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We present a brief review of our recent computational studies of hydrogen bonds (H-bonds) in helical secondary structures of proteins, α-helix and 310-helix, using a Negative Fragmentation Approach with density functional theory. We found that the depolarized electronic structures of the carbonyl oxygen of the ith residue and the amide hydrogen of the (i + 4)th residue cause weaker H-bond in an α-helix than in an isolated H-bond. Our calculations showed that the H-bond energies in the 310-helix were also weaker than those of the isolated H-bonds. In the 310-helices, the adjacent N–H group at the (i + 1)th residue was closer to the C=O group of the H-bond pair than the adjacent C=O group in the 310-helices, whereas the adjacent C=O group at the (i + 1)th residue was close to the H-bond acceptor in α-helices. Therefore, the destabilization of the H-bond is attributed to the depolarization caused by the adjacent residue of the helical backbone connecting the H-bond donor and acceptor. The differences in the change in electron density revealed that such depolarizations were caused by the local electronic interactions in their neighborhood inside the helical structure and redistributed the electron density. We also present the improvements in the force field of classical molecular simulation, based on our findings.

Urease catalyzes the hydrolysis of urea to form ammonia and carbamate, inducing an overall pH increase that affects both human health and agriculture. Inhibition, mutagenesis, and kinetic studies have provided insights into its enzymatic role, but there have been debates on the substrate binding mode as well as the reaction mechanism. In the present study, we report quatum mechanics-only (QM-only) and quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) calculations on urease that mainly investigate the binding mode of urea and the mechanism of the urease-catalyzed hydrolysis reaction. Comparison between the experimental data and our QM(GFN2-xTB)/MM metadynamics results demonstrates that urea hydrolysis via a complex with bidentate-bound urea is much more favorable than via that with monodentate-bound urea for both nucleophilic attack and the subsequent proton transfer steps. We also indicate that the bidentate coordination of urea fits the active site with a closed conformation of the mobile flap and can facilitate the stabilization of transition states and intermediates by forming multiple hydrogen bonds with certain active site residues.

“Plant-type” ferredoxins (Fds) in the thylakoid membranes of plants, algae, and cyanobacteria possess a single [2Fe-2S] cluster in active sites and mediate light-induced electron transfer from Photosystem I reaction centers to various Fd-dependent enzymes. Structural knowledge of plant-type Fds is relatively limited to static structures, and the detailed behavior of oxidized and reduced Fds has not been fully elucidated. It is important that the investigations of the effects of active-center reduction on the structures and dynamics for elucidating electron-transfer mechanisms. In this study, model systems of oxidized and reduced Fds were constructed from the high-resolution crystal structure of Chlamydomonas reinhardtii Fd1, and three 200 ns molecular dynamics simulations were performed for each system. The force field parameters of the oxidized and reduced active centers were independently obtained using quantum chemical calculations. There were no substantial differences in the global conformations of the oxidized and reduced forms. In contrast, active-center reduction affected the hydrogen-bond network and compactness of the surrounding residues, leading to the increased flexibility of the side chain of Phe61, which is essential for the interaction between Fd and the target protein. These computational results will provide insight into the electron-transfer mechanisms in the Fds.

Structure–function relationships in proteins have been one of the crucial scientific topics in recent research. Heme proteins have diverse and pivotal biological functions. Therefore, clarifying their structure–function correlation is significant to understand their functional mechanism and is informative for various fields of science. In this study, we constructed convolutional neural network models for predicting protein functions from the tertiary structures of heme-binding sites (active sites) of heme proteins to examine the structure–function correlation. As a result, we succeeded in the classification of oxygen-binding protein (OB), oxidoreductase (OR), proteins with both functions (OB–OR), and electron transport protein (ET) with high accuracy. Although the misclassification rate for OR and ET was high, the rates between OB and ET and between OB and OR were almost zero, indicating that the prediction model works well between protein groups with quite different functions. However, predicting the function of proteins modified with amino acid mutation(s) remains a challenge. Our findings indicate a structure–function correlation in the active site of heme proteins. This study is expected to be applied to the prediction of more detailed protein functions such as catalytic reactions.

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) P G –O 3B bond cleavage; 3) four concomitant events: W1–PO 3 − formation, OH − and proton cleavage, nucleophilic attack by the OH − against P G , and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi–bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.

Hydrogen-bond (H-bond) energies in 310-helices of short alanine peptides were systematically examined by precise DFT calculations with the negative fragmentation approach (NFA), a modified method based on the molecular tailoring approach. The contribution of each H-bond was evaluated in detail from the 310-helical conformation of total energies (whole helical model, WH3-10 model), and the results were compared with the property of H-bond in α-helix from our previous study. The H-bond energies of the WH3-10 model exhibited tendencies different from those exhibited by the α-helix in that they depended on the helical position of the relevant H-bond pair. H-bond pairs adjacent to the terminal H-bond pairs were observed to be strongly destabilized. The analysis of electronic structures indicated that structural characteristics cause the destabilization of the H-bond in 310-helices. We also found that the longer the helix length, the more stable the H-bond in the terminal pairs of the WH3-10 model, suggesting the action of H-bond cooperativity.

Guanine is the most susceptible base to oxidation damage induced by reactive oxygen species including singlet oxygen (1O2, 1Δg). We clarify whether the first step of guanine oxidation in B−DNA proceeds via either a zwitterionic or a diradical intermediate. The free energy profiles are calculated by means of a combined quantum mechanical and molecular mechanical (QM/MM) method coupled with the adaptive biasing force (ABF) method. To describe the open‐shell electronic structure of 1O2 correctly, the broken‐symmetry spin‐unrestricted density functional theory (BS−UDFT) with an approximate spin projection (AP) correction is applied to the QM region. We find that the effect of spin contamination on the activation and reaction free energies is up to ∼8 kcal mol−1, which is too large to be neglected. The QM(AP−ULC−BLYP)/MM‐based free energy calculations also reveal that the reaction proceeds through a diradical transition state, followed by a conversion to a zwitterionic intermediate. Our computed activation energy of 5.2 kcal mol−1 matches experimentally observed range (0∼6 kcal mol−1)., Realistic (free energy) profile: We demonstrate how singlet oxygen reacts with guanine in B−DNA by means of the spin‐projected QM(AP−ULC−BLYP)/MM free energy simulations. The present approach gives a realistic free energy profile, which agrees well with experimental findings. The rate‐limiting syn‐addition of 1O2 is found to proceed via a diradical transition state, followed by the smooth conversion from the open‐shell surface to the closed‐shell one.

Hydrogen-bond (H-bond) energies in 3

A laminated paper-based analytical device (LPAD) for histidine detection was fabricated from a chromatography filtration paper and laminate films. Histidine recognition was effected by histidyl-tRNA synthetase (HisRS), and its detection was signaled colorimetrically based on the molybdenum blue reaction. The analytical conditions and detectable concentration range of histidine were examined. The method provided selective quantification from 1 to 100 μM histidine. LPAD fabrication is considerably simple, involving only the craft-cutting of the chromatography filtration paper and laminate film, and is cost-effective.

The stereoinversion of amino acid residues in proteins is considered to trigger various age-related diseases. Serine (Ser) residues are relatively prone to stereoinversion. It is assumed that threonine (Thr) residues also undergo stereoinversion, which results in the formation of the d

Heme is located in the active site of proteins and has diverse and important biological functions, such as electron transfer and oxygen transport and/or storage. The distortion of heme porphyrin is considered an important factor for the diverse functions of heme because it correlates with the physical properties of heme, such as oxygen affinity and redox potential. Therefore, clarification of the relationship between heme distortion and the protein environment is crucial in protein science. Here, we analyzed the fluctuation in heme distortion in the protein environment for hemoglobin and myoglobin using molecular dynamics (MD) simulations and quantum mechanical (QM) calculations. We also investigated the protein structures of hemoglobin and myoglobin stored in Protein Data Bank and found that heme is distorted along the doming mode, which correlates with its oxygen affinity, more prominently in the protein environment than in the isolated state, and the magnitude of distortion is different between hemoglobin and myoglobin. This tendency was also observed in the results of MD simulations and QM calculations. These results suggest that heme distortion is affected by its protein environment and fluctuates around its fitted conformation, leading to physical properties that are appropriate for protein functions.

In plants, many invading microbial pathogens are recognized by cell-surface pattern recognition receptors (PRRs), inducing defense responses; yet how PRRs perceive pathogen sphingolipids remains unclear. Here, we show that the ceramide Pi-Cer D from a plant pathogenic oomycetePhytophthora infestanstriggers defense responses in Arabidopsis. Pi-Cer D is cleaved by an Arabidopsis apoplastic ceramidase, NCER2, and the resulting 9-methyl-branched sphingoid base is recognized by a plasma membrane lectin receptor-like kinase, RDA2. Importantly, 9-methyl-branched sphingoid base, which is unique to microbes, induces plant immune responses by interacting with RDA2. Loss ofRDA2orNCER2function compromised Arabidopsis resistance against an oomycete pathogen, indicating that these are crucial for defense. We provide new insights that help elucidate the recognition mechanisms of pathogen-derived lipid molecules in plants.One Sentence SummaryOomycete-derived ceramide is cleaved into sphingoid base by ceramidase and recognized by an Arabidopsis receptor kinase.

Heme proteins play diverse and important biological roles, from electron transfer and chemical catalysis, to oxygen transport and/or storage. Although the distortion of heme porphyrin correlates with the physical properties of heme, such as the redox potential and oxygen affinity, the relationship between heme distortion and the heme protein environment is unclear. We here tested the hypothesis that the protein environment of the heme-binding pocket determines heme distortion (conformation). We analyzed the correlations between the amino acid composition of the heme-binding pocket and the magnitude of heme distortion along 12 vibrational modes by using machine learning. A correlation was detected in the three lowest-vibrational modes. Analysis of heme distortions in nearly the same environments of the heme-binding pocket supported this notion. Our analyses indicate that the heme-binding pocket environment is a major factor impacting the distortion of heme porphyrin along the three lowest-vibrational modes. In addition, statistical analysis of the distortion of heme porphyrin revealed that the peaks of distributions of the ruffling and breathing distortions are shifted from 0 (the equilibrium structure). Both the ruffling and breathing distortions are correlated with the redox potential of heme, so that heme molecules with these distortions have a lower redox potential than planar molecules. These findings explain the structure–function relationship of heme and can inform protein function engineering.

The performance of the approximate spin projected (AP) coupled cluster singles and doubles (CCSD) methods based on the spin-unrestricted Hartree-Fock and Brueckner determinants, namely AP-UCCSD and...

Nitric oxide (NO) reductase from the fungus Fusarium oxysporum is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (N(2)O) in the global nitrogen cycle. In this enzymatic reaction, the heme-bound NO is activated by the direct hydride transfer from NADH to generate a short-lived intermediate (I), a key state to promote N–N bond formation and N–O bond cleavage. This study applied time-resolved (TR) techniques in conjunction with photolabile-caged NO to gain direct experimental results for the characterization of the coordination and electronic structures of I. TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe–NO coordination in I, with an elongated Fe–NO bond length (Fe–NO = 1.91 Å, Fe–N–O = 138°) in the absence of NAD(+). TR-infrared (IR) spectroscopy detects the formation of I with an N–O stretching frequency of 1,290 cm(−1) upon hydride transfer from NADH to the Fe(3+)–NO enzyme via the dissociation of NAD(+) from a transient state, with an N–O stretching of 1,330 cm(−1) and a lifetime of ca. 16 ms. Quantum mechanics/molecular mechanics calculations, based on these crystallographic and IR spectroscopic results, demonstrate that the electronic structure of I is characterized by a singly protonated Fe(3+)–NHO(•−) radical. The current findings provide conclusive evidence for the N(2)O generation mechanism via a radical–radical coupling of the heme nitroxyl complex with the second NO molecule.

Individual plants can produce leaves that differ substantially in size, morphology and many other traits. However, leaves that play a specific role in reproduction have rarely been reported. Here, we report leaves specialized to enclose fruit clusters and enhance seed production in an annual vine, Schizopepon bryoniifolius. Enclosure leaves were produced at the end of the growing season in late autumn. They were different in greenness and structure from other leaves. Under solar radiation, the ambient temperature inside an intact enclosure was up to 4.6°C higher than that near a fruit cluster whose enclosure leaves had been removed. We found that enclosures were thicker at colder sites. Removal of enclosing leaves negatively affected fruit survival and/or growth, but we could not identify the exact mechanism. The results suggested that enclosures allow the plant to produce seeds under the cold weather the plant encounters at the end of its life. Vegetative and reproductive traits of plants have usually been studied separately. This study indicates how they can dynamically interact, as shown by an examination of associations among leaf and reproductive trait changes according to life stages.

Members of theColletotrichum gloeosporioidesspecies complex are causal agents of anthracnose in a wide range of commercially important plants. To provide an in-depth overview of its diversity, we sequenced the genomes of fungi belonging to this group, including multiple strains ofC. fructicola(Cf) andC. siamense(Cs), as well as representatives of three previously unsequenced species,C. aenigma(Ca),C. tropicaleandC. viniferum. Comparisons between multipleCfandCsstrains led to the identification of accessory regions that show variable conservation in both lineages. These accessory regions encode effector candidate genes, including homologs of previously characterized effectors, organized in clusters of conserved synteny with copy number variations in different strains ofCf, CsandCa. Analysis of highly contiguous assemblies ofCf, CsandCastrains revealed the association of such accessory effector gene clusters with subtelomeric regions and repeat-rich minichromosomes and provided evidence of gene transfer between these two genomic compartments. In addition, expression analysis indicated that paralogs associated with clusters of conserved synteny showed a tendency for correlated gene expression. These data highlight the importance of subtelomeric regions and repeat-rich chromosomes to the genome plasticity ofColletotrichumfungi.

We studied the effects of heme distortions on the chemical properties in two protein classes, oxidoreductases and oxygen carrier proteins, in the Protein Data Bank by a combination of statistics and quantum chemistry. A statistical analysis of heme distortion provided the characteristic distortions. They were reasonably correlated with the redox potentials and oxygen affinities evaluated by quantum chemical calculations, implying that heme proteins utilize these distortions to enhance their functions. The combination of statistical analysis and quantum chemical calculations is useful for the elucidation of the structure-function relationships.

We present a reparameterization of PM6 (rPM6) for Fe in a similar procedure to that for Mn in our previous work [T. Saito, Y. Takano, Chem. Lett. 2017, 46, 1567]. The performance of the spin unrest...