Protonation Heterogeneity Modulates the Ultrafast Photocycle Initiation Dynamics of Phytochrome Cph1

Kirpich, Julia S.; Mix, L. Tyler; Martin, Shelley S.; Rockwell, Nathan C.; Lagarias, J. Clark; Larsen, Delmar S.

doi:10.1021/acs.jpclett.8b01133

Cited by 28 publications

(49 citation statements)

References 54 publications

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“…Such signal splitting is not confined to this ring but also seen in several regions of the chromophore, such as C15 associated with the C – D methine bridge, the B -ring propionate (C8 1 , C8 2 , and C8 3 ) and also C17 and C18 1 H of ring D . This observation further supports the notion that both native phytochromes and CBCRs in their Pr dark states are heterogeneous population mixtures that can be distinguished structurally [4,6,18,35,44,45,46,47], biologically [48,49], spectrally [50,51,52,53,54,55] and photochemically [56,57,58,59,60,61]. The apparent A -ring signal splitting is inherently linked to the heterogeneous attachment of the chromophore to C138 and the variable packing of ring A .…”

Section: Discussionsupporting

confidence: 81%

MAS NMR on a Red/Far-Red Photochromic Cyanobacteriochrome All2699 from Nostoc

Bielytskyi

Otis

et al. 2019

IJMS

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Unlike canonical phytochromes, the GAF domain of cyanobacteriochromes (CBCRs) can bind bilins autonomously and is sufficient for functional photocycles. Despite the astonishing spectral diversity of CBCRs, the GAF1 domain of the three-GAF-domain photoreceptor all2699 from the cyanobacterium Nostoc 7120 is the only CBCR-GAF known that converts from a red-absorbing (Pr) dark state to a far-red-absorbing (Pfr) photoproduct, analogous to the more conservative phytochromes. Here we report a solid-state NMR spectroscopic study of all2699g1 in its Pr state. Conclusive NMR evidence unveils a particular stereochemical heterogeneity at the tetrahedral C31 atom, whereas the crystal structure shows exclusively the R-stereochemistry at this chiral center. Additional NMR experiments were performed on a construct comprising the GAF1 and GAF2 domains of all2699, showing a greater precision in the chromophore–protein interactions in the GAF1-2 construct. A 3D Pr structural model of the all2699g1-2 construct predicts a tongue-like region extending from the GAF2 domain (akin to canonical phytochromes) in the direction of the chromophore, shielding it from the solvent. In addition, this stabilizing element allows exclusively the R-stereochemistry for the chromophore-protein linkage. Site-directed mutagenesis performed on three conserved motifs in the hairpin-like tip confirms the interaction of the tongue region with the GAF1-bound chromophore.

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Section: Discussionsupporting

confidence: 81%

MAS NMR on a Red/Far-Red Photochromic Cyanobacteriochrome All2699 from Nostoc

Bielytskyi

Otis

et al. 2019

IJMS

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“…In this case, the H260 itself was observed to undergo deprotonation in the Pr state with a p K a of 7.5 accompanied by a decrease in Q band absorbance. This equilibrium between the cationic H260 and the one singly protonated at Nε is a natural origin of the heterogeneity in the Pr ZZZ state of Cph1 , also observed in complex excited‐state dynamics . There is, however, no evidence for H260 deprotonation in this pH range in Dr ‐PSM; for example, in Y263F, there is no change in contribution of Comp1 or Comp2 until after pH 9 (Fig.…”

Section: Discussionmentioning

confidence: 80%

“…We show that about 20% of WT has a deprotonated Y263 under biological conditions at pH 7.8. This observation may explain the source of heterogeneity of the Pr state, observed in several phytochrome species . There may be aspects of the low p K a of Y263 that are important for phytochrome function, for example, in regard to the proposed proton transfer reaction during photoisomerization , which then may influence the isomerization yield of the chromophore.…”

Section: Discussionmentioning

confidence: 96%

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UV‐Vis Spectroscopy Reveals a Correlation Between Y263 and BV Protonation States in Bacteriophytochromes

Rumfeldt

Takala

Liukkonen

et al. 2019

Photochem & Photobiology

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Red-light photosensory proteins, phytochromes, link light activation to biological functions by interconverting between two conformational states. For this, they undergo large-scale secondary and tertiary changes which follow small-scale Z to E bond photoisomerization of the covalently bound bilin chromophore. The complex network of amino acid interactions in the chromophore-binding pocket plays a central role in this process. Highly conserved Y263 and H290 have been found to be important for the photoconversion yield, while H260 has been identified as important for bilin protonation and proton transfer steps. Here, we focus on the roles these amino acids are playing in preserving the chemical properties of bilin in the resting Pr state of the photosensory unit of a bacteriophytochrome from Deinococcus radiodurans. By using pH-dependent UV-Vis spectroscopy and spectral decomposition modeling, we confirm the importance of H260 for biliverdin protonation. Further, we demonstrate that in the canonical bacteriophytochromes, the pK a value of the phenol group of the Y263 is uncommonly low. This directly influences the protonation of the bilin molecule and likely the functional properties of the protein. Our study expands the understanding of the tight interplay between the nearby amino acids and bilin in the phytochrome family. Abbreviations: BphP, bacteriophytochrome; BV, biliverdin IXa; BV-H 3 , neutral BV with three of four pyrrole nitrogens protonated; BV-H 4 + , cationic BV with all four pyrrole nitrogens protonated; CBD, chromophore-binding domain; Com-p1, component one; Comp2, component two; Comp3, component three; Dr, Deinococcus radiodurans; Dr-PSM, photosensory module from Deinococcus radiodurans; Pfr, the far-red-light absorbing state of the phytochrome molecule; Pr, the red-light absorbing state of the phytochrome molecule; Pr ZZE , the bilin chromophore in the Pfr state conformation ZZE; Pr ZZZ , the bilin chromophore in the Pr state conformation ZZZ; PSM, photosensory module which includes PAS, GAF and PHY domains; WT, wild type.

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“…It is well established that both 15E and 15Z chromophores of Cph1 are inherently heterogeneous (44)(45)(46)(47). Such heterogeneity might be due to 15,syn vs. 15,anti chromophore configurations, αvs.…”

Section: Ph Plays a Critical Role In The 15e Pyo: 15e Pr Photoproductmentioning

confidence: 99%

Spectral and photochemical diversity of tandem cysteine cyanobacterial phytochromes

Song

Lee

Yang

et al. 2020

Journal of Biological Chemistry

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The atypical trichromatic cyanobacterial phytochrome NpTP1 from Nostoc punctiforme ATCC 29133 is a linear tetrapyrrole (bilin)-binding photoreceptor protein that possesses tandem-cysteine residues responsible for shifting its light-sensing maximum to the violet spectral region. Using bioinformatics and phylogenetic analyses, here we established that tandem-cysteine cyanobacterial phytochromes (TCCPs) compose a well-supported monophyletic phytochrome lineage distinct from prototypical red/far-red cyanobacterial phytochromes. To investigate the light-sensing diversity of this family, we compared the spectroscopic properties of NpTP1 (here renamed NpTCCP) with those of three phylogenetically diverged TCCPs identified in the draft genomes of Tolypothrix sp. PCC7910, Scytonema sp. PCC10023, and Gloeocapsa sp. PCC7513. Recombinant photosensory core modules of ToTCCP, ScTCCP, and GlTCCP exhibited violet-blue–absorbing dark-states consistent with dual thioether-linked phycocyanobilin (PCB) chromophores. Photoexcitation generated singly-linked photoproduct mixtures with variable ratios of yellow-orange and red-absorbing species. The photoproduct ratio was strongly influenced by pH and by mutagenesis of TCCP- and phytochrome-specific signature residues. Our experiments support the conclusion that both photoproduct species possess protonated 15E bilin chromophores, but differ in the ionization state of the noncanonical “second” cysteine sulfhydryl group. We found that the ionization state of this and other residues influences subsequent conformational change and downstream signal transmission. We also show that tandem-cysteine phytochromes present in eukaryotes possess similar amino acid substitutions within their chromophore-binding pocket, which tune their spectral properties in an analogous fashion. Taken together, our findings provide a roadmap for tailoring the wavelength specificity of plant phytochromes to optimize plant performance in diverse natural and artificial light environments.

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Protonation Heterogeneity Modulates the Ultrafast Photocycle Initiation Dynamics of Phytochrome Cph1

Cited by 28 publications

References 54 publications

MAS NMR on a Red/Far-Red Photochromic Cyanobacteriochrome All2699 from Nostoc

MAS NMR on a Red/Far-Red Photochromic Cyanobacteriochrome All2699 from Nostoc

UV‐Vis Spectroscopy Reveals a Correlation Between Y263 and BV Protonation States in Bacteriophytochromes

Spectral and photochemical diversity of tandem cysteine cyanobacterial phytochromes

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