Papers - Nakasako, Masayoshi

Cryo-EM and crystal structure analyses reveal the indirect role played by Trp89 in glutamate dehydrogenase enzymatic reactions.

Wakabayashi, T., Matsui, Y. and Nakasako, M.

The FEBS Journal (Wiley & Sons)  in press ( in press ) in press 2025.02

Research paper (scientific journal), Joint Work, Corresponding author, Accepted

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Improvement in positional accuracy of neural-network predicted hydration sites of proteins by incorporating atomic details of water-protein interactions and site-searching algorithm

Kochi Sato and Masayoshi Nakasako

Biophysics and Physicobiology (The Biophysical Society of Japan)  22   e220004(1) - e220004(17) 2025.01

Research paper (scientific journal), Joint Work, Corresponding author, Accepted

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CryoEM-sampling of metastable conformations appearing in cofactor-ligand association and catalysis of glutamate dehydrogenase

Taiki Wakabayashi, Mao Oide and Masayoshi Nakasako

Scientific Reports (Springer-Nature)  14   11165(1) - 11165(16) 2024.05

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

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Protocol using similarity score and improved shrink-wrap algorithm for better convergence of phase-retrieval calculation in X-ray diffraction imaging

Syouyo Yoshida Kosei Harada So Uezu, Yuki Takayama and Masayoshi Nakasako

Journal of Synchrotron Radiation (Wiley & Sons)  31   113 - 128 2023.11

Research paper (scientific journal), Joint Work, Corresponding author, Accepted

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Similarity score for screening phase-retrieved maps in X-ray diffraction imaging – characterization in reciprocal space

Yuki Takayama and Masayoshi Nakasako

Journal of Synchrotron Radiation (Wiley & Sons)  31   95 - 112 2023.11

Research paper (scientific journal), Joint Work, Corresponding author, Accepted

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Coenzyme-binding pathway on glutamate dehydrogenase suggested from multiple-binding sites visualized by cryo-electron microscopy

Taiki Wakabayashi, Mao Oide, Takayuki Kato and Masayoshi Nakasako

The FEBS Journal (Wiley & Sons)  290   5514 - 5535 2023.09

Research paper (scientific journal), Corresponding author, Accepted

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Ultrastructure and fractal property of chromosomes in close‑to‑native yeast nuclei visualized using X‑ray laser diffraction

So Uezu, TakahiroYamamoto, Mao Oide, YukiTakayama, Koji Okajima, Amane Kobayashi, Masaki Yamamoto, Masayoshi Nakasako

Scientifc Reports (Springer-Nature)  13   10802(1) - 10802(14) 2023.07

Research paper (scientific journal), Joint Work, Corresponding author, Accepted

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Prediction of hydrophilic and hydrophobic hydration structure of protein by neural network optimized using experimental data

Kochi Sato, Mao Oide, Masayoshi Nakasako

Scientifc Reports | (Springer-Nature)  13   2183 (1-15) 2023.02

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

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Deducing Free-Energy Landscape of Biomolecular Dynamics from Cryo-Electron Microscopy Images

Mao Oide and Masayoshi Nakasako

Journal of the Crystallographic Society of Japan ( The Crystallographic Society of Japan)  64 ( 4 ) 300 - 305 2022.12

Research paper (scientific journal), Joint Work, Last author, Accepted

Development of Coherent X-ray Diffraction Imaging at SPring-8

Yuki TAKAYAMA and Masayoshi NAKASAKO

日本結晶学会誌 (日本結晶学会)   64 ( 1 ) 41 - 49 2022.03

Research paper (scientific journal), Last author, Corresponding author, Accepted

Common architectures in cyanobacteria Prochlorococcus cells visualized by X-ray diffraction imaging using X-ray free-electron laser

A. Kobayashi, Y. Takayama, T. Hirakawa, K. Okajima, M. Oide, T. Oroguchi, Y. Inui, M. Yamamoto, S. Matsunaga and M. Nakasako

Scientific Reports (Springer-Nature)  in press ( 1 ) in press 2021.02

Research paper (scientific journal), Joint Work, Accepted

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Visualization of intracellular structures and their spatial organization inside cells without any modification is essential to understand the mechanisms underlying the biological functions of cells. Here, we investigated the intracellular structure of cyanobacteria Prochlorococcus in the interphase by X-ray diffraction imaging using X-ray free-electron laser. A number of diffraction patterns from single cells smaller than 1 µm in size were collected with high signal-to-noise ratio with a resolution of up to 30 nm. From diffraction patterns, a set of electron density maps projected along the direction of the incident X-ray were retrieved with high reliability. The most characteristic structure found to be common among the cells was a C-shaped arrangement of 100-nm sized high-density spots, which surrounded a low-density area of 100 nm. Furthermore, a three-dimensional map reconstructed from the projection maps of individual cells was non-uniform, indicating the presence of common structures among cyanobacteria cells in the interphase. By referring to the fluorescent images for distributions of thylakoid membranes, nucleoids, and carboxysomes, we inferred and represented their spatial arrangements in the three-dimensional map. The arrangement allowed us to discuss the relevance of the intracellular organization to the biological functions of cyanobacteria.

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Red light‑induced structure changes in phytochrome A from Pisum sativum

Mao Oide and Masayoshi Nakasako

Scientific Reports (Springer-Nature)  11 ( 1 ) 2827(1) - 2827(11) 2021.02

Research paper (scientific journal), Joint Work, Accepted

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Phytochrome A (phyA) is a photoreceptor protein of plants that regulates the red/far-red light photomorphogenic responses of plants essential for growth and development. PhyA, composed of approximately 1100 amino acid residues, folds into photosensory and output signaling modules. The photosensory module covalently binds phytochromobilin as a chromophore for photoreversible interconversion between inactive red light-absorbing (Pr) and active far-red light-absorbing (Pfr) forms to act as a light-driven phosphorylation enzyme. To understand the molecular mechanism in the initial process of photomorphogenic response, we studied the molecular structures of large phyA (LphyA) from Pisum sativum, which lacks the 52 residues in the N-terminal, by small-angle X-ray scattering combined with multivariate analyses applied to molecular models predicted from the scattering profiles. According to our analyses, Pr was in a dimer and had a four-leaf shape, and the subunit was approximated as a bent rod of 175 × 50 Å. The scattering profile of Pfr was calculated from that recorded for a mixture of Pr and Pfr under red-light irradiation by using their population determined from the absorption spectrum. The Pfr dimer exhibited a butterfly shape composed of subunits with a straight rod of 175 × 50 Å. The shape differences between Pr and Pfr indicated conformational changes in the Pr/Pfr interconversion which would be essential to the interaction with protein molecules involved in transcriptional control.

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Assessment of Force Field Accuracy Using Cryogenic Electron Microscopy Data of Hyper-thermostable Glutamate Dehydrogenase

Oroguchi T., Oide M., Wakabayashi T., Nakasako M.

Journal of Physical Chemistry B (Journal of Physical Chemistry B)  124 ( 39 ) 8479 - 8494 2020.10

ISSN  15206106

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© 2020 American Chemical Society. Molecular dynamics (MD) simulations in biophysically relevant time scales of microseconds is a powerful tool for studying biomolecular processes, but results often display force field dependency. Therefore, assessment of force field accuracy using experimental data of biomolecules in solution is essential for simulation studies. Here, we propose the use of structural models obtained via cryo-electron microscopy (cryoEM), which provides biomolecular structures in vitreous ice mimicking the environment in solution. The accuracy of the AMBER (ff99SB-ILDN-NMR, ff14SB, ff15ipq, and ff15FB) and CHARMM (CHARMM22 and CHARMM36m) force fields was assessed by comparing their MD trajectories with the cryoEM data of thermostable hexameric glutamate dehydrogenase (GDH), which included a cryoEM map at a resolution of approximately 3 Å and structure models of subunits reflecting metastable conformations in domain motion occurring in GDH. In the assessment, we validated the force fields with respect to the reproducibility and stability of secondary structures and intersubunit interactions in the cryoEM data. Furthermore, we evaluated the force fields regarding the reproducibility of the energy landscape in the domain motion expected from the cryoEM data. As a result, among the six force fields, ff15FB and ff99SB-ILDN-NMR displayed good agreement with the experiment. The present study demonstrated the advantages of the high-resolution cryoEM map and suggested the optimal force field to reproduce experimentally observed protein structures.

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Domain organization in plant blue-light receptor phototropin2 of arabidopsis thaliana studied by small-angle x-ray scattering

Nakasako M., Oide M., Takayama Y., Oroguchi T., Okajima K.

International Journal of Molecular Sciences (International Journal of Molecular Sciences)  21 ( 18 ) 6638(1) - 6638(21) 2020.09

ISSN  16616596

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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Phototropin2 (phot2) is a blue-light (BL) receptor protein that regulates the BL-dependent activities of plants for efficient photosynthesis. Phot2 is composed of two light-oxygen-voltage sensing domains (LOV1 and LOV2) to absorb BL, and a kinase domain. Photo-activated LOV domains, especially LOV2, play a major role in photo-dependent increase in the phosphorylation activity of the kinase domain. The atomic details of the overall structure of phot2 and the intramolecular mechanism to convert BL energy to a phosphorylation signal remain unknown. We performed structural studies on the LOV fragments LOV1, LOV2, LOV2-linker, and LOV2-kinase, and full-length phot2, using small-angle X-ray scattering (SAXS). The aim of the study was to understand structural changes under BL irradiation and discuss the molecular mechanism that enhance the phosphorylation activity under BL. SAXS is a suitable technique for visualizing molecular structures of proteins in solution at low resolution and is advantageous for monitoring their structural changes in the presence of external physical and/or chemical stimuli. Structural parameters and molecular models of the recombinant specimens were obtained from SAXS profiles in the dark, under BL irradiation, and after dark reversion. LOV1, LOV2, and LOV2-linker fragments displayed minimal structural changes. However, BL-induced rearrangements of functional domains were noted for LOV2-kinase and full-length phot2. Based on the molecular model together with the absorption measurements and biochemical assays, we discuss the intramolecular interactions and domain motions necessary for BL-enhanced phosphorylation activity of phot2.

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Energy landscape of domain motion in glutamate dehydrogenase deduced from cryo-electron microscopy

Oide M., Kato T., Oroguchi T., Nakasako M.

FEBS Journal (FEBS Journal)  287 ( 16 ) 3472 - 3493 2020.08

ISSN  1742464X

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© 2020 Federation of European Biochemical Societies Analysis of the conformational changes of protein is important to elucidate the mechanisms of protein motions correlating with their function. Here, we studied the spontaneous domain motion of unliganded glutamate dehydrogenase from Thermococcus profundus using cryo-electron microscopy and proposed a novel method to construct free-energy landscape of protein conformations. Each subunit of the homo-hexameric enzyme comprises nucleotide-binding domain (NAD domain) and hexamer-forming core domain. A large active-site cleft is situated between the two domains and varies from open to close according to the motion of a NAD domain. A three-dimensional map reconstructed from all cryo-electron microscopy images displayed disordered volumes of NAD domains, suggesting that NAD domains in the collected images adopted various conformations in domain motion. Focused classifications on NAD domain of subunits provided several maps of possible conformations in domain motion. To deduce what kinds of conformations appeared in EM images, we developed a novel analysis method that describe the EM maps as a linear combination of representative conformations appearing in a 200-ns molecular dynamics simulation as reference. The analysis enabled us to estimate the appearance frequencies of the representative conformations, which illustrated a free-energy landscape in domain motion. In the open/close domain motion, two free-energy basins hindered the direct transformation from open to closed state. Structure models constructed for representative EM maps in classifications demonstrated the correlation between the energy landscape and conformations in domain motion. Based on the results, the domain motion in glutamate dehydrogenase and the analysis method to visualize conformational changes and free-energy landscape were discussed. Database: The EM maps of the four conformations were deposited to Electron Microscopy Data Bank (EMDB) as accession codes EMD-9845 (open), EMD-9846 (half-open1), EMD-9847 (half-open2), and EMD-9848 (closed), respectively. In addition, the structural models built for the four conformations were deposited to the Protein Data Bank (PDB) as accession codes 6JN9 (open), 6JNA (half-open1), 6JNC (half-open2), and 6JND (closed), respectively.

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Methods and application of coherent X-ray diffraction imaging of noncrystalline particles

Nakasako M., Kobayashi A., Takayama Y., Asakura K., Oide M., Okajima K., Oroguchi T., Yamamoto M.

Biophysical Reviews (Biophysical Reviews)  12 ( 2 ) 541 - 567 2020.04

ISSN  18672450

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© 2020, International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature. Microscopic imaging techniques have been developed to visualize events occurring in biological cells. Coherent X-ray diffraction imaging is one of the techniques applicable to structural analyses of cells and organelles, which have never been crystallized. In the experiment, a single noncrystalline particle is illuminated by an X-ray beam with almost complete spatial coherence. The structure of the particle projected along the direction of the beam is, in principle, retrieved from a finely recorded diffraction pattern alone by using iterative phase-retrieval algorithms. Here, we describe fundamental theory and experimental methods of coherent X-ray diffraction imaging and the recent application in structural studies of noncrystalline specimens by using X-rays available at Super Photon Ring of 8-Gev and SPring-8 Angstrom Compact Free Electron Laser in Japan.

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特別企画 試料環境シリーズ(7) 第4回「温度」 4-1 蛋白質結晶の低温X線回折実験 ～低温 1～

中迫雅由、高山裕貴、山本雅貴

放射光 (日本放射光学会)  33 ( 2 ) 101 - 111 2020.01

Research paper (scientific journal), Joint Work, Accepted

Molecular shape under far-red light and red light-induced association of Arabidopsis phytochrome B

Mao Oide, Takaaki Hikima, Tomotaka Oroguchi, Takayuki Kato, Yuhki Yamaguchi, Shizue Yoshihara, Masaki Yamamoto, Masayoshi Nakasako and Koji Okajima

The FEBS Journal (Federation of European Biochemical Societies)  287 ( 8 ) 1612 - 1625 2019.10

Research paper (scientific journal), Joint Work, Accepted,  ISSN  1742464X

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© 2019 Federation of European Biochemical Societies Phytochrome B (phyB) is a plant photoreceptor protein that regulates various photomorphogenic responses to optimize plant growth and development. PhyB exists in two photoconvertible forms: a red light-absorbing (Pr) and a far-red light-absorbing (Pfr) form. Therefore, to understand the mechanism of phototransformation, the structural characterization of full-length phyB in these two forms is necessary. Here, we report the molecular structure of Arabidopsis thaliana phyB in Pr form and the molecular properties of the Pfr form determined by small-angle X-ray scattering coupled with size-exclusion chromatography. In solution, the Pr form associated as a dimer with a radius of gyration of 50 Å. The molecular shape was a crossed shape, in which the orientation of the photosensory modules differed from that in the crystal structure of dimeric photosensory module. PhyB exhibited structural reversibility in the Pfr-to-Pr phototransformation and thermal reversion from Pfr to Pr in the dark. In addition, Pfr only exhibited nonspecific association, which distinguished molecular properties of Pfr form from those of the inactive Pr form.

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Dipole–dipole interactions between tryptophan side chains and hydration water molecules dominate the observed dynamic Stokes shift of lysozyme

Asahi Fukuda, Tomotaka Oroguchi and Masayoshi Nakasako

Biochimica et Biophysica Acta - General Subjects (Elsevier)  1864 ( 2 ) 129406(1) - 129406(11) 2019.08

Research paper (scientific journal), Joint Work, Accepted,  ISSN  03044165

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© 2019 Elsevier B.V. The fluorescence intensity of tryptophan residues in hen egg-white lysozyme was measured up to 500 ps after the excitation by irradiation pulses at 290 nm. From the time-dependent variation of fluorescence intensity in a wavelength range of 320–370 nm, the energy relaxation in the dynamic Stokes shift was reconstructed as the temporal variation in wavenumber of the estimated fluorescence maximum. The relaxation was approximated by two exponential curves with decay constants of 1.2 and 26.7 ps. To interpret the relaxation, a molecular dynamics simulation of 75 ns was conducted for lysozyme immersed in a water box. From the simulation, the energy relaxation in the electrostatic interactions of each tryptophan residue was evaluated by using a scheme derived from the linear response theory. Dipole–dipole interactions between each of the Trp62 and Trp123 residues and hydration water molecules displayed an energy relaxation similar to that experimentally observed regarding time constants and magnitudes. The side chains of these residues were partly or fully exposed to the solvent. In addition, by inspecting the variation in dipole moments of the hydration water molecules around lysozyme, it was suggested that the observed relaxation could be attributed to the orientational relaxation of hydration water molecules participating in the hydrogen-bond network formed around each of the two tryptophan residues.

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高分子材料における異方性のテラヘルツセンシング

渡邉 紳一, 岡野 真人, 中迫 雅由

レーザー研究 (一般社団法人レーザー学会)  47 ( 1月 ) 21 - 26 2019.01

Research paper (bulletin of university, research institution), Joint Work, Accepted