Fluorescence investigation of the recombinant cyanobacterial phytochrome (Cph1) and its C-terminally truncated monomeric species (Cph1Δ2): implication for holoprotein assembly, chromophore–apoprotein interaction and photochemistry

Sineshchekov, V.A.; Koppel, L.; Esteban, Berta; Hughes, Jon; Lamparter, Tilman

doi:10.1016/s1011-1344(02)00282-8

Cited by 37 publications

(77 citation statements)

References 19 publications

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“…1B). This scheme fits the photochemical data for both plant phytochromes and the more recently discovered cyanobacterial phytochrome 1 (Cph1) (7,13). The local protein environment of the bilin chromophore of phytochromes thus appears to have been preserved throughout billions of years of evolution.…”

supporting

confidence: 62%

Harnessing phytochrome's glowing potential

Fischer

Lagarias

2004

Proc. Natl. Acad. Sci. U.S.A.

164

217

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Directed evolution of a cyanobacterial phytochrome was undertaken to elucidate the structural basis of its light sensory activity by remodeling the chemical environment of its linear tetrapyrrole prosthetic group. In addition to identifying a small region of the apoprotein critical for maintaining phytochrome's native spectroscopic properties, our studies revealed a tyrosine-to-histidine mutation that transformed phytochrome into an intensely red fluorescent biliprotein. This tyrosine is conserved in all members of the phytochrome superfamily, implicating direct participation in the primary photoprocess of phytochromes. Fluorescent phytochrome mutants also hold great promise to expand the present repertoire of genetically encoded fluorescent proteins into the near infrared.biliprotein ͉ light signaling ͉ tetrapyrrole ͉ photoisomerization ͉ fluorescence

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supporting

confidence: 62%

Harnessing phytochrome's glowing potential

Fischer

Lagarias

2004

Proc. Natl. Acad. Sci. U.S.A.

164

217

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“…palustris RpBphP2 showed two kinetic components (39). Discrepancies between Pr fluorescence excitation and absorbance spectra of Cph1Δ2 have also been noted and associated with Pr heterogeneity (40). Local doubling of 13 C signals was also detected in our previous DARR NMR experiments on Synechocystis Cph1Δ2 and a recombinant N-terminal fragment of phytochrome A oat (65 kDa, phyA65), implying the coexistence of two Pr isoforms in solution (18).…”

Section: Resultsmentioning

confidence: 59%

Two ground state isoforms and a chromophore D -ring photoflip triggering extensive intramolecular changes in a canonical phytochrome

Song

Psakis

Lang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

160

299

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Phytochrome photoreceptors mediate light responses in plants and in many microorganisms. Here we report studies using 1 H-13 C magic-angle spinning NMR spectroscopy of the sensor module of cyanobacterial phytochrome Cph1. Two isoforms of the red-light absorbing Pr ground state are identified. Conclusive evidence that photoisomerization occurs at the C15-methine bridge leading to a β-facial disposition of the ring D is presented. In the far-red-light absorbing Pfr state, strong hydrogen-bonding interactions of the D-ring carbonyl group to Tyr-263 and of N24 to Asp-207 hold the chromophore in a tensed conformation. Signaling is triggered when Asp-207 is released from its salt bridge to Arg-472, probably inducing conformational changes in the tongue region. A second signal route is initiated by partner swapping of the B-ring propionate between Arg-254 and Arg-222.chromophore-protein interaction | signal transduction | solid-state NMR | photomorphogenesis P hytochrome was first demonstrated in plants as a red-lightdependent photoreceptor regulating numerous photomorphogenic processes (1, 2). Phytochromes are, however, also now known in photosynthetic prokaryotes including cyanobacteria (3, 4), nonphotosynthetic bacteria (5), and fungi (6). Generally, the phytochrome apoprotein binds an open-chain tetrapyrrole as a chromophore (7,8) to form the red-light absorbing Pr ground state (λ max ≈ 658 nm in case of cyanobacterial phytchrome Cph1 from Synechocystis 6803). Red light absorption photoactivates the molecule to form the photoactivated far-red-light absorbing Pfr state (λ max ≈ 702 nm for Cph1) via a series of intermediates (8-10). Photoactivation is thought to be initiated by a double bond isomerization of the chromophore (10, 11). Early NMR spectroscopic studies on proteolytic phytochrome fragments (12, 13) indicated that this isomerization occurs at the C15═C16 double bond (for numbering, see Fig. 1A), a geometrical change in line with vibrational spectroscopic investigations (14-16) and results from recent 13 C solid-state NMR (17, 18) in which the most significant changes during the light-triggered conversions are confined to rings C and D. Exact geometries of the chromophore in the Pr state have been resolved as periplanar ZZZssa configurations in bacteriophytochromes from Deinococcus radiodurans (19) and Rhodopseudomonas palustris (20) as well as in the more plant-phytochrome-like Cph1 from the cyanobacterium Synechocystis 6803 (21). On the other hand, the crystal structure of the unusual bacteriophytochrome PaBphP Pseudomonas aeruginosa (22) whose ground state is Pfr shows a ZZEssa conformation, consistent with the expected primary photochemistry at the C15═C16 double bond (Fig. 1 B vs. C). Very recently, however, Ulijasz et al. presented structural simulations based on liquid NMR data of a 20-kDa GAF (cGMP phosphodiesterase/ adenylyl cyclase/FhlA) domain fragment of "SyB-Cph1" phytochrome from the thermotolerant cyanobacterium Synechococcus OSB′ (23). Surprisingly, they concluded that photoisomerization occ...

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“…23,65 To minimize spectral artifacts deriving from self-absorption and scattering, we used a protein concentration of approximately 0.1 mg/ml. Emission spectra were measured using excitation at 610 nm, slit bandwidth of 0.3 nm and a red interference filter (λ = 610 nm, T max = 40%), that is, with low intensity excitation light to minimize phytochrome photoconversion during the scan.…”

Section: Uv-vis Fluorescence and CD Spectroscopymentioning

confidence: 99%

“…The D-ring isomerization and the formation of the lumi-R intermediate is considered to be the first step of the photocycle. 17,18 This initial photoreaction and the subsequent intermediates have been investigated using time-resolved UV-Vis absorbance [18][19][20][21] and fluorescence 22,23 and infrared difference 24,25 and resonance Raman 19,26-33 spectroscopies. Upon photon absorption, the excited state relaxes to give a short-lived hot state, which can lead to an unstable pre-lumi-R via an activation energy barrier: this step occurs with a quantum efficiency close to unity.…”

Section: Introductionmentioning

confidence: 99%