57 research outputs found
Conserved histidine and tyrosine determine spectral responses through the water network in Deinococcus radiodurans phytochrome
Funding Information: This work was supported by Academy of Finland grants 285461 (H.T.), 330678 (H.T., J.R.), 277194 (H.L.), and 290677 (S.M.). We acknowledge the European Synchrotron Radiation Facility (ESRF) for providing synchrotron access for crystal data collection. We thank Prof. Janne Ihalainen (University of Jyväskylä) for all the help in all aspects of the paper, Prof. Gerrit Groenhof (University of Jyväskylä) for support, and Prof. Nikolai V. Tkachenko (Tampere University) for help and facilities for time-resolved absorption spectroscopy. We also thank M.Sc. Alli Liukkonen (University of Jyväskylä) and Dr. Heikki Häkkänen (University of Jyväskylä) for the assistance in laboratory and spectroscopy, respectively. Funding Information: This work was supported by Academy of Finland grants 285461 (H.T.), 330678 (H.T., J.R.), 277194 (H.L.), and 290677 (S.M.). We acknowledge the European Synchrotron Radiation Facility (ESRF) for providing synchrotron access for crystal data collection. We thank Prof. Janne Ihalainen (University of Jyväskylä) for all the help in all aspects of the paper, Prof. Gerrit Groenhof (University of Jyväskylä) for support, and Prof. Nikolai V. Tkachenko (Tampere University) for help and facilities for time-resolved absorption spectroscopy. We also thank M.Sc. Alli Liukkonen (University of Jyväskylä) and Dr. Heikki Häkkänen (University of Jyväskylä) for the assistance in laboratory and spectroscopy, respectively. Publisher Copyright: © 2022, The Author(s).Phytochromes are red light-sensing photoreceptor proteins that bind a bilin chromophore. Here, we investigate the role of a conserved histidine (H260) and tyrosine (Y263) in the chromophore-binding domain (CBD) of Deinococcus radiodurans phytochrome (DrBphP). Using crystallography, we show that in the H260A variant, the missing imidazole side chain leads to increased water content in the binding pocket. On the other hand, Y263F mutation reduces the water occupancy around the chromophore. Together, these changes in water coordination alter the protonation and spectroscopic properties of the biliverdin. These results pinpoint the importance of this conserved histidine and tyrosine, and the related water network, for the function and applications of phytochromes.Peer reviewe
Conserved histidine and tyrosine determine spectral responses through the water network in Deinococcus radiodurans phytochrome
Phytochromes are red light-sensing photoreceptor proteins that bind a bilin chromophore. Here, we investigate the role of a conserved histidine (H260) and tyrosine (Y263) in the chromophore-binding domain (CBD) of Deinococcus radiodurans phytochrome (DrBphP). Using crystallography, we show that in the H260A variant, the missing imidazole side chain leads to increased water content in the binding pocket. On the other hand, Y263F mutation reduces the water occupancy around the chromophore. Together, these changes in water coordination alter the protonation and spectroscopic properties of the biliverdin. These results pinpoint the importance of this conserved histidine and tyrosine, and the related water network, for the function and applications of phytochromes.</p
On the (un)coupling of the chromophore, tongue interactions, and overall conformation in a bacterial phytochrome
Phytochromes are photoreceptors in plants, fungi, and various microorganisms and cycle between metastable red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Their light responses are thought to follow a conserved structural mechanism that is triggered by isomerization of the chromophore. Downstream structural changes involve refolding of the so-called tongue extension of the phytochrome-specific GAF-related (PHY) domain of the photoreceptor. The tongue is connected to the chromophore by conserved DIP and PRXSF motifs and a conserved tyrosine, but the role of these residues in signal transduction is not clear. Here, we examine the tongue interactions and their interplay with the chromophore by substituting the conserved tyrosine (Tyr(263)) in the phytochrome from the extremophile bacterium Deinococcus radiodurans with phenylalanine. Using optical and FTIR spectroscopy, X-ray solution scattering, and crystallography of chromophore-binding domain (CBD) and CBD-PHY fragments, we show that the absence of the Tyr(263) hydroxyl destabilizes the -sheet conformation of the tongue. This allowed the phytochrome to adopt an -helical tongue conformation regardless of the chromophore state, hence distorting the activity state of the protein. Our crystal structures further revealed that water interactions are missing in the Y263F mutant, correlating with a decrease of the photoconversion yield and underpinning the functional role of Tyr(263) in phytochrome conformational changes. We propose a model in which isomerization of the chromophore, refolding of the tongue, and globular conformational changes are represented as weakly coupled equilibria. The results also suggest that the phytochromes have several redundant signaling routes.Peer reviewe
Rekommendation och checklista för öppen vetenskap inom ömsesidig forsknings-, utvecklings- och innovationsverksamhet mellan forskningsorganisationer och företag
Detta är en rekommendation för genomförande av den öppna vetenskapens verksamhetsformer i forsknings-, utvecklings- och innovationsverksamhet mellan finländska forskningsorganisationer och företag (FUI-verksamhet) . FUI-verksamhet genomförs ofta via FUI-projekt. I denna rekommendation redogörs för hur forskningsresultat och -material som uppkommer inom företagssamarbetet kan utnyttjas och/eller spridas på ett säkert, effektivt och så öppet sätt som möjligt. Rekommendationen har skrivits i samarbete mellan Samordningen av öppen vetenskap och forskning, arbetsgruppen för öppenhet i företagssamarbete, det finländska vetenskapssamfundet och näringslivet. Den är förenlig med Deklarationen för öppen vetenskap och forskning 2020–2025, Policyn för öppen forskningskultur och andra nationella rekommendationer som uppkommit i samordningen av öppen vetenskap och forskning.publishedVersio
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Structural photoactivation of a full-length bacterial phytochrome
Phytochromes are light sensor proteins found in plants, bacteria, and fungi. They function by converting a photon absorption event into a conformational signal that propagates from the chromophore through the entire protein. However, the structure of the photoactivated state and the conformational changes that lead to it are not known. We report time-resolved x-ray scattering of the full-length phytochrome from Deinococcus radiodurans on micro- and millisecond time scales. We identify a twist of the histidine kinase output domains with respect to the chromophore-binding domains as the dominant change between the photoactivated and resting states. The time-resolved data further show that the structural changes up to the microsecond time scales are small and localized in the chromophore-binding domains. The global structural change occurs within a few milliseconds, coinciding with the formation of the spectroscopic meta-Rc state. Our findings establish key elements of the signaling mechanism of full-length bacterial phytochromes
Avoimen toimintakulttuurin palveluiden itsearviointityökalu
Toimintakulttuurin avoimen linjauksen tueksi on kehitetty palveluiden itsearviointityökalu, joka huomioi kaikkien aiempien avoimen tieteen kansallisten linjausten suositukset. Työkalun tarkoituksena on auttaa tutkimusorganisaatioita palveluiden itsearvioinnissa, kehittämisessä ja saataville asettamisessa.</p
The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography
Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 angstrom resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 angstrom resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.Peer reviewe
Fast Photochemistry of Prototypical Phytochromes : A Species vs. Subunit Specific Comparison
Phytochromes are multi-domain red light photosensor proteins, which convert red
light photons to biological activity utilizing the multitude of structural and chemical
reactions. The steady increase in structural information obtained from various
bacteriophytochromes has increased understanding about the functional mechanism
of the photochemical processes of the phytochromes. Furthermore, a number of
spectroscopic studies have revealed kinetic information about the light-induced
reactions. The spectroscopic changes are, however, challenging to connect with the
structural changes of the chromophore and the protein environment, as the excited state
properties of the chromophores are very sensitive to the small structural and chemical
changes of their environment. In this article, we concentrate on the results of ultra-fast
spectroscopic experiments which reveal information about the important initial steps of
the photoreactions of the phytochromes. We survey the excited state properties obtained
during the last few decades. The differences in kinetics between different research
laboratories are traditionally related to the differences of the studied species. However,
we notice that the variation in the excited state properties depends on the subunit
composition of the protein as well. This observation illustrates a feedback mechanism
from the other domains to the chromophore. We propose that two feedback routes exist
in phytochromes between the chromophore and the remotely located effector domain.
The well-known connection between the subunits is the so-called tongue region, which
changes its secondary structure while changing the light-activated state of the system.
The other feedback route which we suggest is less obvious, it is made up of several water
molecules ranging from the dimer interface to the vicinity of the chromophore, allowing
even proton transfer reactions nearby the chromophore.peerReviewe
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