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.
We present the structure of a photoactivated animal (6-4) photolyase in its radical pair state, captured by serial crystallography. We observe how a conserved asparigine moves towards the semiquinone FAD...
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 β-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.
(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 Å 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 Å 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.
Charge transfer reactions in proteins are fundamentally important for life, but it is currently not clear how protein structural dynamics control these electron transfer reactions. Photolyases/cryptochromes, which repair DNA and signal in all kingdoms of life, have a paradigm electron transfer cascade. Here, photoreduction of the flavin cofactor initiates charge transfer along a chain of four conserved tryptophans. We report femtosecond X-ray crystallographic snap-shots for the Drosophila melanogaster (6-4) photolyase, revealing protein structural changes while electron transfer occurs. At femto- and picosecond delays, photoreduction of the flavin by the first tryptophan causes directed structural responses at key residue asparagine 403, at a conserved salt bridge, and by rearrangements of nearby water molecules. Along the tryptophan cascade, we detect charge-induced protein structural changes close to the second tryptophan from 1 ps to 20 ps, thereby identifying methionine 408 as an active participant in the redox chain, and from 300 ps around the fourth tryptophan. The data reveal that the protein undergoes highly directed and carefully timed adaptations of its structure to facilitate electron transfer. This suggests that evolution has optimized fast protein fluctuations for optimal function.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.