent with our previous resonance Raman spectroscopy and QM/MM simulation experiment in RcaE (53). Deprotonation in the NB nitrogen is also consistent with solid-state NMR of in vitro-prepared AnPixJ proteins harboring deprotonated PCB fraction for its Pg photoproduct state (54).…”

Section: Discussionsupporting

confidence: 90%

“…The C15-Z,anti structure for the Pg state of RcaE (Fig. 4, left panel) is supported by recent our resonance Raman spectroscopy of RcaE (53). That study showed that the observed resonance Raman spectra in the Pg state of RcaE were well reproduced by the simulated spectra of C15-simulation (53).…”

Section: Discussionsupporting

confidence: 80%

“…4, left panel) is supported by recent our resonance Raman spectroscopy of RcaE (53). That study showed that the observed resonance Raman spectra in the Pg state of RcaE were well reproduced by the simulated spectra of C15-simulation (53). The C15-Z,anti structure is also supported by the recent in virtro reconstitution experiment (18).…”

Section: Discussionsupporting

confidence: 73%

“…Site-directed mutagenesis of RcaE identified the protochromic triad residues, Glu217, Leu249 and K261, required for the marked shift in the bilin pK a (18). Resonance Raman spectroscopy of the C15-Z Pg dark state of RcaE (44) was highly consistent with B-ring-deprotonated bilin system that adopts a C15-Z,anti structure based upon QM/MM calculations using the structure of C15-Z,anti Pr dark state of AnPixJ as template (45). However, structural information and direct evidence of the chemical environments of bilin in the green/red CBCR subfamily have not yet been reported, preventing deeper understanding of the molecular basis of the protochromic photocycle.…”

Section: Introductionmentioning

confidence: 98%

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Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE

Nagae

Unno

Koizumi

et al. 2020

Preprint

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Cyanobacteriochromes (CBCRs) are bilin-binding photosensors of the phytochrome superfamily that show remarkable spectral diversity. The green/red CBCR subfamily is important for regulating chromatic acclimation of photosynthetic antenna in cyanobacteria and is applied for optogenetic control of gene expression in synthetic biology. They are suggested to combine the bilin C15-Z/C15-E photoisomerization with a change in the bilin protonation state to drive their absorption changes. However, structural information and direct evidence of the bilin protonation state are lacking. Here we report a high-resolution (1.63Å) crystal structure of the bilin-binding domain of the chromatic acclimation sensor RcaE in the red-absorbing photoproduct state. The bilin is buried within a “pan” consisting of hydrophobic residues, where the bilin configuration/conformation is C5-Z,syn/C10-Z,syn/C15-E,syn with the A–C rings co-planar and the D-ring tilted. Three pyrrole nitrogens of the A–C rings are covered in the α-face with a hydrophobic lid of Leu249 influencing the bilin pKa, whereas they are directly hydrogen-bonded in the β-face with the carboxyl group of Glu217. Glu217 is further connected to a cluster of waters forming a hole in the pan, which are in exchange with solvent waters in molecular dynamics simulation. We propose that the “holey pan” structure functions as a proton-exit/influx pathway upon photoconversion. NMR analysis demonstrated that the four pyrrole nitrogen atoms are indeed fully protonated in the red-absorbing state, but one of them, most likely the B-ring nitrogen, is deprotonated in the green-absorbing state. These findings deepen our understanding of the diverse spectral tuning mechanisms present in CBCRs.Significance StatementGreen/red CBCRs are one of the most important CBCR subfamilies owing to their physiological roles in cyanobacteria phylum and optogenetic applications. They are known to utilize a change in the bilin protonation state to drive the marked change in green/red absorption, but the structural basis of the protochromic green/red photocycle are not well understood. Here, we have determined the crystal structure of the chromatic acclimation sensor RcaE of this subfamily in the photoproduct state, demonstrating a unique conformation of the bilin and its interacting residues. In addition, we provide direct evidence of the protonation state of the bilin via NMR analysis. These findings bring insight to our understanding of the molecular mechanisms underlying the spectral diversity of CBCRs.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionsupporting

confidence: 80%

Section: Discussionsupporting

confidence: 73%

Section: Introductionmentioning

confidence: 98%

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Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE

Nagae

Unno

Koizumi

et al. 2020

Preprint

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“…In contrast, green/red CBCRs have a reversed photocycle: the greenabsorbing 15Z dark state photoconverts to yield a red-absorbing 15E photoproduct. This subfamily uses a protochromic mechanism, whereby photoconversion triggers a proton transfer to the chromophore and induces a spectral red shift (2,38).…”

Section: /Bodymentioning

confidence: 99%

Molecular Basis of Far-red Sensing in Cyanobacteriochrome

Bandara

Rockwell

Zeng

et al. 2020

Preprint

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Author contributionsXY designed the project; SB, SSM and XZ carried out cloning, purification, and crystallization; SB, XZ, SSM and NR conducted mutagenesis and spectroscopic experiments; SB, XZ, ZR, HS and XY collected monochromatic diffraction data; HS, CW and ZR collected Laue diffraction data; ZR processed the Laue data and carried out singlecrystal spectroscopic experiments; SB and XY determined, refined and analyzed crystal structures; NR carried out phylogenetic analysis; SB, XY, NR, MVM, and JCL interpreted the data; XY, NR, and JCL wrote the paper with inputs from all authors. Abstract (156 words)Cyanobacteriochromes are small, panchromatic photoreceptors in the phytochrome superfamily that regulate diverse light-mediated adaptive processes in cyanobacteria. The molecular basis of far-red (FR) light perception by cyanobacteriochromes is currently unknown. Here we report the crystal structure of a far-red-sensing cyanobacteriochrome from Anabaena cylindrica PCC 7122, which exhibits a reversible far-red/orange photocycle.The 2.7 Å structure of its FR-absorbing dark state, determined by room temperature serial crystallography and cryo-crystallography, reveals an all-Z,syn configuration of its bound linear tetrapyrrole (bilin) chromophore that is less extended than the bilin chromophores of all known phytochromes. Based on structural comparisons with other bilin-binding proteins and extensive spectral analyses on mutants, we identify key proteinchromophore interactions that enable far-red sensing in bilin-binding proteins. We propose that FR-CBCRs employ two distinct tuning mechanisms, which work together to produce a large batho-chromatic shift. Findings of this work have important implications for development and improvement of photoproteins with far-red absorption and fluorescence. Significance Statement (99 words)Phytochromes are well known far-red-light sensors found in plants that trigger adaptive responses to facilitate competition for light capture with neighboring plants. Red-and farred-sensing are critical to cyanobacteria living in the far-red-enriched shade of plants.Here we report the crystal structure of a far-red-sensing cyanobacteriochrome, a distant cyanobacterial relative of phytochrome. These studies shed insight into the poorly understood molecular basis of far-red-sensing by phytobilin-based photoreceptors.Owing to the deep tissue penetration of far-red light, far-red-sensing photoreceptors offer promising protein scaffolds for developing gene-based photoswitches, optoacoustic contrast agents and fluorescent probes for in situ imaging and optogenetic applications.

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Hemin‐phytic Acid Functionalized Porous Conducting Polymer Hydrogel With Good Biocompatibility for Electrochemical Detection of H₂O₂ Released From Living Cells

Shang

et al. 2021

Electroanalysis

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A novel hemin/phytic acid doped polyaniline (PA‐PANI) hydrogel composite was prepared through a simple chemical and self‐assembly method, which was modified onto electrode for electrochemical detection of H2O2 released from living cells. It showed good analytical performance with high sensitivity, selectivity and a rapid response for the analysis of H2O2 in the range of 2 to 102 μM, with the detection limit of 1.2 μM. The favorable results mainly originated from both the high conductivity of PA‐PANI hydrogel and its network structure preventing hemin from self‐dimerization to provide active catalytic species. Furthermore, PA‐PANI with good biocompatibility allowed living cells to adhere and resulted in a short diffusion distance between H2O2 released from cells and electrode.

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Identification of the Deprotonated Pyrrole Nitrogen of the Bilin-Based Photoreceptor by Raman Spectroscopy with an Advanced Computational Analysis

Cited by 31 publications

References 37 publications

Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE

Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE

Molecular Basis of Far-red Sensing in Cyanobacteriochrome

Hemin‐phytic Acid Functionalized Porous Conducting Polymer Hydrogel With Good Biocompatibility for Electrochemical Detection of H₂O₂ Released From Living Cells

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Identification of the Deprotonated Pyrrole Nitrogen of the Bilin-Based Photoreceptor by Raman Spectroscopy with an Advanced Computational Analysis

Cited by 31 publications

References 37 publications

Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE

Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE

Molecular Basis of Far-red Sensing in Cyanobacteriochrome

Hemin‐phytic Acid Functionalized Porous Conducting Polymer Hydrogel With Good Biocompatibility for Electrochemical Detection of H2O2 Released From Living Cells

Contact Info

Product

Resources

About

Hemin‐phytic Acid Functionalized Porous Conducting Polymer Hydrogel With Good Biocompatibility for Electrochemical Detection of H₂O₂ Released From Living Cells