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Cryptochromes are essential flavoproteins for circadian rhythms and avian magnetoreception. Flavin adenine dinucleotide (FAD), a chromophore within cryptochromes, absorbs blue light, initiating electron transfer processes that lead to a biological signaling cascade. A key step in this cascade is the formation of the FAD semiquinone radical (FADHâ¢), characterized through a specific red-light absorption. The absorption spectra of FADH⢠in cryptochromes are, however, significantly different from those recorded for the cofactor in solution, primarily due to protein-induced shifts in the absorption peaks. This study employs a multiscale approach, combining molecular dynamics (MD) simulations with quantum mechanical/molecular mechanical (QM/MM) methodologies, to investigate the influence of protein dynamics on embedded FADH⢠absorption. We emphasize the role of the protein's polarizable environment in the shaping of the absorption spectrum, crucial for accurate spectral predictions in cryptochromes. Our findings provide valuable insights into the absorption process, advancing our understanding of cryptochrome functioning.
Assuntos
Arabidopsis , Criptocromos , Flavina-Adenina Dinucleotídeo , Simulação de Dinâmica Molecular , Teoria Quântica , Criptocromos/química , Criptocromos/metabolismo , Arabidopsis/metabolismo , Arabidopsis/química , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismoRESUMO
The Earth's magnetic field can provide reliable directional information, allowing migrating animals to orient themselves using a magnetic compass or estimate their position relative to a target using map-based orientation. Here we show for the first time that young, inexperienced herring (Clupea harengus, Ch) have a magnetic compass when they migrate hundreds of kilometres to their feeding grounds. In birds, such as the European robin (Erithacus rubecula), radical pair-based magnetoreception involving cryptochrome 4 (ErCRY4) was demonstrated; the molecular basis of magnetoreception in fish is still elusive. We show that cry4 expression in the eye of herring is upregulated during the migratory season, but not before, indicating a possible use for migration. The amino acid structure of herring ChCRY4 shows four tryptophans and a flavin adenine dinucleotide-binding site, a prerequisite for a magnetic receptor. Using homology modelling, we successfully reconstructed ChCRY4 of herring, DrCRY4 of zebrafish (Danio rerio) and StCRY4 of brown trout (Salmo trutta) and showed that ChCRY4, DrCRY4 and ErCRY4a, but not StCRY4, exhibit very comparable dynamic behaviour. The electron transfer could take place in ChCRY4 in a similar way to ErCRY4a. The combined behavioural, transcriptomic and simulation experiments provide evidence that CRY4 could act as a magnetoreceptor in Atlantic herring.
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Criptocromos , Peixes , Animais , Criptocromos/metabolismo , Criptocromos/química , Peixes/fisiologia , Migração Animal/fisiologia , Campos Magnéticos , Proteínas de Peixes/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/química , Orientação/fisiologiaRESUMO
This roadmap reviews the new, highly interdisciplinary research field studying the behavior of condensed matter systems exposed to radiation. The Review highlights several recent advances in the field and provides a roadmap for the development of the field over the next decade. Condensed matter systems exposed to radiation can be inorganic, organic, or biological, finite or infinite, composed of different molecular species or materials, exist in different phases, and operate under different thermodynamic conditions. Many of the key phenomena related to the behavior of irradiated systems are very similar and can be understood based on the same fundamental theoretical principles and computational approaches. The multiscale nature of such phenomena requires the quantitative description of the radiation-induced effects occurring at different spatial and temporal scales, ranging from the atomic to the macroscopic, and the interlinks between such descriptions. The multiscale nature of the effects and the similarity of their manifestation in systems of different origins necessarily bring together different disciplines, such as physics, chemistry, biology, materials science, nanoscience, and biomedical research, demonstrating the numerous interlinks and commonalities between them. This research field is highly relevant to many novel and emerging technologies and medical applications.
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It is still a puzzle that has not been entirely solved how migratory birds utilize the Earth's magnetic field for biannual migration. The most consistent explanation thus far is rooted in the modulation of the biological function of the cryptochrome 4 (Cry4) protein by an external magnetic field. This phenomenon is closely linked with the flavin adenine dinucleotide (FAD) cofactor that is noncovalently bound in the protein. Cry4 is activated by blue light, which is absorbed by the FAD cofactor. Subsequent electron and proton transfers trigger radical pair formation in the protein, which is sensitive to the external magnetic field. An important long-lasting redox state of the FAD cofactor is the signaling (FADHâ¢) state, which is present after the transient electron transfer steps have been completed. Recent experimental efforts succeeded in crystallizing the Cry4 protein from Columbia livia (ClCry4) with all of the important residues needed for protein photoreduction. This specific crystallization of Cry4 protein so far is the only avian cryptochrome crystal structure available, which, however, has great similarity to the Cry4 proteins of night migratory birds. The previous experimental studies of the ClCry4 protein included the absorption properties of the protein in its different redox states. The absorption spectrum of the FADH⢠state demonstrated a peculiar red shift compared to the photoabsorption properties of the FAD cofactor in its FADH⢠state in other Cry proteins from other species. The aim of this study is to understand this red shift by employing the tools of computational microscopy and, in particular, a QM/MM approach that relies on the polarizable embedding approximation.
Assuntos
Criptocromos , Flavina-Adenina Dinucleotídeo , Criptocromos/química , Criptocromos/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Animais , Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , OxirreduçãoRESUMO
The tick-borne encephalitis virus (TBE) is a neurotrophic disease that has spread more rapidly throughout Europe and Asia in the past few years. At the same time, no cure or specific therapy is known to battle the illness apart from vaccination. To find a pharmacologically relevant drug, a computer-aided drug screening was initiated. Such a procedure probes a possible binding of a drug to the RNA Polymerase of TBE. The crystal structure of the receptor, however, includes missing and partially modeled regions, which rendered the structure incomplete and of questionable use for a thorough drug screening procedure. The quality of the receptor model was addressed by studying three putative structures created. We show that the choice of receptor models greatly influences the binding affinity of potential drug molecules and that the binding location could also be significantly impacted. We demonstrate that some drug candidates are unsuitable for one model but show decent results for another. Without any prejudice on the three employed receptor models, the study reveals the imperative need to investigate the receptor structure before drug binding is probed whether experimentally or computationally.
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Marine fish migrate long distances up to hundreds or even thousands of kilometers for various reasons that include seasonal dependencies, feeding, or reproduction. The ability to perceive the geomagnetic field, called magnetoreception, is one of the many mechanisms allowing some fish to navigate reliably in the aquatic realm. While it is believed that the photoreceptor protein cryptochrome 4 (Cry4) is the key component for the radical pair-based magnetoreception mechanism in night migratory songbirds, the Cry4 mechanism in fish is still largely unexplored. The present study aims to investigate properties of the fish Cry4 protein in order to understand the potential involvement in a radical pair-based magnetoreception. Specifically, a computationally reconstructed atomistic model of Cry4 from the Atlantic herring (Clupea harengus) was studied employing classical molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) methods to investigate internal electron transfers and the radical pair formation. The QM/MM simulations reveal that electron transfers occur similarly to those found experimentally and computationally in Cry4 from European robin (Erithacus rubecula). It is therefore plausible that the investigated Atlantic herring Cry4 has the physical and chemical properties to form radical pairs that in turn could provide fish with a radical pair-based magnetic field compass sensor.
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Cryptochrome is currently the major contender of a protein to underpin magnetoreception, the ability to sense the Earth's magnetic field. Among various types of cryptochromes, cryptochrome 4 has been identified as the likely magnetoreceptor in migratory birds. All-atom molecular dynamics (MD) studies have offered first insights into the structural dynamics of cryptochrome but are limited to a short time scale due to large computational demands. Here, we employ coarse-grained MD simulations to investigate the emergence of long-lived states and conformational changes in pigeon cryptochrome 4. Our coarse-grained simulations complete the picture by permitting observation on a significantly longer time scale. We observe conformational transitions in the phosphate-binding loop of pigeon cryptochrome 4 upon activation and identify prominent motions in residues 440-460, suggesting a possible role as a signaling state of the protein or as a gated interaction site for forming protein complexes that might facilitate downstream processes. The findings highlight the importance of considering longer time scales in studying cryptochrome dynamics and magnetoreception. Coarse-grained MD simulations offer a valuable tool to unravel the complex behavior of cryptochrome proteins and shed new light on the mechanisms underlying their role in magnetoreception. Further exploration of these conformational changes and their functional implications may contribute to a deeper understanding of the molecular mechanisms of magnetoreception in birds.
Assuntos
Columbidae , Criptocromos , Oxirredução , Animais , Columbidae/genética , Columbidae/metabolismo , Criptocromos/química , Criptocromos/metabolismo , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Migratory birds possess remarkable accuracy in orientation and navigation, which involves various compass systems including the magnetic compass. Identifying the primary magnetosensor remains a fundamental open question. Cryptochromes (Cry) have been shown to be magnetically sensitive, and Cry4a from a migratory songbird seems to show enhanced magnetic sensitivity in vitro compared to Cry4a from resident species. We investigate Cry and their potential involvement in magnetoreception in a phylogenetic framework, integrating molecular evolutionary analyses with protein dynamics modelling. Our analysis is based on 363 bird genomes and identifies different selection regimes in passerines. We show that Cry4a is characterized by strong positive selection and high variability, typical characteristics of sensor proteins. We identify key sites that are likely to have facilitated the evolution of an optimized sensory protein for night-time orientation in songbirds. Additionally, we show that Cry4 was lost in hummingbirds, parrots and Tyranni (Suboscines), and thus identified a gene deletion, which might facilitate testing the function of Cry4a in birds. In contrast, the other avian Cry (Cry1 and Cry2) were highly conserved across all species, indicating basal, non-sensory functions. Our results support a specialization or functional differentiation of Cry4 in songbirds which could be magnetosensation.
Assuntos
Aves Canoras , Animais , Filogenia , Aves Canoras/fisiologia , Criptocromos/metabolismo , Campos Magnéticos , Migração Animal/fisiologiaRESUMO
Gram-negative bacteria possess a complex structural cell envelope that constitutes a barrier for antimicrobial peptides that neutralize the microbes by disrupting their cell membranes. Computational and experimental approaches were used to study a model outer membrane interaction with an antimicrobial peptide, melittin. The investigated membrane included di[3-deoxy-d-manno-octulosonyl]-lipid A (KLA) in the outer leaflet and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in the inner leaflet. Molecular dynamics simulations revealed that the positively charged helical C-terminus of melittin anchors rapidly into the hydrophilic headgroup region of KLA, while the flexible N-terminus makes contacts with the phosphate groups of KLA, supporting melittin penetration into the boundary between the hydrophilic and hydrophobic regions of the lipids. Electrochemical techniques confirmed the binding of melittin to the model membrane. To probe the peptide conformation and orientation during interaction with the membrane, polarization modulation infrared reflection absorption spectroscopy was used. The measurements revealed conformational changes in the peptide, accompanied by reorientation and translocation of the peptide at the membrane surface. The study suggests that melittin insertion into the outer membrane affects its permeability and capacitance but does not disturb the membrane's bilayer structure, indicating a distinct mechanism of the peptide action on the outer membrane of Gram-negative bacteria.
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Peptídeos Antimicrobianos , Lipopolissacarídeos , Lipopolissacarídeos/química , Meliteno/química , Peptídeos/química , Bactérias Gram-Negativas/metabolismoRESUMO
Transition metal complexes featuring redox-active ligands often exhibit multiple redox states, influenced by the interplay between the metal center and the ligand. This study delves into the electronic structures of two mononuclear complexes of copper with two similar redox-active urea azine ligands. The ligands differ by the replacement of an NCH3 moiety by an S atom in the ligand backbone. Experimental analysis yields pronounced electronic structural disparities between these complexes, observable in both the solution and solid phases. Conventional quantum chemical methods, such as density functional theory using different functionals (B3LYP, TPSSh, and CAM-B3LYP), remain inadequate to rationalize the observed spectroscopic anomalies. However, a multiconfigurational approach elucidates the disparate behaviors of these complexes. Multireference perturbation theory, based on complete active space self-consistent field computations, identifies Cu(I) in the case of the complex with the NCH3 containing ligands and a state with substantial Cu(II) contributions in the case of the complex with the S atom containing ligands. In contrast, DFT indicates Cu(I) in both scenarios.
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The Automated Ligand Searcher (ALISE) is designed as an automated computational drug discovery tool. To approximate the binding free energy of ligands to a receptor, ALISE includes a three-stage workflow, with each stage involving an increasingly sophisticated computational method: molecular docking, molecular dynamics, and free energy perturbation, respectively. To narrow the number of potential ligands, poorly performing ligands are gradually segregated out. The performance and usability of ALISE are benchmarked for a case study containing known active ligands and decoys for the HIV protease. The example illustrates that ALISE filters the decoys successfully and demonstrates that the automation, comprehensiveness, and user-friendliness of the software make it a valuable tool for improved and faster drug development workflows.
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Simulação de Dinâmica Molecular , Software , Ligantes , Simulação de Acoplamento Molecular , Descoberta de Drogas , Ligação ProteicaRESUMO
Cryptochromes (CRYs) are a structurally conserved but functionally diverse family of proteins that can confer unique sensory properties to organisms. In the marine bristle worm Platynereis dumerilii, its light receptive cryptochrome L-CRY (PdLCry) allows the animal to discriminate between sunlight and moonlight, an important requirement for synchronizing its lunar cycle-dependent mass spawning. Using cryo-electron microscopy, we show that in the dark, PdLCry adopts a dimer arrangement observed neither in plant nor insect CRYs. Intense illumination disassembles the dimer into monomers. Structural and functional data suggest a mechanistic coupling between the light-sensing flavin adenine dinucleotide chromophore, the dimer interface, and the C-terminal tail helix, with a likely involvement of the phosphate binding loop. Taken together, our work establishes PdLCry as a CRY protein with inverse photo-oligomerization with respect to plant CRYs, and provides molecular insights into how this protein might help discriminating the different light intensities associated with sunlight and moonlight.
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Criptocromos , Luz , Animais , Criptocromos/metabolismo , Microscopia CrioeletrônicaRESUMO
Protein glycosylation is a common post-translational modification on extracellular proteins. The conformational dynamics of several glycoproteins have been characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS). However, it is, in most cases, not possible to extract information about glycan conformation and dynamics due to the general difficulty of separating the deuterium content of the glycan from that of the peptide (in particular, for O-linked glycans). Here, we investigate whether the fragmentation of protonated glycopeptides by collision-induced dissociation (CID) can be used to determine the solution-specific deuterium content of the glycan. Central to this concept is that glycopeptides can undergo a facile loss of glycans upon CID, thereby allowing for the determination of their masses. However, an essential prerequisite is that hydrogen and deuterium (H/D) scrambling can be kept in check. Therefore, we have measured the degree of scrambling upon glycosidic bond cleavage in glycopeptides that differ in the conformational flexibility of their backbone and glycosylation pattern. Our results show that complete scrambling precedes the glycosidic bond cleavage in normal glycopeptides derived from a glycoprotein; i.e., all labile hydrogens have undergone positional randomization prior to loss of the glycan. In contrast, the glycosidic bond cleavage occurs without any scrambling in the glycopeptide antibiotic vancomycin, reflecting that the glycan cannot interact with the peptide moiety due to a conformationally restricted backbone as revealed by molecular dynamics simulations. Scrambling is also inhibited, albeit to a lesser degree, in the conformationally restricted glycopeptides ristocetin and its pseudoaglycone, demonstrating that scrambling depends on an intricate interplay between the flexibility and proximity of the glycan and the peptide backbone.
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Glicopeptídeos , Hidrogênio , Glicopeptídeos/química , Deutério , Peptídeos/química , Glicoproteínas/química , Polissacarídeos/químicaRESUMO
Cryptochromes are proteins that are highly conserved across species and in many instances bind the flavin adenine dinucleotide (FAD) cofactor within their photolyase-homology region (PHR) domain. The FAD cofactor has multiple redox states that help catalyze reactions, and absorbs photons at about 450 nm, a feature linked to the light-related functions of cryptochrome proteins. Reactive oxygen species (ROS) are produced from redox reactions involving molecular oxygen and are involved in a myriad of biological processes. Superoxide O2â¢- is an exemplary ROS that may be formed through electron transfer from FAD to O2, generating an electron radical pair. Although the formation of a superoxide-FAD radical pair has been speculated, it is still unclear if the required process steps could be realized in cryptochrome. Here, we present results from molecular dynamics (MD) simulations of oxygen interacting with the PHR domain of Arabidopsis thaliana cryptochrome 1 (AtCRY1). Using MD simulation trajectories, oxygen binding locations are characterized through both the O2-FAD intermolecular distance and the local protein environment. Oxygen unbinding times are characterized through replica simulations of the bound oxygen. Simulations reveal that oxygen molecules can localize at certain sites within the cryptochrome protein for tens of nanoseconds, and superoxide molecules can localize for significantly longer. This relatively long-duration molecule binding suggests the possibility of an electron-transfer reaction leading to superoxide formation. Estimates of electron-transfer rates using the Marcus theory are performed for the identified potential binding sites. Molecular oxygen binding results are compared with recent results demonstrating long-time oxygen binding within the electron-transfer flavoprotein (ETF), another FAD binding protein.
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Arabidopsis , Superóxidos , Superóxidos/química , Superóxidos/metabolismo , Criptocromos/química , Criptocromos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Arabidopsis/metabolismo , Oxigênio/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas Transferidoras de Elétrons/químicaRESUMO
The ability of migratory birds to sense magnetic fields has been known for decades, although the understanding of the underlying mechanism is still elusive. Currently, the strongest magnetoreceptor candidate in birds is a protein called cryptochrome 4a. The cryptochrome 4a protein has changed through evolution, apparently endowing some birds with a more pronounced magnetic sensitivity than others. Using phylogenetic tools, we show that a specific tryptophan tetrad and a tyrosine residue predicted to be essential for cryptochrome activation are highly conserved in the avian clade. Through state-of-the-art molecular dynamics simulations and associated analyses, we also studied the role of these specific residues and the associated mutants on the overall dynamics of the protein. The analyses of the single residue mutations were used to judge how far a local change in the protein structure can impact specific dynamics of European robin cryptochrome 4a. We conclude that the replacements of each of the tryptophans one by one with a phenylalanine do not compromise the overall stability of the protein.
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Homo-dimer formation is important for the function of many proteins. Although dimeric forms of cryptochromes (Cry) have been found by crystallography and were recently observed in vitro for European robin Cry4a, little is known about the dimerization of avian Crys and the role it could play in the mechanism of magnetic sensing in migratory birds. Here, we present a combined experimental and computational investigation of the dimerization of robin Cry4a resulting from covalent and non-covalent interactions. Experimental studies using native mass spectrometry, mass spectrometric analysis of disulfide bonds, chemical cross-linking, and photometric measurements show that disulfide-linked dimers are routinely formed, that their formation is promoted by exposure to blue light, and that the most likely cysteines are C317 and C412. Computational modeling and molecular dynamics simulations were used to generate and assess a number of possible dimer structures. The relevance of these findings to the proposed role of Cry4a in avian magnetoreception is discussed.
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Criptocromos , Aves Canoras , Animais , Criptocromos/química , Dimerização , Aves Canoras/metabolismo , LuzRESUMO
Night-migratory songbirds have a light-dependent magnetic compass sense, the mechanism of which is thought to depend on the photochemical formation of radical pairs in cryptochrome (Cry) proteins located in the retina. The finding that weak radiofrequency (RF) electromagnetic fields can prevent birds from orienting in the Earth's magnetic field has been regarded as a diagnostic test for this mechanism and as a potential source of information on the identities of the radicals. The maximum frequency that could cause such disorientation has been predicted to lie between 120 and 220 MHz for a flavin-tryptophan radical pair in Cry. Here we show that the magnetic orientation capabilities of Eurasian blackcaps (Sylvia atricapilla) are not affected by RF noise in the frequency bands 140 to 150 MHz and 235 to 245 MHz. From a consideration of its internal magnetic interactions, we argue that RF field effects on a flavin-containing radical-pair sensor should be approximately independent of frequency up to 116 MHz and that birds' sensitivity to RF disorientation should fall by about two orders of magnitude when the frequency exceeds 116 MHz. Taken together with our earlier finding that 75 to 85 MHz RF fields disrupt the magnetic orientation of blackcaps, these results provide compelling evidence that the magnetic compass of migratory birds operates by a radical pair mechanism.
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Aves Canoras , Resposta Táctica , Animais , Aves Canoras/metabolismo , Processos Fotoquímicos , Migração Animal , Campos Magnéticos , Criptocromos/metabolismoRESUMO
This short review reports the surprising phenomenon of nuclear hyperpolarization occurring in chemical reactions, which is called CIDNP (chemically induced dynamic nuclear polarization) or photo-CIDNP if the chemical reaction is light-driven. The phenomenon occurs in both liquid and solid-state, and electron transfer systems, often carrying flavins as electron acceptors, are involved. Here, we explain the physical and chemical properties of flavins, their occurrence in spin-correlated radical pairs (SCRP) and the possible involvement of flavin-carrying SCRPs in animal magneto-reception at earth's magnetic field.
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Flavoproteínas , Campos Magnéticos , Animais , Transporte de Elétrons , Flavinas/químicaRESUMO
The primary step in the mechanism by which migratory birds sense the Earth's magnetic field is thought to be the light-induced formation of long-lived magnetically sensitive radical pairs within cryptochrome flavoproteins located in the birds' retinas. Blue-light absorption by the non-covalently bound flavin chromophore triggers sequential electron transfers along a chain of four tryptophan residues toward the photoexcited flavin. The recently demonstrated ability to express cryptochrome 4a from the night-migratory European robin (Erithacus rubecula), ErCry4a, and to replace each of the tryptophan residues by a redox-inactive phenylalanine offers the prospect of exploring the roles of the four tryptophans. Here, we use ultrafast transient absorption spectroscopy to compare wild type ErCry4a and four mutants having a phenylalanine at different positions in the chain. We find that each of the three tryptophan residues closest to the flavin adds a distinct relaxation component (time constants: 0.5, 30, and 150 ps) in the transient absorption data. The dynamics of the mutant containing a phenylalanine at the fourth position, furthest from the flavin, are very similar to those of wild type ErCry4a, except for a reduced concentration of long-lived radical pairs. The experimental results are evaluated and discussed in the framework of real-time quantum mechanical/molecular mechanical electron transfer simulations based on the density functional-based tight binding approach. This comparison between simulation results and experimental measurements provides a detailed microscopic insight into the sequential electron transfers along the tryptophan chain. Our results offer a route to the study of spin transport and dynamical spin correlations in flavoprotein radical pairs.
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Criptocromos , Triptofano , Criptocromos/química , Triptofano/química , Elétrons , Transporte de Elétrons , Campos Magnéticos , Flavinas/metabolismoRESUMO
Spin relaxation is an important aspect of the spin dynamics of free radicals and can have a significant impact on the outcome of their spin-selective reactions. Examples range from the use of radicals as spin qubits in quantum information processing to the radical pair reactions in proteins that may allow migratory birds to sense the direction of the Earth's magnetic field. Accurate modeling of spin relaxation, however, is non-trivial. Bloch-Redfield-Wangsness theory derives a quantum mechanical master equation from system-bath interactions in the Markovian limit that provides a comprehensive framework for describing spin relaxation. Unfortunately, the construction of the master equation is system-specific and often resource-heavy. To address this challenge, we introduce a generalized and efficient implementation of BRW theory as a new feature of the spin dynamics toolkit MolSpin which offers an easy-to-use approach for studying systems of reacting radicals of varying complexity.