研究者詳細 - 内橋　貴之

論文 - 内橋　貴之

分割表示 237 件中 141 - 160 件目／全件表示 >>

Construction of a Quadrangular Tetramer and a Cage-Like Hexamer from Three-Helix Bundle-Linked Fusion Proteins 査読有り国際誌

Miyamoto, T; Hayashi, Y; Yoshida, K; Watanabe, H; Uchihashi, T; Yonezawa, K; Shimizu, N; Kamikubo, H; Hirota, S

ACS SYNTHETIC BIOLOGY 8 巻 ( 5 ) 頁： 1112 - 1120 2019年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACS Synthetic Biology

Self-assembled protein nanostructures have gained interest, owing to their potential applications in biomaterials; however, successful design and construction of protein nanostructures are limited. Herein, we constructed fusion protein 1 by linking the C-terminus of a dimerization domain and the N-terminus of another dimerization domain with a three-helix bundle protein, where it self-assembled mainly into tetramers. By replacing the C-terminal dimerization domain of 1 with a trimerization domain (fusion protein 2), hexamers were mainly obtained. According to ab initio structural models reconstructed from the small-angle X-ray scattering data, the tetramer of 1 and hexamer of 2 adopted quadrangle and cage-like structures, respectively, although they were combinations of different conformations. High-speed atomic force microscopy observations indicated that the tetramer and hexamer exhibit conformational dynamics. These results show that the present method utilizing three-helix bundle-linked fusion proteins is useful in the construction of protein nanostructures.

DOI： 10.1021/acssynbio.9b00019

Inner lumen proteins stabilize doublet microtubules in cilia and flagella 査読有り国際誌 Open Access

Owa, M; Uchihashi, T; Yanagisawa, H; Yamano, T; Iguchi, H; Fukuzawa, H; Wakabayashi, K; Ando, T; Kikkawa, M

NATURE COMMUNICATIONS 10 巻 ( 1 ) 頁： 1143 - 1143 2019年3月

記述言語：英語出版者・発行元：Nature Communications

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.

DOI： 10.1038/s41467-019-09051-x

A ring-shaped hemoprotein trimer thermodynamically controlled by the supramolecular heme-heme pocket interaction

Oohora, K; Kajihara, R; Fujimaki, N; Uchihashi, T; Hayashi, T

CHEMICAL COMMUNICATIONS 55 巻 ( 11 ) 頁： 1544 - 1547 2019年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Chemical Communications

Engineered cytochrome b562, a small hemoprotein, with an externally-attached heme moiety via a moderately long linker at a suitable position predominantly forms a thermodynamically stable ring-shaped trimer in dilute solution. In an equilibrium between supramolecular polymerization and depolymerization, the ring-shaped trimer is kinetically trapped even in a concentrated solution.

DOI： 10.1039/c8cc09314h

Microtubule self- healing and defect creation investigated by in- line force measurements during high- speed atomic force microscopy imaging 査読有り

Ganser, C; Uchihashi, T

NANOSCALE 11 巻 ( 1 ) 頁： 125 - 135 2019年1月

記述言語：英語出版者・発行元：Royal Society of Chemistry ({RSC})

Microtubules are biopolymers composed of tubulin and play diverse roles in a wide variety of biological processes such as cell division, migration and intracellular transport in eukaryotic cells. To perform their functions, microtubules are mechanically stressed and, thereby, susceptible to structural defects. Local variations in mechanical properties caused by these defects modulate their biological functions, including binding and transportation of microtubule-associated proteins. Therefore, assessing the local mechanical properties of microtubules and analyzing their dynamic response to mechanical stimuli provide insight into fundamental processes. It is, however, not trivial to control defect formation, gather mechanical information at the same time, and subsequently image the result at a high temporal resolution at the molecular level with minimal delay. In this work, we describe the so-called in-line force curve mode based on high-speed atomic force microscopy. This method is directly applied to create defects in microtubules at the level of tubulin dimers and monitor the following dynamic processes around the defects. Furthermore, force curves obtained during defect formation provide quantitative mechanical information to estimate the bonding energy between tubulin dimers.

DOI： 10.1039/c8nr07392a

Single-Unit Imaging of Membrane Protein-Embedded Nanodiscs from Two Oriented Sides by High-Speed Atomic Force Microscopy 査読有り Open Access

Haruyama, T; Sugano, Y; Kodera, N; Uchihashi, T; Ando, T; Tanaka, Y; Konno, H; Tsukazaki, T

STRUCTURE 27 巻 ( 1 ) 頁： 152 - + 2019年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Structure

Membrane proteins play important roles in various cellular functions. To analyze membrane proteins, nanodisc technology using membrane scaffold proteins allows single membrane protein units to be embedded into the lipid bilayer disc without detergents. Recent advancements in high-speed atomic force microscopy (HS-AFM) have enabled us to monitor the real-time dynamics of proteins in solution at the nanometer scale. In this study, we report HS-AFM imaging of membrane proteins reconstituted into nanodiscs using two membrane protein complexes, SecYEG complex and MgtE dimer. The observed images showed single particles of membrane protein-embedded nanodiscs in an end-up orientation whereby the membrane was fixed parallel to the supporting solid surface and in a side-on orientation whereby the membrane plane was vertically fixed to the solid surface, enabling the elucidation of domain fluctuations in membrane proteins. This technique provides a basic method for the high-resolution imaging of single membrane proteins by HS-AFM.

DOI： 10.1016/j.str.2018.09.005

Metastable asymmetrical structure of a shaftless V<sub>1</sub> motor 査読有り Open Access

Maruyama, S; Suzuki, K; Imamura, M; Sasaki, H; Matsunami, H; Mizutani, K; Saito, Y; Imai, FL; Ishizuka-Katsura, Y; Kimura-Someya, T; Shirouzu, M; Uchihashi, T; Ando, T; Yamato, I; Murata, T

SCIENCE ADVANCES 5 巻 ( 1 ) 頁： eaau8149 - eaau8149 2019年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Science Advances

V 1 -ATPase is an ATP-driven rotary motor that is composed of a ring-shaped A 3 B 3 complex and a central DF shaft. The nucleotide-free A 3 B 3 complex of Enterococcus hirae, composed of three identical A 1 B 1 heterodimers, showed a unique asymmetrical structure, probably due to the strong binding of the N-terminal barrel domain, which forms a crown structure. Here, we mutated the barrel region to weaken the crown, and performed structural analyses using high-speed atomic force microscopy and x-ray crystallography of the mutant A 3 B 3 . The nucleotide-free mutant A 3 B 3 complex had a more symmetrical open structure than the wild type. Binding of nucleotides produced a closely packed spiral-like structure with a disrupted crown. These findings suggest that wild-type A 3 B 3 forms a metastable (stressed) asymmetric structure composed of unstable A 1 B 1 conformers due to the strong constraint of the crown. The results further the understanding of the principle of the cooperative transition mechanism of rotary motors.

DOI： 10.1126/sciadv.aau8149

高速原子間力顕微鏡による生体・合成高分子の動態イメージング招待有り

内橋貴之

高分子 68 巻 ( 01 ) 頁： 564 - 568 2019年

Development of Wide-area Tip-scanning High-speed Atomic Force Microscopy

Watanabe Hiroki, Uchihashi Takayuki

2019 IEEE INTERNATIONAL CONFERENCE ON MANIPULATION, MANUFACTURING AND MEASUREMENT ON THE NANOSCALE (IEEE 3M-NANO) 頁： 281 - 285 2019年

Real-time Nanoscale Visualization of Biological Molecules at Work with High-speed Atomic Force Microscopy

Uchihashi Takayuki

2019 IEEE INTERNATIONAL CONFERENCE ON MANIPULATION, MANUFACTURING AND MEASUREMENT ON THE NANOSCALE (IEEE 3M-NANO) 頁： 253 - 256 2019年

Development of Wide-area Tip-scanning High-speed Atomic Force Microscopy

Hiroki Watanabe, Takayuki Uchihashi

2019 IEEE INTERNATIONAL CONFERENCE ON MANIPULATION, MANUFACTURING AND MEASUREMENT ON THE NANOSCALE (IEEE 3M-NANO) 頁： 281 - 285 2019年

記述言語：英語掲載種別：研究論文（国際会議プロシーディングス）出版者・発行元：IEEE

Recently a tip-scanning type high-speed atomic force microscopy (HS-AFM), which is combined with total internal reflection fluorescence microscope (TIRFM), has been developed. Using this system, the topographic and fluorescence images of biological samples in action have been simultaneously visualized. However, the maximum scanning range of the combined system was limited to similar to 20 mu m and similar to 3 mu m in X- and Y-directions respectively. This limitation makes it impossible to visualize the dynamics of much larger samples, including bacteria or mammalian cells. Here, we improved a laser-tracking system and expand the tracking range in X- and Y-directions to similar to 50 mu m and similar to 32 mu m, respectively by increasing an angular variation of a mirror-tilter unit changing the arrangement and the adhesive area between piezoactuators and base plate of the unit. The Shift of the X-direction of the scanner with large displacement of the Z-scanner is eliminated by modifying an input triangular signal to the mirror-tilter unit. We also developed a scanner, which could cover in a wider imaging area, for the expanded mirror-tilter unit in the tip-scanning HS-AFM system. The capability of this expanded system is demonstrated by measuring the laser-spot displacement, large-area imaging of a square grating sample and eukaryotic cells.

DOI： 10.1109/3m-nano46308.2019.8947429

Versatility of RNA-Binding Proteins in Living Cells 査読有り

Riki Kurokawa, Akihide Takeuchi, Nobuyuki Shiina, Masato Katahira, Takefumi Yamashita, Yoko Matsuno, Keisuke Hitachi, Shinsuke Ishigaki, Nesreen Haamad, Ryoma Yoneda, Naomi Ueda, Kei Iida, Motoyasu Hosokawa, Masatoshi Hagiwara, Mamiko Iida, Tsukasa Mashima, Yudai Yamaoki, Masatomo So, Takashi Nagata, Gen Sobue, Keiko Kondo, Hiroki Watanabe, Takayuki Uchihashi

Biomedical Sciences 5 巻 ( 1 ) 頁： 7 - 7 2019年

Direct Observation and Manipulation of Supramolecular Polymerization by High-Speed Atomic Force Microscopy

Fukui, T; Uchihashi, T; Sasaki, N; Watanabe, H; Takeuchi, M; Sugiyasu, K

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 57 巻 ( 47 ) 頁： 15465 - 15470 2018年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Angewandte Chemie - International Edition

Despite recent advances in mechanistic understanding and controlled-synthesis methodologies regarding synthetic supramolecular assemblies, it has remained challenging to capture the molecular-level phenomena in real time, thus hindering further progress in this research field. In this study, we applied high-speed atomic-force microscopy (AFM), which has extraordinary spatiotemporal resolution (1 nm and sub-100 ms), to capture dynamic events occurring during synthetic molecular self-assembly. High-speed AFM permitted the visualization of unique dynamic behavior, such as seeded growth and self-repair in real time. Furthermore, scanning-probe AFM permitted the site-specific manipulation and functionalization of a molecular self-assembly. This powerful combination of bottom-up and top-down approaches at the molecular level should enable targeted syntheses of unprecedented functional nanoarchitectures.

DOI： 10.1002/anie.201809165

Monitoring Thermoresponsive Morphological Changes in Individual Hydrogel Microspheres 査読有り国際誌 Open Access

Matsui, S; Nishizawa, Y; Uchihashi, T; Suzuki, D

ACS OMEGA 3 巻 ( 9 ) 頁： 10836 - 10842 2018年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACS Omega

Real-time morphology/structure changes in individual hydrogel microspheres (microgels) were directly visualized at high spatiotemporal resolution using high-speed atomic force microscopy (HS-AFM) under temperature control ranging from room temperature to ∼40 °C. The recorded HS-AFM movies demonstrate that the size and morphology of thermoresponsive poly(N-isopropyl acrylamide)-based microgels change with increasing temperature at the individual microgel level. Specifically, the height of the microgels gradually decreases and domain structures appeared even below the volume phase transition temperature. Moreover, the domain structure is retained, even after the microgels have fully collapsed. The present study thus demonstrates that temperature-controlled HS-AFM is a useful tool for monitoring stimulus-responsiveness of microgels. In the near future, it should furthermore be possible to extend this temperature-controlled HS-AFM to other stimulus-responsive materials, including autonomously oscillating microgels.

DOI： 10.1021/acsomega.8b01770

Supramolecular Hemoprotein Assembly with a Periodic Structure Showing Heme-Heme Exciton Coupling

Oohora, K; Fujimaki, N; Kajihara, R; Watanabe, H; Uchihashi, T; Hayashi, T

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 140 巻 ( 32 ) 頁： 10145 - 10148 2018年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of the American Chemical Society

A supramolecular assembly of units of cytochrome b562 with externally attached heme having intermolecular linkages formed via the heme-heme pocket interaction was investigated in an effort to construct a well-defined structure. The engineered site for surface attachment of heme at Cys80 in an N80C mutant of cytochrome b562 provides the primary basis for the formation of the periodic assembly structure, which is characterized herein by circular dichroism (CD) spectroscopy and high-speed atomic force microscopy (AFM). This assembly represents the first example of the observation of a split-type Cotton effect by heme-heme exciton coupling in an artificial hemoprotein assembly system. Molecular dynamics simulations validated by simulated CD spectra, AFM images, and mutation experiments reveal that the assembly has a periodic helical structure with 3 nm pitches, suggesting the formation of the assembled structure is driven not only by the heme-heme pocket interaction but also by additional secondary hydrogen bonding and/or electrostatic interactions at the protein interfaces of the assembly.

DOI： 10.1021/jacs.8b06690

Revealing circadian mechanisms of integration and resilience by visualizing clock proteins working in real time Open Access

Mori, T; Sugiyama, S; Byrne, M; Johnson, CH; Uchihashi, T; Ando, T

NATURE COMMUNICATIONS 9 巻 ( 1 ) 頁： 3245 2018年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Communications

The circadian clock proteins KaiA, KaiB, and KaiC reconstitute a remarkable circa-24 h oscillation of KaiC phosphorylation that persists for many days in vitro. Here we use high-speed atomic force microscopy (HS-AFM) to visualize in real time and quantify the dynamic interactions of KaiA with KaiC on sub-second timescales. KaiA transiently interacts with KaiC, thereby stimulating KaiC autokinase activity. As KaiC becomes progressively more phosphorylated, KaiA’s affinity for KaiC weakens, revealing a feedback of KaiC phosphostatus back onto the KaiA-binding events. These non-equilibrium interactions integrate high-frequency binding and unbinding events, thereby refining the period of the longer term oscillations. Moreover, this differential affinity phenomenon broadens the range of Kai protein stoichiometries that allow rhythmicity, explaining how the oscillation is resilient in an in vivo milieu that includes noise. Therefore, robustness of rhythmicity on a 24-h scale is explainable by molecular events occurring on a scale of sub-seconds.

DOI： 10.1038/s41467-018-05438-4

Scanning probe microscopy

Takahashi T., Fukui K.I., Kageshima M., Komeda T., Nakajima K., Nakayama T., Sumitomo K., Uchihashi T.

Japanese Journal of Applied Physics 57 巻 ( 8 ) 2018年8月

Quantum-dot antibody conjugation visualized at the single-molecule scale with high-speed atomic force microscopy

Umakoshi, T; Udaka, H; Uchihashi, T; Ando, T; Suzuki, M; Fukuda, T

COLLOIDS AND SURFACES B-BIOINTERFACES 167 巻頁： 267 - 274 2018年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Colloids and Surfaces B: Biointerfaces

Conjugates of semiconductor quantum dots (QDs) and antibodies have emerged as a promising bioprobes due to their great combination of QD's efficient fluorescence and the high specificity of antigen-antibody reactions. For further developments in this field, it is essential to understand the molecular conformation of the QD-antibody conjugates at the single-molecule scale. Here, we report on the direct imaging of QD-antibody conjugates at the single-molecule scale by using high-speed atomic force microscopy (HS-AFM). Owing to the high spatiotemporal resolution of HS-AFM, we observed the dynamic splitting of individual antibodies during the conjugation process. QD-antibody conjugates were also clearly visualized at the single-molecule scale details. Several important features were even discovered through dynamic observation of the QD-antibody conjugates. We observed an intermediate state of conjugation, where the antibodies attached and detached to QDs repeatedly. We also revealed that the attached antibodies were not steady but drastically fluctuated in their recognition areas due to the Brownian motion. We also demonstrated that HS-AFM observation is useful for the quantitative analysis of fabricated conjugates.

DOI： 10.1016/j.colsurfb.2018.04.015

Translating MOF chemistry into supramolecular chemistry: soluble coordination nanofibers showing efficient photon upconversion

Hosoyamada, M; Yanai, N; Okumura, K; Uchihashi, T; Kimizuka, N

CHEMICAL COMMUNICATIONS 54 巻 ( 50 ) 頁： 6828 - 6831 2018年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Chemical Communications

A method for synthesizing coordination nanofibers by extracting the structural motifs of metal-organic frameworks (MOFs) is demonstrated. In these soluble nanofibers, multiple chromophores with largely different sizes and shapes can be arranged at desired compositions, and excited triplet energy migrates among the densely assembled chromophore arrays, showing an efficient photon upconversion even at very low concentration.

DOI： 10.1039/c8cc01594e

Dynamic structural states of CIpB involved in its disaggregation function Open Access

Uchihashi, T; Watanabe, Y; Nakazaki, Y; Yamasaki, T; Watanabe, H; Maruno, T; Ishii, K; Uchiyama, S; Song, CH; Murata, K; Iino, R; Ando, T

NATURE COMMUNICATIONS 9 巻 ( 1 ) 頁： 2147 2018年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Communications

The ATP-dependent bacterial protein disaggregation machine, ClpB belonging to the AAA+ superfamily, refolds toxic protein aggregates into the native state in cooperation with the cognate Hsp70 partner. The ring-shaped hexamers of ClpB unfold and thread its protein substrate through the central pore. However, their function-related structural dynamics has remained elusive. Here we directly visualize ClpB using high-speed atomic force microscopy (HS-AFM) to gain a mechanistic insight into its disaggregation function. The HS-AFM movies demonstrate massive conformational changes of the hexameric ring during ATP hydrolysis, from a round ring to a spiral and even to a pair of twisted half-spirals. HS-AFM observations of Walker-motif mutants unveil crucial roles of ATP binding and hydrolysis in the oligomer formation and structural dynamics. Furthermore, repressed and hyperactive mutations result in significantly different oligomeric forms. These results provide a comprehensive view for the ATP-driven oligomeric-state transitions that enable ClpB to disentangle protein aggregates.

DOI： 10.1038/s41467-018-04587-w

Sweeping of Adsorbed Therapeutic Protein on Prefillable Syringes Promotes Micron Aggregate Generation 査読有り Open Access

Maruno, T; Watanabe, H; Yoneda, S; Uchihashi, T; Adachi, S; Arai, K; Sawaguchi, T; Uchiyama, S

JOURNAL OF PHARMACEUTICAL SCIENCES 107 巻 ( 6 ) 頁： 1521 - 1529 2018年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Pharmaceutical Sciences

This study evaluated how differences in the surface properties of prefillable syringe barrels and in-solution sampling methods affect micron aggregates and protein adsorption levels. Three syringe types (glass barrel with silicone oil coating [GLS/SO+], glass barrel without silicone oil coating [GLS/SO−], and cyclo-olefin polymer [COP] barrel syringes) were tested with 3 therapeutic proteins (adalimumab, etanercept, and infliximab) using 2 sampling methods (aspiration or ejection). After quiescent incubation, solutions sampled by aspiration exhibited no significant change in micron aggregate concentration in any syringes, whereas those sampled by ejection exhibited increased micron aggregates in both GLS syringe types. Micron aggregate concentration in ejected solutions generally increased with increasing density of adsorbed proteins. Notably, COP syringes contained the lowest micron aggregate concentrations, which were independent of the sampling method. Correspondingly, the adsorbed protein density on COP syringes was the lowest at 1-2 mg/m <sup>2</sup> , which was much less compared with that on GLS syringes and was calculated to be equivalent to only 1–2 protein layers, as visually confirmed by high-speed atomic force microscopy. These data indicate that low-adsorption prefillable syringes should be used for therapeutic proteins because protein aggregate concentration in the ejected solution is elevated by increased protein adsorption to the syringe surface.

DOI： 10.1016/j.xphs.2018.01.021

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