| 1. |
- Berntsson, Oskar, 1989, et al.
(författare)
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Photoactivation of Drosophila melanogaster cryptochrome through sequential conformational transitions
- 2019
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Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 5:7
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Tidskriftsartikel (refereegranskat)abstract
- Cryptochromes are blue-light photoreceptor proteins, which provide input to circadian clocks. The cryptochrome from Drosophila melanogaster (DmCry) modulates the degradation of Timeless and itself. It is unclear how light absorption by the chromophore and the subsequent redox reactions trigger these events. Here, we use nano- to millisecond time-resolved x-ray solution scattering to reveal the light-activated conformational changes in DmCry and the related (6-4) photolyase. DmCry undergoes a series of structural changes, culminating in the release of the carboxyl-terminal tail (CTT). The photolyase has a simpler structural response. We find that the CTT release in DmCry depends on pH. Mutation of a conserved histidine, important for the biochemical activity of DmCry, does not affect transduction of the structural signal to the CTT. Instead, molecular dynamics simulations suggest that it stabilizes the CTT in the resting-state conformation. Our structural photocycle unravels the first molecular events of signal transduction in an animal cryptochrome.
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| 2. |
- Berntsson, Oskar, 1989, et al.
(författare)
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Sequential conformational transitions and alpha-helical supercoiling regulate a sensor histidine kinase
- 2017
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Sensor histidine kinases are central to sensing in bacteria and in plants. They usually contain sensor, linker, and kinase modules and the structure of many of these components is known. However, it is unclear how the kinase module is structurally regulated. Here, we use nano- to millisecond time-resolved X-ray scattering to visualize the solution structural changes that occur when the light-sensitive model histidine kinase YF1 is activated by blue light. We find that the coiled coil linker and the attached histidine kinase domains undergo a left handed rotation within microseconds. In a much slower second step, the kinase domains rearrange internally. This structural mechanism presents a template for signal transduction in sensor histidine kinases.
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| 3. |
- Berntsson, Oskar, 1989, et al.
(författare)
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Time-Resolved X-Ray Solution Scattering Reveals the Structural Photoactivation of a Light-Oxygen-Voltage Photoreceptor
- 2017
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Ingår i: Structure. - : Elsevier BV. - 0969-2126. ; 25:6
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Tidskriftsartikel (refereegranskat)abstract
- Light-oxygen-voltage (LOV) receptors are sensory proteins controlling a wide range of organismal adaptations in multiple kingdoms of life. Because of their modular nature, LOV domains are also attractive for use as optogenetic actuators. A flavin chromophore absorbs blue light, forms a bond with a proximal cysteine residue, and induces changes in the surroundings. There is a gap of knowledge on how this initial signal is relayed further through the sensor to the effector module. To characterize these conformational changes, we apply time-resolved X-ray scattering to the homodimeric LOV domain from Bacillus subtilis YtvA. We observe a global structural change in the LOV dimer synchronous with the formation of the chromophore photoproduct state. Using molecular modeling, this change is identified as splaying apart and relative rotation of the two monomers, which leads to an increased separation at the anchoring site of the effector modules.
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| 4. |
- Björling, Alexander, 1983, et al.
(författare)
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Structural photoactivation of a full-length bacterial phytochrome
- 2016
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Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 2:8
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Cellini, Andrea, 1991, et al.
(författare)
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The three-dimensional structure of Drosophila melanogaster (6-4) photolyase at room temperature
- 2021
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Ingår i: Acta Crystallographica Section D-Structural Biology. - : International Union of Crystallography (IUCr). - 2059-7983. ; 77, s. 1001-1009
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Tidskriftsartikel (refereegranskat)abstract
- (6-4) photolyases are flavoproteins that belong to the photolyase/cryptochrome family. Their function is to repair DNA lesions using visible light. Here, crystal structures of Drosophila melanogaster (6-4) photolyase [Dm(6-4)photolyase] at room and cryogenic temperatures are reported. The room-temperature structure was solved to 2.27 angstrom resolution and was obtained by serial femtosecond crystallography (SFX) using an X-ray free-electron laser. The crystallization and preparation conditions are also reported. The cryogenic structure was solved to 1.79 angstrom resolution using conventional X-ray crystallography. The structures agree with each other, indicating that the structural information obtained from crystallography at cryogenic temperature also applies at room temperature. Furthermore, UV-Vis absorption spectroscopy confirms that Dm(6-4)photolyase is photoactive in the crystals, giving a green light to time-resolved SFX studies on the protein, which can reveal the structural mechanism of the photoactivated protein in DNA repair.
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| 6. |
- Claesson, Elin, 1989, et al.
(författare)
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The primary structural photoresponse of phytochrome proteins captured by a femtosecond X-ray laser
- 2020
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Ingår i: eLife. - 2050-084X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The data reveal a twist of the D-ring, which leads to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signaling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the observed collective changes are important for the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.
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| 7. |
- Edlund, Petra, et al.
(författare)
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The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Henry, Léocadie, et al.
(författare)
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New Light on the Mechanism of Phototransduction in Phototropin
- 2020
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 59:35, s. 3206-3215
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Tidskriftsartikel (refereegranskat)abstract
- Phototropins are photoreceptor proteins that regulate blue light-dependent biological processes for efficient photosynthesis in plants and algae. The proteins consist of a photosensory domain that responds to the ambient light and an output module that triggers cellular responses. The photosensory domain of phototropin from Chlamydomonas reinhardtii contains two conserved LOV (light-oxygen-voltage) domains with flavin chromophores. Blue light triggers the formation of a covalent cysteine-flavin adduct and upregulates the phototropin kinase activity. Little is known about the structural mechanism that leads to kinase activation and how the two LOV domains contribute to this. Here, we investigate the role of the LOV1 domain from C. reinhardtii phototropin by characterizing the structural changes occurring after blue light illumination with nano- to millisecond time-resolved X-ray solution scattering. By structurally fitting the data with atomic models generated by molecular dynamics simulations, we find that adduct formation induces a rearrangement of the hydrogen bond network from the buried chromophore to the protein surface. In particular, the change in conformation and the associated hydrogen bonding of the conserved glutamine 120 induce a global movement of the beta-sheet, ultimately driving a change in the electrostatic potential on the protein surface. On the basis of the change in the electrostatics, we propose a structural model of how LOV1 and LOV2 domains interact and regulate the full-length phototropin from C. reinhardtii. This provides a rationale for how LOV photosensor proteins function and contributes to the optimal design of optogenetic tools based on LOV domains.
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| 9. |
- Henry, Léocadie, et al.
(författare)
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Real-time tracking of protein unfolding with time-resolved x-ray solution scattering
- 2020
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Ingår i: Structural Dynamics. - : AIP Publishing. - 2329-7778. ; 7:5
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Tidskriftsartikel (refereegranskat)abstract
- The correct folding of proteins is of paramount importance for their function, and protein misfolding is believed to be the primary cause of a wide range of diseases. Protein folding has been investigated with time-averaged methods and time-resolved spectroscopy, but observing the structural dynamics of the unfolding process in real-time is challenging. Here, we demonstrate an approach to directly reveal the structural changes in the unfolding reaction. We use nano- to millisecond time-resolved x-ray solution scattering to probe the unfolding of apomyoglobin. The unfolding reaction was triggered using a temperature jump, which was induced by a nanosecond laser pulse. We demonstrate a new strategy to interpret time-resolved x-ray solution scattering data, which evaluates ensembles of structures obtained from molecular dynamics simulations. We find that apomyoglobin passes three states when unfolding, which we characterize as native, molten globule, and unfolded. The molten globule dominates the population under the conditions investigated herein, whereas native and unfolded structures primarily contribute before the laser jump and 30 mu s after it, respectively. The molten globule retains much of the native structure but shows a dynamic pattern of inter-residue contacts. Our study demonstrates a new strategy to directly observe structural changes over the cause of the unfolding reaction, providing time- and spatially resolved atomic details of the folding mechanism of globular proteins. (C) 2020 Author(s).
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| 10. |
- Takala, H., et al.
(författare)
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On the (un)coupling of the chromophore, tongue interactions, and overall conformation in a bacterial phytochrome
- 2018
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 293:21, s. 8161-8172
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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