研究者詳細 - 内橋　貴之

論文 - 内橋　貴之

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Multistep, site-selective noncovalent synthesis of two-dimensional block supramolecular polymers 査読有り

Sasaki, N; Kikkawa, J; Ishii, Y; Uchihashi, T; Imamura, H; Takeuchi, M; Sugiyasu, K

NATURE CHEMISTRY 15 巻 ( 7 ) 頁： 922 - + 2023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：Nature Chemistry

Although the principles of noncovalent bonding are well understood and form the basis for the syntheses of many intricate supramolecular structures, supramolecular noncovalent synthesis cannot yet achieve the levels of precision and complexity that are attainable in organic and/or macromolecular covalent synthesis. Here we show the stepwise synthesis of block supramolecular polymers from metal–porphyrin derivatives (in which the metal centre is Zn, Cu or Ni) functionalized with fluorinated alkyl chains. These monomers first undergo a one-dimensional supramolecular polymerization and cyclization process to form a toroidal structure. Subsequently, successive secondary nucleation, elongation and cyclization steps result in two-dimensional assemblies with concentric toroidal morphologies. The site selectivity endowed by the fluorinated chains, reminiscent of regioselectivity in covalent synthesis, enables the precise control of the compositions and sequences of the supramolecular structures, as demonstrated by the synthesis of several triblock supramolecular terpolymers. [Figure not available: see fulltext.].

DOI： 10.1038/s41557-023-01216-y

その他リンク： https://www.nature.com/articles/s41557-023-01216-y

High Density of N- and O-Glycosylation Shields and Defines the Structural Dynamics of the Intrinsically Disordered Ectodomain of Receptor-type Protein Tyrosine Phosphatase Alpha 査読有り国際共著

Chien, YC; Wang, YS; Sridharan, D; Kuo, CW; Chien, CT; Uchihashi, T; Kato, K; Angata, T; Meng, TC; Hsu, STD; Khoo, KH

JACS AU 3 巻 ( 7 ) 頁： 1864 - 1875 2023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：JACS Au

The intracellular phosphatase domain of the receptor-type protein tyrosine phosphatase alpha (PTPRA) is known to regulate various signaling pathways related to cell adhesion through c-Src kinase activation. In contrast, the functional significance of its relatively short, intrinsically disordered, and heavily glycosylated ectodomain remains unclear. Through detailed mass spectrometry analyses of a combination of protease and glycosidase digests, we now provide the first experimental evidence for its site-specific glycosylation pattern. This includes the occurrence of O-glycan at the N-glycosylation sequon among the more than 30 O-glycosylation sites confidently identified beside the 7 N-glycosylation sites. The closely spaced N- and O-glycans appear to have mutually limited the extent of further galactosylation and sialylation. An immature smaller form of full-length PTPRA was found to be deficient in O-glycosylation, most likely due to failure to transit the Golgi. N-glycosylation, on the other hand, is dispensable for cell surface expression and contributes less than the extensive O-glycosylation to the overall solution structure of the ectodomain. The glycosylation information is combined with the overall structural features of the ectodomain derived from small-angle X-ray scattering and high-speed atomic force microscopy monitoring to establish a dynamic structural model of the densely glycosylated PTPRA ectodomain. The observed high structural flexibility, as manifested by continuous transitioning from fully to partially extended and fold-back conformations, suggests that the receptor-type phosphatase is anchored to the membrane and kept mostly at a monomeric state through an ectodomain shaped and fully shielded by glycosylation.

DOI： 10.1021/jacsau.3c00124

Closed-loop recycling of microparticle-based polymers 査読有り

Watanabe, T; Minato, H; Sasaki, Y; Hiroshige, S; Suzuki, H; Matsuki, N; Sano, K; Wakiya, T; Nishizawa, Y; Uchihashi, T; Kureha, T; Shibayama, M; Takata, T; Suzuki, D

GREEN CHEMISTRY 25 巻 ( 9 ) 頁： 3418 - 3424 2023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：Green Chemistry

Contemporary polymer science is shifting toward the development of recycling systems to curb global resource depletion and environmental contamination. However, most methods of polymer recycling require cleavage of chemical bonds, which diminishes the quality of the polymers during recycling. Here, we propose a recycling strategy for tough polymers based on microparticles, which allows materials recycling without loss of their properties (‘closed-loop’ recycling). The polymer microparticles can be used to generate tough polymer films by controlling the interparticle physical cross-linking, and subsequently recycled on demand by disassembling into individual microparticles without chemical reactions. Our “microparticle-based concept” for polymer recycling circumvents the infamous trade-off between mechanical stability and degradability of polymers and be expected to open new avenues for closed-loop recycling of polymer materials.

DOI： 10.1039/d3gc00090g

Antiparallel dimer structure of CELSR cadherin in solution revealed by high-speed atomic force microscopy 査読有り

Nishiguchi, S; Kasai, RS; Uchihashi, T

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 120 巻 ( 18 ) 頁： e2302047120 2023年4月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences of the United States of America

Cadherin EGF LAG seven-pass G-type receptors (CELSR) cadherins, members of the cadherin superfamily, and adhesion G-protein-coupled receptors, play a vital role in cell–cell adhesion. The mutual binding of the extracellular domains (ectodomains) of CELSR cadherins between cells is crucial for tissue formation, including the establishment of planar cell polarity, which directs the proper patterning of cells. CELSR cadherins possess nine cadherin ectodomains (EC1–EC9) and noncadherin ectodomains. However, the structural and functional mechanisms of the binding mode of CELSR cadherins have not been determined. In this study, we investigated the binding mode of CELSR cadherins using single-molecule fluorescence microscopy, high-speed atomic force microscopy (HS-AFM), and bead aggregation assay. The fluorescence microscopy analysis results indicated that the trans-dimer of the CELSR cadherin constitutes the essential adhesive unit between cells. HS-AFM analysis and bead aggregation assay results demonstrated that EC1–EC8 entirely overlap and twist to form antiparallel dimer conformations and that the binding of EC1–EC4 is sufficient to sustain bead aggregation. The interaction mechanism of CELSR cadherin may elucidate the variation of the binding mechanism within the cadherin superfamily and physiological role of CELSR cadherins in relation to planar cell polarity.

DOI： 10.1073/pnas.2302047120

Creation of single molecular conjugates of metal-organic cages and DNA 査読有り国際誌

Nakajo T., Kusaka S., Hiraoka H., Nomura K., Matsubara N., Baba R., Yoshida Y., Nakamoto K., Honma M., Iguchi H., Uchihashi T., Abe H., Matsuda R.

Chemical Communications 59 巻 ( 33 ) 頁： 4974 - 4977 2023年4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

Here we report the development of an equimolar conjugate of a metal-organic cage (MOC) and DNA (MOC-DNA). Several MOC-DNA conjugates were assembled into a programmed structure by coordinating with a template DNA having a complementary base sequence. Moreover, conjugation with the MOC drastically enhanced the permeability of DNA through the lipid bilayer, presenting great potential as a drug delivery system.

DOI： 10.1039/d3cc00460k

高速原子間力顕微鏡で明らかになった細胞間接着分子カドヘリンのダイマー形成過程招待有り査読有り

西口茂孝, 古田忠臣, 内橋貴之

生物物理 63 巻 ( 2 ) 頁： 104 - 106 2023年4月

担当区分：責任著者記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：一般社団法人日本生物物理学会

<p>多細胞動物のからだの形作りは，隣り合う細胞と細胞をつなぐカドヘリン間の結合によって制御されているが，その結合機構については不明な点が多かった．高速原子間力顕微鏡を用いて，溶液中におけるカドヘリンの結合過程を一分子のスケールで直接可視化することで，カドヘリンの結合機構を解析した．</p>

DOI： 10.2142/biophys.63.104

3D Structure of Ring-shaped Microtubule Swarms Revealed by High-speed Atomic Force Microscopy 査読有り

Rashid M.R., Ganser C., Akter M., Nasrin S.R., Kabir A.M.R., Sada K., Uchihashi T., Kakugo A.

Chemistry Letters 52 巻 ( 2 ) 頁： 100 - 104 2023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：Chemistry Letters

Biomolecular motor, microtubule (MT) and kinesin based molecular swarm robots are important due to their applications in cooperative task achievements, while the structural details are still unknown. In this work, high-speed atomic force microscopy was used to observe the MT swarm ring structure at nanometerlevel resolution. MTs were observed to pile up in multiple layers to form swarm rings. The number of MTs involved in force generation was estimated which will specifically impact the understanding of the force-generation capability of swarms.

DOI： 10.1246/cl.220491

Disulfide Bond-mediated Oligomerization of a Green Fluorescent Protein in Solution 査読有り

Soon J.W., Oohora K., Uchihashi T., Hayashi T.

Chemistry Letters 52 巻 ( 2 ) 頁： 105 - 109 2023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：Chemistry Letters

A green fluorescent protein (GFP) variant with two cysteine residues added to the protein surface was modified with 2,2′- dipyridyl disulfide and oligomerization of the modified protein and the unmodified variant via the disulfide bond was achieved in solution. The fluorescence anisotropy decay of the obtained GFP oligomer is capable of efficient homo-FRET.

DOI： 10.1246/cl.220495

Biosynthesis of highly branched gold nanoparticles through structural engineering of fatty acids 査読有り

Yue Y., Yokota Y., Uchihashi T.

iScience 26 巻 ( 1 ) 頁： 105864 2023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：iScience

Biomimetic approaches have been used to develop inorganic nanomaterials with complex morphologies and functions. Fatty acids are among the most important and decomposable biomolecules in nature. However, the controlled synthesis of branched gold nanoparticles using these biomolecules has not been reported. Herein, we demonstrate a strategy to produce highly branched gold nanoparticles through structural engineering of fatty acids. Furthermore, we developed a method for tailoring fatty acid molecules by altering their aliphatic chains to facilitate the morphological evolution of gold nanoparticles from spherical to branched shape. It is found that the growth of the nanoparticles is sensitive to characteristics of fatty acids, such as saturation degrees. The growth of the nanoparticle is visualized by high-speed atomic force microscopy. The reaction mechanisms and growth processes of branched gold nanoparticles are proposed. This work may serve as a cornerstone to the design in a biomimetic fashion for the controllable synthesis of metallic nanomaterials.

DOI： 10.1016/j.isci.2022.105864

Decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex 査読有り国際誌

Matsuo, Y; Uchihashi, T; Inada, T

NATURE COMMUNICATIONS 14 巻 ( 1 ) 頁： 79 - 79 2023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：Nature Communications

The collision sensor Hel2 specifically recognizes colliding ribosomes and ubiquitinates the ribosomal protein uS10, leading to noncanonical subunit dissociation by the ribosome-associated quality control trigger (RQT) complex. Although uS10 ubiquitination is essential for rescuing stalled ribosomes, its function and recognition steps are not fully understood. Here, we show that the RQT complex components Cue3 and Rqt4 interact with the K63-linked ubiquitin chain and accelerate the recruitment of the RQT complex to the ubiquitinated colliding ribosome. The CUE domain of Cue3 and the N-terminal domain of Rqt4 bind independently to the K63-linked ubiquitin chain. Their deletion abolishes ribosomal dissociation mediated by the RQT complex. High-speed atomic force microscopy (HS-AFM) reveals that the intrinsically disordered regions of Rqt4 enable the expansion of the searchable area for interaction with the ubiquitin chain. These findings provide mechanistic insight into the decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex.

DOI： 10.1038/s41467-022-35608-4

Introduction of Session 2, "Advanced methods for retinal proteins" 招待有り

Uchihashi, T; Kandori, H

BIOPHYSICS AND PHYSICOBIOLOGY 20 巻 ( Supplemental ) 頁： n/a 2023年

支部だより

古谷祐詞, 内橋貴之, 矢木真穂

生物物理 63 巻 ( 5 ) 頁： 285 - 286 2023年

Ring formation by Vibrio fusion protein composed of FliF and FliG, MS-ring and C-ring component of bacterial flagellar motor in membrane 査読有り

Takahashi K., Nishikino T., Kajino H., Kojima S., Uchihashi T., Homma M.

Biophysics and physicobiology 20 巻 ( 2 ) 頁： n/a 2023年

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

The marine bacterium Vibrio alginolyticus has a single flagellum as a locomotory organ at the cell pole, which is rotated by the Na+-motive force to swim in a liquid. The base of the flagella has a motor composed of a stator and rotor, which serves as a power engine to generate torque through the rotor–stator interaction coupled to Na+ influx through the stator channel. The MS-ring, which is embedded in the membrane at the base of the flagella as part of the rotor, is the initial structure required for flagellum assembly. It comprises 34 molecules of the two-transmembrane protein FliF. FliG, FliM, and FliN form a C-ring just below the MS-ring. FliG is an important rotor protein that interacts with the stator PomA and directly contributes to force generation. We previously found that FliG promotes MS-ring formation in E. coli. In the present study, we constructed a fliF–fliG fusion gene, which encodes an approximately 100 kDa protein, and the successful production of this protein effectively formed the MS-ring in E. coli cells. We observed fuzzy structures around the ring using either electron microscopy or high-speed atomic force microscopy (HS-AFM), suggesting that FliM and FliN are necessary for the formation of a stable ring structure. The HS-AFM movies revealed flexible movements at the FliG region.

DOI： 10.2142/biophysico.bppb-v20.0028

Clarification of Surface Deswelling of Thermoresponsive Microgels by Electrophoresis 査読有り

Nishizawa, Y; Inui, T; Namioka, R; Uchihashi, T; Watanabe, T; Suzuki, D

LANGMUIR 38 巻 ( 51 ) 頁： 16084 - 16093 2022年12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：Langmuir

Although many investigations of thermoresponsive microgels have been reported, their surface properties, which are crucial in colloid science, are still not fully understood. In this study, microgels with surface-localized charged groups were synthesized by precipitation polymerization, and their electrophoretic behaviors were analyzed using a modified version of Ohshima's equation to obtain two surface properties of the soft particles: the softness parameter and the surface charge density. This systematic evaluation allows us to discuss the thermoresponsiveness of the overall microgels and their surfaces separately. Furthermore, the validity of the surface properties obtained from electrophoresis was verified by comparing them with the results of seeded emulsion polymerization in the presence of the microgels and the force-indentation curves obtained via high-speed atomic force microscopy (HS-AFM).

DOI： 10.1021/acs.langmuir.2c02742

Lytic polysaccharide monooxygenase increases cellobiohydrolases activity by promoting decrystallization of cellulose surface 査読有り国際共著

Uchiyama, T; Uchihashi, T; Ishida, T; Nakamura, A; Vermaas, JV; Crowley, MF; Samejima, M; Beckham, GT; Igarashi, K

SCIENCE ADVANCES 8 巻 ( 51 ) 頁： eade5155 2022年12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

Efficient depolymerization of crystalline cellulose requires cooperation between multiple cellulolytic enzymes. Through biochemical approaches, molecular dynamics (MD) simulation, and single-molecule observations using high-speed atomic force microscopy (HS-AFM), we quantify and track synergistic activity for cellobiohydrolases (CBHs) with a lytic polysaccharide monooxygenase (LPMO) from Phanerochaete chrysosporium. Increasing concentrations of LPMO (AA9D) increased the activity of a glycoside hydrolase family 6 CBH, Cel6A, whereas the activity of a family 7 CBH (Cel7D) was enhanced only at lower concentrations of AA9D. MD simulation suggests that the result of AA9D action to produce chain breaks in crystalline cellulose can oxidatively disturb the crystalline surface by disrupting hydrogen bonds. HS-AFM observations showed that AA9D increased the number of Cel7D molecules moving on the substrate surface and increased the processivity of Cel7D, thereby increasing the depolymerization performance, suggesting that AA9D not only generates chain ends but also amorphizes the crystalline surface, thereby increasing the activity of CBHs.

DOI： 10.1126/sciadv.ade5155

Tip-scan high-speed atomic force microscopy with a uniaxial substrate stretching device for studying dynamics of biomolecules under mechanical stress 査読有り

Chan, FY; Kurosaki, R; Ganser, C; Takeda, T; Uchihashi, T

REVIEW OF SCIENTIFIC INSTRUMENTS 93 巻 ( 11 ) 頁： 113703 - 113703 2022年11月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Review of Scientific Instruments

High-speed atomic force microscopy (HS-AFM) is a powerful tool for studying the dynamics of biomolecules in vitro because of its high temporal and spatial resolution. However, multi-functionalization, such as combination with complementary measurement methods, environment control, and large-scale mechanical manipulation of samples, is still a complex endeavor due to the inherent design and the compact sample scanning stage. Emerging tip-scan HS-AFM overcame this design hindrance and opened a door for additional functionalities. In this study, we designed a motor-driven stretching device to manipulate elastic substrates for HS-AFM imaging of biomolecules under controllable mechanical stimulation. To demonstrate the applicability of the substrate stretching device, we observed a microtubule buckling by straining the substrate and actin filaments linked by α-actinin on a curved surface. In addition, a BAR domain protein BIN1 that senses substrate curvature was observed while dynamically controlling the surface curvature. Our results clearly prove that large-scale mechanical manipulation can be coupled with nanometer-scale imaging to observe biophysical effects otherwise obscured.

DOI： 10.1063/5.0111017

Scanning Probe Microscopy 招待有り

Nakajima K., Sumitomo K., Nakayama T., Ken-Ichi Fukui , Kageshima M., Kawai S., Komeda T., Takahashi T., Uchihashi T.

Japanese Journal of Applied Physics 61 巻 ( SL ) 2022年9月

Mechanism of the Irreversible Transition from Pentamer to Monomer at pH 2 in a Blue Proteorhodopsin

Sumikawa, M; Abe-Yoshizumi, R; Uchihashi, T; Kandori, H

BIOCHEMISTRY 61 巻 ( 18 ) 頁： 1936 - 1944 2022年8月

記述言語：英語出版者・発行元：Biochemistry

Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria, and thousands of PRs are classified as blue-absorbing PRs (BPR; λmax∼490 nm) and green-absorbing PRs (GPR; λmax∼525 nm). We previously converted BPR into GPR using an anomalous pH effect, which was achieved by an irreversible process at around pH 2. Recent size-exclusion chromatography (SEC) and atomic force microscopy (AFM) analyses of BPR from Vibrio califitulae (VcBPR) revealed the anomalous pH effect owing to the irreversible transition from pentamer to monomer. Different pKavalues of the Schiff base counterion between pentamer and monomer lead to different colors at the same pH. Here, we incorporate systematic mutation into VcBPR and examine the anomalous pH effect. The anomalous pH effect was observed for the mutants of key residues near the retinal chromophore such as D76N, D206N, and Q84L, indicating that the Schiff base counterions and the L/Q switch do not affect the irreversible transition from pentamer to monomer at pH ∼2. We then focus on the two specific interactions at the intermonomer interface in a pentamer, E29/R30/D31 and W13/H54. Single mutants such as E29Q, R30A, W13A, and H54A and the wild type (WT) exhibited an anomalous pH effect. In contrast, the anomalous pH effect was lost for E29Q/H54A, R30A/H54A, and W13A/E29Q. Size-exclusion chromatography (SEC) and atomic force microscopy (AFM) measurements showed monomer forms in the original states of the double mutants, being a clear contrast to the pentamer forms of all single mutants in the original states. It was concluded that the pentamer structure of VcBPR was stabilized by an electrostatic interaction in the E29/R30/D31 region and a hydrogen-bonding interaction in the W13/H54 region, which was disrupted at pH 2 and converted into monomers.

DOI： 10.1021/acs.biochem.2c00328

Multiple dimeric structures and strand-swap dimerization of E-cadherin in solution visualized by high-speed atomic force microscopy 査読有り国際誌

Nishiguchi, S; Furuta, T; Uchihashi, T

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 119 巻 ( 30 ) 頁： e2208067119 2022年7月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences of the United States of America

Classical cadherins play key roles in cell–cell adhesion. The adhesion process is thought to comprise mainly two steps: X-dimer and strand-swap (SS-) dimer formation of the extracellular domains (ectodomains) of cadherins. The dimerization mechanism of this two-step process has been investigated for type I cadherins, including E-cadherin, of classical cadherins, whereas other binding states also have been proposed, raising the possibility of additional binding processes required for the cadherin dimerization. However, technical limitations in observing single-molecule structures and their dynamics have precluded the investigation of the dynamic binding process of cadherin. Here, we used high-speed atomic force microscopy (HS-AFM) to observe full-length ectodomains of E-cadherin in solution and identified multiple dimeric structures that had not been reported previously. HS-AFM revealed that almost half of the cadherin dimers showed S- (or reverse S-) shaped conformations, which had more dynamic properties than the SS- and X-like dimers. The combined HS-AFM, mutational, and molecular modeling analyses showed that the S-shaped dimer was formed by membrane-distal ectodomains, while the binding interface was different from that of SS- and X-dimers. Furthermore, the formation of the SS-dimer from the S-shaped and X-like dimers was directly visualized, suggesting the processes of SS-dimer formation from S-shaped and X-dimers during cadherin dimerization.

DOI： 10.1073/pnas.2208067119

Protein Needles Designed to Self-Assemble through Needle Tip Engineering 査読有り

Kikuchi, K; Fukuyama, T; Uchihashi, T; Furuta, T; Maeda, YT; Ueno, T

SMALL 18 巻 ( 10 ) 頁： 2106401 - 2106401 2022年3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：Small

The dynamic process of formation of protein assemblies is essential to form highly ordered structures in biological systems. Advances in structural and synthetic biology have led to the construction of artificial protein assemblies. However, development of design strategies exploiting the anisotropic shape of building blocks of protein assemblies has not yet been achieved. Here, the 2D assembly pattern of protein needles (PNs) is controlled by regulating their tip-to-tip interactions. The PN is an anisotropic needle-shaped protein composed of β-helix, foldon, and His-tag. Three different types of tip-modified PNs are designed by deleting the His-tag and foldon to change the protein–protein interactions. Observing their assembly by high-speed atomic force microscopy (HS-AFM) reveals that PN, His-tag deleted PN, and His-tag and foldon deleted PN form triangular lattices, the monomeric state with nematic order, and fiber assemblies, respectively, on a mica surface. Their assembly dynamics are observed by HS-AFM and analyzed by the theoretical models. Monte Carlo (MC) simulations indicate that the mica-PN interactions and the flexible and multipoint His-tag interactions cooperatively guide the formation of the triangular lattice. This work is expected to provide a new strategy for constructing supramolecular protein architectures by controlling directional interactions of anisotropic shaped proteins.

DOI： 10.1002/smll.202106401

その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/smll.202106401

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