研究者詳細 - 内橋　貴之

Structural properties determining low K+ affinity of the selectivity filter in the TWIK1 K+ channel

Tsukamoto, H; Higashi, M; Motoki, H; Watanabe, H; Ganser, C; Nakajo, K; Kubo, Y; Uchihashi, T; Furutani, Y

JOURNAL OF BIOLOGICAL CHEMISTRY 293 巻 ( 18 ) 頁： 6969 - 6984 2018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：Journal of Biological Chemistry

Canonical K channels are tetrameric and highly K-selective, whereas two-pore– domain K (K2P) channels form dimers, but with a similar pore architecture. A two-pore– domain potassium channel TWIK1 (KCNK1 or K2P1) allows permeation of Na and other monovalent ions, resulting mainly from the presence of Thr-118 in the P1 domain. However, the mechanistic basis for this reduced selectivity is unclear. Using ion-exchange–induced difference IR spectroscopy, we analyzed WT TWIK1 and T118I (highly K-selective) and L228F (substitution in the P2 domain) TWIK1 variants and found that in the presence of K ions, WT and both variants exhibit an amide-I band at 1680 cm1. This band corresponds to interactions of the backbone carbonyls in the selectivity filter with K, a feature very similar to that of the canonical K channel KcsA. Computational analysis indicated that the relatively high frequency for the amide-I band is well explained by impairment of hydrogen bond formation with water molecules. Moreover, concentration-dependent spectral changes indicated that the K affinity of the WT selectivity filter was much lower than those of the variants. Furthermore, only the variants displayed a higher frequency shift of the 1680-cm1 band upon changes from K to Rb or Cs conditions. High-speed atomic force microscopy disclosed that TWIK1’s surface morphology largely does not change in K and Na solutions. Our results reveal the local conformational changes of the TWIK1 selectivity filter and suggest that the amide-I bands may be useful “molecular fingerprints” for assessing the properties of other K channels.

DOI： 10.1074/jbc.RA118.001817

Construction of a Triangle-Shaped Trimer and a Tetrahedron Using an -Helix-Inserted Circular Permutant of Cytochrome c555

Oda, A; Nagao, S; Yamanaka, M; Ueda, I; Watanabe, H; Uchihashi, T; Shibata, N; Higuchi, Y; Hirota, S

CHEMISTRY-AN ASIAN JOURNAL 13 巻 ( 8 ) 頁： 964 - 967 2018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：Chemistry - An Asian Journal

Highly-ordered protein structures have gained interest for future uses for biomaterials. Herein, we constructed a building block protein (BBP) by the circular permutation of the hyperthermostable Aquifex aeolicus cytochrome (cyt) c555, and assembled BBP into a triangle-shaped trimer and a tetrahedron. The angle of the intermolecular interactions of BBP was controlled by cleaving the domain-swapping hinge loop of cyt c555 and connecting the original N- and C-terminal α-helices with an α-helical linker. We obtained BBP oligomers up to ≈40 mers, with a relatively large amount of trimers. According to the X-ray crystallographic analysis of the BBP trimer, the N-terminal region of one BBP molecule interacted intermolecularly with the C-terminal region of another BBP molecule, resulting in a triangle-shaped structure with an edge length of 68 Å. Additionally, four trimers assembled into a unique tetrahedron in the crystal. These results demonstrate that the circular permutation connecting the original N- and C-terminal α-helices with an α-helical linker may be useful for constructing organized protein structures.

DOI： 10.1002/asia.201800252

Insight into structural remodeling of the FlhA ring responsible for bacterial flagellar type III protein export

Terahara, N; Inoue, Y; Kodera, N; Morimoto, YV; Uchihashi, T; Imada, K; Ando, T; Namba, K; Minamino, T

SCIENCE ADVANCES 4 巻 ( 4 ) 頁： eaao7054 2018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：Science Advances

The bacterial flagellum is a supramolecular motility machine. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. A carboxyl-terminal cytoplasmic domain of FlhA (FlhAC) forms a nonameric ring structure in the flagellar type III protein export apparatus and coordinates flagellar protein export with assembly. However, the mechanism of this process remains unknown. We report that a flexible linker of FlhAC (FlhAL) is required not only for FlhAC ring formation but also for substrate specificity switching of the protein export apparatus from the hook protein to the filament protein upon completion of the hook structure. FlhAL was required for cooperative ring formation of FlhAC. Alanine substitutions of residues involved in FlhAC ring formation interfered with the substrate specificity switching, thereby inhibiting filament assembly at the hook tip. These observations lead us to propose a mechanistic model for export switching involving structural remodeling of FlhAC.

DOI： 10.1126/sciadv.aao7054

Front Cover: Construction of a Triangle-Shaped Trimer and a Tetrahedron Using an α-Helix-Inserted Circular Permutant of Cytochrome c 555 (Chem. Asian J. 10/2018) 査読有り

Akiya Oda, Satoshi Nagao, Masaru Yamanaka, Ikki Ueda, Hiroki Watanabe, Takayuki Uchihashi, Naoki Shibata, Yoshiki Higuchi, Shun Hirota

Chemistry - An Asian Journal 13 巻 ( 10 ) 頁： 1229 2018年4月

High-speed AFM reveals detail understanding in adsorption of soft hydrogel microspheres onto solid substrate in aqueous solution

Matsui, S; Uchihashi, T; Suzuki, D

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 255 巻頁： . 2018年3月

Real-time adsorption behavior of hydrogel microspheres onto solid/liquid interface

Matsui, S; Uchihashi, T; Suzuki, D

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 255 巻頁： . 2018年3月

High-speed AFM reveals detail understanding in adsorption of soft hydrogel microspheres onto solid substrate in aqueous solution

Matsui Shusuke, Uchihashi Takayuki, Suzuki Daisuke

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 255 巻頁： . 2018年3月

Real-time adsorption behavior of hydrogel microspheres onto solid/liquid interface

Matsui Shusuke, Uchihashi Takayuki, Suzuki Daisuke

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 255 巻頁： . 2018年3月

Negatively Charged Lipids Are Essential for Functional and Structural Switch of Human 2-Cys Peroxiredoxin II 査読有り

Haruyama, T; Uchihashi, T; Yamada, Y; Kodera, N; Ando, T; Konno, H

JOURNAL OF MOLECULAR BIOLOGY 430 巻 ( 5 ) 頁： 602 - 610 2018年3月

Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis 国際誌

Nakamura, A; Tasaki, T; Okuni, Y; Song, CH; Murata, K; Kozai, T; Hara, M; Sugimoto, H; Suzuki, K; Watanabe, T; Uchihashi, T; Noji, H; Iino, R

PHYSICAL CHEMISTRY CHEMICAL PHYSICS 20 巻 ( 5 ) 頁： 3010 - 3018 2018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：Physical Chemistry Chemical Physics

Serratia marcescens chitinase A is a linear molecular motor that hydrolyses crystalline chitin in a processive manner. Here, we quantitatively determined the rate constants of elementary reaction steps, including binding (kon), translational movement (ktr), and dissociation (koff) with single-molecule fluorescence imaging. The kon for a single chitin microfibril was 2.1 × 109 M-1 μm-1 s-1. The koff showed two components, kfastoff (3.2 s-1, 78%) and kslowoff (0.38 s-1, 22%), corresponding to bindings to different crystal surfaces. From the kon, kfastoff, kslowoff and ratio of fast and slow dissociations, dissociation constants for low and high affinity sites were estimated as 2.0 × 10-9 M μm and 8.1 × 10-10 M μm, respectively. The ktr was 52.5 nm s-1, and processivity was estimated as 60.4. The apparent inconsistency between high turnover (52.5 s-1) calculated from ktr and biochemically determined low kcat (2.6 s-1) is explained by a low ratio (4.8%) of productive enzymes on the chitin surface (52.5 s-1 × 0.048 = 2.5 s-1). Our results highlight the importance of single-molecule analysis in understanding the mechanism of enzymes acting on a solid-liquid interface.

DOI： 10.1039/c7cp04606e

Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis (vol 20, pg 3010, 2018) 国際誌

Nakamura, A; Tasaki, T; Okuni, Y; Song, C; Murata, K; Kozai, T; Hara, M; Sugimoto, H; Suzuki, K; Watanabe, T; Uchihashi, T; Noji, H; Iino, R

PHYSICAL CHEMISTRY CHEMICAL PHYSICS 20 巻 ( 5 ) 頁： 3844 - 3844 2018年2月

Dynamic Observation of Kai Proteins by HS-AFM Reveals a Mechanism of the Robustness in the Cyanobacterial Circadian Oscillator

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

BIOPHYSICAL JOURNAL 114 巻 ( 3 ) 頁： 68A - 68A 2018年2月

Dynamic Observation of Kai Proteins by HS-AFM Reveals a Mechanism of the Robustness in the Cyanobacterial Circadian Oscillator

Sugiyama Shogo, Mori Tetsya, Byrne Mark, Uchihashi Takayuki, Johnson Carl H, Ando Toshio

BIOPHYSICAL JOURNAL 114 巻 ( 3 ) 頁： 68A-68A 2018年2月

Applications of high-speed atomic force microscopy to real-time visualization of dynamic biomolecular processes

Uchihashi, T; Scheuring, S

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1862 巻 ( 2 ) 頁： 229 - 240 2018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：Biochimica et Biophysica Acta - General Subjects

Background Many biological processes in a living cell are consequences of sequential and hierarchical dynamic events of biological macromolecules such as molecular interactions and conformational changes. Hence, knowledge of structures, assembly and dynamics of proteins is the foundation for understanding how biological molecules work. Among several techniques to analyze dynamics of proteins, high-speed atomic force microscopy (HS-AFM) is unique to provide direct information about both structure and dynamics of single proteins at work. Scope of review The scope of this review is overviewing recent progresses of HS-AFM for studying dynamic processes of biomolecular systems. In the technical descriptions, key developments enabling fast and non-invasive imaging of biological samples are briefly mentioned. Then recent successful applications of HS-AFM are overviewed to showcase the power of HS-AFM in biological research. Major conclusions We discuss examples where HS-AFM movies captured important dynamic biological processes, including conformational dynamics of membrane proteins, processive movements of enzymes, assembly and disassembly processes of protein supramolecular structures, and dynamics in a two-dimensional protein crystal. These examples demonstrate the usability of HS-AFM to reveal biomolecular processes at high spatiotemporal (nanometer and subsecond) resolution. General significance Real-time movies of unlabeled proteins at work captured by HS-AFM allowed us to directly gain insights into mechanisms of molecular actions. Together with further functional extensions, HS-AFM will enable researchers to investigate more complex biological systems involving multiple proteins and will become an indispensable technique for life science. This article is part of a Special Issue entitled “Biophysical Exploration of Dynamical Ordering of Biomolecular Systems” Guest Editor: Dr., Professor Koichi Kato.

DOI： 10.1016/j.bbagen.2017.07.010

Dynamic clustering of dynamin-amphiphysin helices regulates membrane constriction and fission coupled with GTP hydrolysis 査読有り

Takeda, T; Kozai, T; Yang, H; Ishikuro, D; Seyama, K; Kumagai, Y; Abe, T; Yamada, H; Uchihashi, T; Ando, T; Takei, K

ELIFE 7 巻 2018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：eLife

Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin-mediated endocytosis. Dynamin forms helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulate membrane remodeling during the fission, but its precise mechanism remains elusive. In this study, we analyzed structural changes of dynamin-amphiphysin complexes during the membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin helices are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our approaches using combination of EM and HS-AFM clearly demonstrate new mechanistic insights into the dynamics of dynamin-amphiphysin complexes during membrane fission.

DOI： 10.7554/eLife.30246

Conversion of functionally undefined homopentameric protein PbaA into a proteasome activator by mutational modification of its C-terminal segment conformation 査読有り国際誌

Yagi-Utsumi, M; Sikdar, A; Kozai, T; Inoue, R; Sugiyama, M; Uchihashi, T; Yagi, H; Satoh, T; Kato, K

PROTEIN ENGINEERING DESIGN & SELECTION 31 巻 ( 1 ) 頁： 29 - 36 2018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：Protein Engineering, Design and Selection

Recent bioinformatic analyses identified proteasome assembly chaperone-like proteins, PbaA and PbaB, in archaea. PbaB forms a homotetramer and functions as a proteasome activator, whereas PbaA does not interact with the proteasome despite the presence of an apparent C-terminal proteasome activation motif. We revealed that PbaA forms a homopentamer predominantly in the closed conformation with its C-terminal segments packed against the core domains, in contrast to the PbaB homotetramer with projecting C-terminal segments. This prompted us to create a novel proteasome activator based on a well-characterized structural framework. We constructed a panel of chimeric proteins comprising the homopentameric scaffold of PbaA and C-terminal segment of PbaB and subjected them to proteasome-activating assays as well as small-angle X-ray scattering and high-speed atomic force microscopy. The results indicated that the open conformation and consequent proteasome activation activity could be enhanced by replacement of the crystallographically disordered C-terminal segment of PbaA with the corresponding disordered segment of PbaB. Moreover, these effects can be produced just by incorporating two glutamate residues into the disordered C-terminal segment of PbaA, probably due to electrostatic repulsion among the negatively charged segments. Thus, we successfully endowed a functionally undefined protein with proteasome-activating activity by modifying its C-terminal segment.

DOI： 10.1093/protein/gzx066

Visualization of Protein Dynamics using High-Speed Atomic Force Microscopy and Image Analysis

Uchihashi Takayuki

JOURNAL OF COMPUTER CHEMISTRY-JAPAN 17 巻 ( 1 ) 頁： 20-30 - 30 2018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：日本コンピュータ化学会

最近の高速原子間力顕微鏡(Atomic Force Microscopy: AFM)の進展によって, 生理溶液中でのタンパク質の構造とその時間発展，分子の離散集合などダイナミクスをリアルタイムで可視化できるようになってきた. これまで様々な計測手法による実験事実の積み重ねから推定されてきた事実を動画として提示することで, タンパク質の動的振る舞いを介した機能発現の分子機構を直接的かつ直感的に把握できるようになった. 動画は静止画に比べて極めて多くの情報を与えるが, 一方で, 単に動画鑑賞に終わることなく, そこから有益な情報を客観的な指標を基に抽出すること, すなわち動画解析が次のステップには重要である. 本稿では, これまで高速AFMで観察されてきた代表的なタンパク質の動的現象をi)一分子の構造変化, ii)分子の直進運動と速度解析, iii)二分子間の結合と解離, の3つに大別し, それぞれについて筆者らの実験結果をデータの解釈と解析の効率化のために開発した画像解析法とともに解説する.

DOI： 10.2477/jccj.2018-0001

Optimum Substrates for Imaging Biological Molecules with High-Speed Atomic Force Microscopy

CiNii Research

Uchihashi, T; Watanabe, H; Kodera, N

NANOSCALE IMAGING: METHODS AND PROTOCOLS 1814 巻頁： 159 - 179 2018年

記述言語：英語出版者・発行元：Methods in Molecular Biology

Recent progresses in high-speed atomic force microscopy (HS-AFM) have enabled us to directly visualize dynamic processes of various proteins in liquid conditions. One of the key factors leading to successful HS-AFM observations is the selection of an appropriate substrate depending on molecules to be observed. For the HS-AFM imaging, a target molecule must be absorbed on a substrate by controlling its orientation without impairing the dynamics or physiological function of the molecule. In this chapter, we describe protocols for preparation of substrates that have been used for HS-AFM and then introduce observation examples on dynamic processes of biological molecules.

DOI： 10.1007/978-1-4939-8591-3_10

High-speed scanning near-field optical microscopy

Umakoshi T., Fukuda S., Uchihashi T., Verma P., Ando T.

Optics InfoBase Conference Papers Part F125-JSAP 2018 巻 2018年

記述言語：日本語出版者・発行元：Optics InfoBase Conference Papers

Scanning near-field optical microscopy (SNOM) has been recognized as a powerful technique for super-resolution optical imaging [1]. It has been even more unique compared with other super-resolution fluorescent imaging techniques because it utilizes near-field light at a metallic tip as a nano-light-source, which realizes super-resolution not only in fluorescence but also in any other optical signals such as Raman scattering, infrared absorption and photoluminescence. Such a strong advantage has been stimulating biological fields as a new class of analytical techniques. However, it has been yet highly challenging to apply SNOM for biological samples. Although there have been several issues for biological applications, one of the major problems is its low imaging speed. Because it requires to physically scan a metallic tip, the imaging rate was typically limited to several minutes per frame, which is too slow to capture dynamic biological samples.