TOHOKU UNIVERSITY Researchers - Satoshi Takahashi

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Satoshi Takahashi

Section

Institute of Multidisciplinary Research for Advanced Materials

Job title

Professor

Degree

PhD (The Graduate University for Advanced Studies)

researchmap

https://researchmap.jp/read0149961

Research History 6

2003/04 - 2009/03

大阪大学蛋白質研究所　助教授および准教授
1996/08 - 2003/03

京都大学大学院工学研究科分子工学専攻　助手
1995/11 - 1996/07

理化学研究所　基礎科学特別研究員
1993/02 - 1995/10

AT&T Bell Laboratories　博士研究員
1992/04 - 1993/01

岡崎共同研究機構分子科学研究所　日本学術振興会特別研究員
2009/04 -

東北大学多元物質科学研究所　教授

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Education 2

The Graduate University for Advanced Studies　Graduate School, Division of Mathematical and Physical Science　機能分子科学専攻

- 1992/03/31
Tohoku University　Faculty of Science　化学

- 1987/03/31

Committee Memberships 5

The Biophysical Society of Japan　President

2023/06 - 2025/06
日本生物物理学会　日本生物物理学会学会誌「生物物理」編集委員長

2020/01 - 2021/12
日本生物物理学会　第59回日本生物物理学会年会・年会長

- 2021/12
The Protein Society　Executive Council Member

2016/01 - 2018/12
日本生物物理学会　日本生物物理学会・副会長

2015/06 - 2017/05

Professional Memberships 5

Protein Society

2016 - Present
Biophysical Society of Japan

1996 - Present
Biophysical Society

1993 - Present
Protein Science Society Japan

- Present
日本化学会

- Present

Research Interests 4

Physical Chemistry
Protein Science
Molecular Spectroscopy
Biophysics

Research Areas 5

Natural sciences / Bio-, chemical, and soft-matter physics /
Life sciences / Functional biochemistry /
Life sciences / Structural biochemistry /
Nanotechnology/Materials / Basic physical chemistry /
Life sciences / Biophysics /

Papers 97

Single-molecule microscopy reveals that importin α slides along DNA while transporting cargo molecules

Trishit Banerjee, Kazuya Jibiki, Hinata Sugasawa, Saori Kanbayashi, Taiki Niikura, Eriko Mano, Shigeru Chaen, Takashi S. Kodama, Satoshi Takahashi, Noriko Yasuhara, Kiyoto Kamagata

Biochemical and Biophysical Research Communications　748　151320-151320　2025/02
Publisher: Elsevier BV
DOI： 10.1016/j.bbrc.2025.151320 　

ISSN： 0006-291X
A single dimer of the SARS-CoV-2 N protein can associate with multiple fragments of single-stranded and stem-loop RNAs

Naoya Kaneda, Leo Suzuki, Shun Endo, Syamil Muharror Ahsanul Husna, Shion Ishikawa, Hiroto Takahashi, Hiroyuki Oikawa, Yuji Itoh, Satoshi Takahashi

2025/01/24
Publisher: Cold Spring Harbor Laboratory
DOI： 10.1101/2025.01.23.634602 　

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Abstract The nucleocapsid (N) protein of SARS-CoV-2 associates with the viral genomic RNA (gRNA) and forms the ribonucleoprotein (RNP) granules. However, the detailed molecular structures of RNP and their formation mechanism are largely unknown. We used circular dichroism (CD) spectroscopy, fluorescence correlation spectroscopy (FCS) and single-molecule Förster resonance energy transfer (sm-FRET) spectroscopy to understand the interaction between the N protein and different structural units of RNA. We chose polyadenylate chains with 40, 30 and 20 bases possessing a single-stranded structure and three stem loops with 50, 41 and 29 bases selected from gRNA, and labeled their 5’ and 3’ ends by Alexa488 and Alexa647, respectively, for the FCS and sm-FRET measurements. We found that the N protein started to bind to the single-stranded RNAs at the concentrations between 10 and 100 nM. The binding of the N protein to one of the stem loops occurred at the concentration less than 10 nM without melting the stem loop. For all the samples, the binding of multiple molecules of the RNA fragments to a single dimer of the N protein was observed. These results demonstrate that the N protein acts as a non-specific binder to both single-stranded and stem-loop structures of RNA, and that the N protein might contract a long RNA chain by bridging its multiple segments. We propose that the RNP granules might be folded by the association of the numerous stem loops of gRNA triggered by the assembly of the N protein.
Thermostable nucleoid protein Cren7 slides along DNA and rapidly dissociates from DNA while not inhibiting the sliding of other DNA-binding protein. International-journal

Trishit Banerjee, K Geethika, Saori Kanbayashi, Satoshi Takahashi, Soumit S Mandal, Kiyoto Kamagata

Journal of molecular biology　168803-168803　2024/09/24

DOI： 10.1016/j.jmb.2024.168803 　

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A nucleoid protein Cren7 compacts DNA, contributing to the living of Crenarchaeum in high temperature environment. In this study, we investigated the dynamic behavior of Cren7 on DNA and its functional relation using single-molecule fluorescence microscopy. We found two mobility modes of Cren7, sliding along DNA and pausing on it, and the rapid dissociation kinetics from DNA. The salt dependence analysis suggests a sliding with continuous contact to DNA, rather than hopping/jumping. The mutational analysis demonstrates that Cren7 slides along DNA while Trp (W26) residue interacts with the DNA. Furthermore, Cren7 does not impede the target search by a model transcription factor p53, implying no significant interference to other DNA-binding proteins on DNA. At high concentration of Cren7, the molecules form large clusters on DNA via bridging, which compacts DNA. We discuss how the dynamic behavior of Cren7 on DNA enables DNA-compaction and protein-bypass functions.
Simple and Efficient Detection Scheme of Two-Color Fluorescence Correlation Spectroscopy for Protein Dynamics Investigation from Nanoseconds to Milliseconds. International-journal Peer-reviewed

Yutaka Sano, Yuji Itoh, Supawich Kamonprasertsuk, Leo Suzuki, Atsuhito Fukasawa, Hiroyuki Oikawa, Satoshi Takahashi

ACS physical chemistry Au　4　(1)　85-93　2024/01/24

DOI： 10.1021/acsphyschemau.3c00040 　

eISSN： 2694-2445
Citrullinated fibrinogen-SAAs complex causes vascular metastagenesis. International-journal Peer-reviewed

Yibing Han, Takeshi Tomita, Masayoshi Kato, Norihiro Ashihara, Yumiko Higuchi, Hisanori Matoba, Weiyi Wang, Hikaru Hayashi, Yuji Itoh, Satoshi Takahashi, Hiroshi Kurita, Jun Nakayama, Nobuo Okumura, Sachie Hiratsuka

Nature communications　14　(1)　4960-4960　2023/08/24

DOI： 10.1038/s41467-023-40371-1 　

eISSN： 2041-1723
Hyper-mobile Water and Raman 2900 cm-1 Peak Band of Water Observed around Backbone Phosphates of Double Stranded DNA by High-Resolution Spectroscopies and MD Structural Feature Analysis of Water. International-journal Peer-reviewed

Makoto Suzuki, Akira Tsuchiko, Yoshiyuki Tanaka, Nobuyuki Matubayasi, George Mogami, Nobuyuki Uozumi, Satoshi Takahashi

The journal of physical chemistry. B　127　(1)　285-299　2022/12/27

DOI： 10.1021/acs.jpcb.2c06952 　

ISSN： 1520-6106

eISSN： 1520-5207
Flexible Target Recognition of the Intrinsically Disordered DNA-Binding Domain of CytR Monitored by Single-Molecule Fluorescence Spectroscopy International-journal Peer-reviewed

Shrutarshi Mitra, Hiroyuki Oikawa, Divya Rajendran, Toshiyuki Kowada, Shin Mizukami, Athi N. Naganathan, Satoshi Takahashi

The Journal of Physical Chemistry B　126　(33)　6136-6147　2022/08/25
Publisher: American Chemical Society (ACS)
DOI： 10.1021/acs.jpcb.2c02791 　

ISSN： 1520-6106

eISSN： 1520-5207
Quantification of Entropic Excluded Volume Effects Driving Crowding-Induced Collapse and Folding of a Disordered Protein Peer-reviewed

Divya Rajendran, Shrutarshi Mitra, Hiroyuki Oikawa, Kulkarni Madhurima, Ashok Sekhar, Satoshi Takahashi, Athi N. Naganathan

The Journal of Physical Chemistry Letters　13　(13)　3112-3120　2022/04/07
Publisher: American Chemical Society (ACS)
DOI： 10.1021/acs.jpclett.2c00316 　

ISSN： 1948-7185

eISSN： 1948-7185
Molecular principles of recruitment and dynamics of guest proteins in liquid droplets. International-journal Peer-reviewed

Kiyoto Kamagata, Nanako Iwaki, Milan Kumar Hazra, Saori Kanbayashi, Trishit Banerjee, Rika Chiba, Seiji Sakomoto, Virginie Gaudon, Bertrand Castaing, Hiroto Takahashi, Michiko Kimura, Hiroyuki Oikawa, Satoshi Takahashi, Yaakov Levy

Scientific reports　11　(1)　19323-19323　2021/09/29

DOI： 10.1038/s41598-021-98955-0 　

eISSN： 2045-2322
Extracellular mRNA transported to the nucleus exerts translation-independent function. International-journal Peer-reviewed

Takeshi Tomita, Masayoshi Kato, Taishi Mishima, Yuta Matsunaga, Hideki Sanjo, Ken-Ichi Ito, Kentaro Minagawa, Toshimitsu Matsui, Hiroyuki Oikawa, Satoshi Takahashi, Toshifumi Takao, Noriki Iwai, Takashi Mino, Osamu Takeuchi, Yoshiro Maru, Sachie Hiratsuka

Nature communications　12　(1)　3655-3655　2021/06/16

DOI： 10.1038/s41467-021-23969-1 　

eISSN： 2041-1723
The HMGB chromatin protein Nhp6A can bypass obstacles when traveling on DNA International-journal Peer-reviewed

Kiyoto Kamagata, Kana Ouchi, Cheng Tan, Eriko Mano, Sridhar Mandali, Yining Wu, Shoji Takada, Satoshi Takahashi, Reid C Johnson

Nucleic Acids Research　48　(19)　10820-10831　2020/11/04
Publisher: Oxford University Press (OUP)
DOI： 10.1093/nar/gkaa799 　

ISSN： 0305-1048

eISSN： 1362-4962
Unique Electronic Structures of the Highly Ruffled Hemes in Heme-Degrading Enzymes of Staphylococcus aureus, IsdG and IsdI, by Resonance Raman and Electron Paramagnetic Resonance Spectroscopies International-journal Peer-reviewed

Satoshi Takahashi, Shusuke Nambu, Toshitaka Matsui, Hiroshi Fujii, Haruto Ishikawa, Yasuhisa Mizutani, Kouhei Tsumoto, Masao Ikeda-Saito

Biochemistry　59　(40)　3918-3928　2020/10/13
Publisher: American Chemical Society (ACS)
DOI： 10.1021/acs.biochem.0c00731 　

ISSN： 0006-2960

eISSN： 1520-4995
Transient binding and jumping dynamics of p53 along DNA revealed by sub-millisecond resolved single-molecule fluorescence tracking. International-journal Peer-reviewed

Dwiky Rendra Graha Subekti, Agato Murata, Yuji Itoh, Satoshi Takahashi, Kiyoto Kamagata

Scientific reports　10　(1)　13697-13697　2020/08/13

DOI： 10.1038/s41598-020-70763-y 　

eISSN： 2045-2322
Liquid-like droplet formation by tumor suppressor p53 induced by multivalent electrostatic interactions between two disordered domains Peer-reviewed

Kamagata K, Kanbayashi S, Honda M, Itoh Y, Takahashi H, Kameda T, Nagatsugi F, Takahashi S

Scientific Reports　10　(1)　580　2020/01

DOI： 10.1038/s41598-020-57521-w 　

ISSN： 2045-2322

eISSN： 2045-2322
Intrinsically disordered domain of tumor suppressor p53 facilitates target search by ultrafast transfer between different DNA strands. International-journal Peer-reviewed

Yuji Itoh, Agato Murata, Satoshi Takahashi, Kiyoto Kamagata

Nucleic acids research　46　(14)　7261-7269　2018/08/21

DOI： 10.1093/nar/gky586 　

ISSN： 0305-1048

eISSN： 1362-4962
Hypothesis: structural heterogeneity of the unfolded proteins originating from the coupling of the local clusters and the long-range distance distribution Invited Peer-reviewed

Satoshi Takahashi, Aya Yoshida, Hiroyuki Oikawa

Biophysical Reviews　10　(2)　363-373　2018/04/01
Publisher: Springer Verlag
DOI： 10.1007/s12551-018-0405-8 　

ISSN： 1867-2469 1867-2450

eISSN： 1867-2469
Microsecond resolved single-molecule FRET time series measurements based on the line confocal optical system combined with hybrid photodetectors Peer-reviewed

Hiroyuki Oikawa, Takumi Takahashi, Supawich Kamonprasertsuk, Satoshi Takahashi

Physical Chemistry Chemical Physics　20　(5)　3277-3285　2018
Publisher: Royal Society of Chemistry (RSC)
DOI： 10.1039/c7cp06268k 　

ISSN： 1463-9076

eISSN： 1463-9084
Non-Native alpha-Helices in the Initial Folding Intermediate Facilitate the Ordered Assembly of the beta-Barrel in beta-Lactoglobulin Peer-reviewed

Kazumasa Sakurai, Masanori Yagi, Tsuyoshi Konuma, Satoshi Takahashi, Chiaki Nishimura, Yuji Goto

BIOCHEMISTRY　56　(36)　4799-4807　2017/09

DOI： 10.1021/acs.biochem.7b00458 　

ISSN： 0006-2960

eISSN： 1520-4995
The Disordered Linker in p53 Participates in Nonspecific Binding to and One-Dimensional Sliding along DNA Revealed by Single-Molecule Fluorescence Measurements Peer-reviewed

Dwiky Rendra Graha Subekti, Agato Murata, Yuji Itoh, Satoshi Fukuchi, Hiroto Takahashi, Saori Kanbayashi, Satoshi Takahashi, Kiyoto Kamagata

BIOCHEMISTRY　56　(32)　4134-4144　2017/08

DOI： 10.1021/acs.biochem.7b00292 　

ISSN： 0006-2960

eISSN： 1520-4995
Probing native metal ion association sites through quenching of fluorophores in the nucleotide-binding domains of the ABC transporter MsbA Peer-reviewed

Daiki Tatsumi, Kei Nanatani, Yuto Koike, Kiyoto Kamagata, Satoshi Takahashi, Ayumu Konno, Tadaomi Furuta, Minoru Sakurai, Nobuyuki Uozumi

BIOCHEMICAL JOURNAL　474　(12)　1993-2007　2017/06

DOI： 10.1042/BCJ20161051 　

ISSN： 0264-6021

eISSN： 1470-8728
Highly Heterogeneous Nature of the Native and Unfolded States of the B Domain of Protein A Revealed by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy Peer-reviewed

Takuhiro Otosu, Kunihiko Ishii, Hiroyuki Oikawa, Munehito Arai, Satoshi Takahashi, Tahei Tahara

JOURNAL OF PHYSICAL CHEMISTRY B　121　(22)　5463-5473　2017/06

DOI： 10.1021/acs.jpcb.7b00546 　

ISSN： 1520-6106

eISSN： 1520-5207
One-Dimensional Search Dynamics of Tumor Suppressor p53 Regulated by a Disordered C-Terminal Domain Peer-reviewed

Agato Murata, Yuji Itoh, Eriko Mano, Saori Kanbayashi, Chihiro Igarashi, Hiroto Takahashi, Satoshi Takahashi, Kiyoto Kamagata

BIOPHYSICAL JOURNAL　112　(11)　2301-2314　2017/06

DOI： 10.1016/j.bpj.2017.04.038 　

ISSN： 0006-3495

eISSN： 1542-0086
Characterization of facilitated diffusion of tumor suppressor p53 along DNA using single-molecule fluorescence imaging Invited Peer-reviewed

Kiyoto Kamagata, Agato Murata, Yuji Itoh, Satoshi Takahashi

JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY C-PHOTOCHEMISTRY REVIEWS　30　36-50　2017/03

DOI： 10.1016/j.jphotochemrev.2017.01.004 　

ISSN： 1389-5567

eISSN： 1873-2739
Raman optical activity of tetra-alanine in the poly(L-proline) II type peptide conformation Peer-reviewed

Masakazu Furuta, Tomotsumi Fujisawa, Hiroyasu Urago, Takahiro Eguchi, Takahito Shingae, Satoshi Takahashi, Ewan W. Blanch, Masashi Unno

PHYSICAL CHEMISTRY CHEMICAL PHYSICS　19　(3)　2078-2086　2017/01

DOI： 10.1039/c6cp07828a 　

ISSN： 1463-9076

eISSN： 1463-9084
DNA Garden: A Simple Method for Producing Arrays of Stretchable DNA for Single-Molecule Fluorescence Imaging of DNA-Binding Proteins Peer-reviewed

Chihiro Igarashi, Agato Murata, Yuji Itoh, Dwiky Rendra, Graha Subekti, Satoshi Takahashi, Kiyoto Kamagata

BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN　90　(1)　34-43　2017/01

DOI： 10.1246/bcsj.20160298 　

ISSN： 0009-2673

eISSN： 1348-0634
Significant Heterogeneity and Slow Dynamics of the Unfolded Ubiquitin Detected by the Line Confocal Method of Single-Molecule Fluorescence Spectroscopy Peer-reviewed

Masataka Saito, Supawich Kamonprasertsuk, Satomi Suzuki, Kei Nanatani, Hiroyuki Oikawa, Keiichiro Kushiro, Madoka Takai, Po-ting Chen, Eric H. -L. Chen, Rita P. -Y. Chen, Satoshi Takahashi

JOURNAL OF PHYSICAL CHEMISTRY B　120　(34)　8818-8829　2016/09

DOI： 10.1021/acs.jpcb.6b05481 　

ISSN： 1520-6106

eISSN： 1520-5207
Activation of p53 Facilitates the Target Search in DNA by Enhancing the Target Recognition Probability Peer-reviewed

Yuji Itoh, Agato Murata, Seiji Sakamoto, Kei Nanatani, Takehiko Wada, Satoshi Takahashi, Kiyoto Kamagata

JOURNAL OF MOLECULAR BIOLOGY　428　(14)　2916-2930　2016/07

DOI： 10.1016/j.jmb.2016.06.001 　

ISSN： 0022-2836

eISSN： 1089-8638
Unique coupling of mono- and dioxygenase chemistries in a single active site promotes heme degradation Peer-reviewed

Toshitaka Matsui, Shusuke Nambu, Celia W. Goulding, Satoshi Takahashi, Hiroshi Fujii, Masao Ikeda-Saito

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　113　(14)　3779-3784　2016/04

DOI： 10.1073/pnas.1523333113 　

ISSN： 0027-8424

eISSN： 1091-6490
Where the complex things are: single molecule and ensemble spectroscopic investigations of protein folding dynamics Invited Peer-reviewed

Satoshi Takahashi, Kiyoto Kamagata, Hiroyuki Oikawa

CURRENT OPINION IN STRUCTURAL BIOLOGY　36　1-9　2016/02

DOI： 10.1016/j.sbi.2015.11.006 　

ISSN： 0959-440X

eISSN： 1879-033X
Highly Collapsed Conformation of the Initial Folding Intermediates of beta-Lactoglobulin with Non-Native alpha-Helix Peer-reviewed

Tsuyoshi Konuma, Kazumasa Sakurai, Masanori Yagi, Yuji Goto, Tetsuro Fujisawa, Satoshi Takahashi

JOURNAL OF MOLECULAR BIOLOGY　427　(19)　3158-3165　2015/09

DOI： 10.1016/j.jmb.2015.07.018 　

ISSN： 0022-2836

eISSN： 1089-8638
One-Dimensional Sliding of p53 Along DNA Is Accelerated in the Presence of Ca2+ or Mg2+ at Millimolar Concentrations Peer-reviewed

Agato Murata, Yuji Ito, Risa Kashima, Saori Kanbayashi, Kei Nanatani, Chihiro Igarashi, Masaki Okumura, Kenji Inaba, Takashi Tokino, Satoshi Takahashi, Kiyoto Kamagata

JOURNAL OF MOLECULAR BIOLOGY　427　(16)　2663-2678　2015/08

DOI： 10.1016/j.jmb.2015.06.016 　

ISSN： 0022-2836

eISSN： 1089-8638
Complexity of the Folding Transition of the B Domain of Protein A Revealed by the High-Speed Tracking of Single-Molecule Fluorescence Time Series Peer-reviewed

Hiroyuki Oikawa, Kiyoto Kamagata, Munehito Arai, Satoshi Takahashi

JOURNAL OF PHYSICAL CHEMISTRY B　119　(20)　6081-6091　2015/05

DOI： 10.1021/acs.jpcb.5b00414 　

ISSN： 1520-6106

eISSN： 1520-5207
Tip-enhanced Raman spectroscopy of 4,4 '-bipyridine and 4,4 '-bipyridine N,N '-dioxide adsorbed on gold thin films Peer-reviewed

I. I. Rzeznicka, H. Horino, N. Kikkawa, S. Sakaguchi, A. Morita, S. Takahashi, T. Komeda, H. Fukumura, T. Yamada, M. Kawai

SURFACE SCIENCE　617　1-9　2013/11

DOI： 10.1016/j.susc.2013.08.010 　

ISSN： 0039-6028

eISSN： 1879-2758
Serum amyloid A3 binds MD-2 to activate p38 and NF-κB pathways in a MyD88-dependent manner Peer-reviewed

Atsuko Deguchi, Takeshi Tomita, Tsutomu Omori, Akiko Komatsu, Umeharu Ohto, Satoshi Takahashi, Natsuko Tanimura, Sachiko Akashi-Takamura, Kensuke Miyake, Yoshiro Maru

Journal of Immunology　191　(4)　1856-1864　2013/08/15

DOI： 10.4049/jimmunol.1201996 　

ISSN： 0022-1767 1550-6606

eISSN： 1550-6606
Microsecond dynamics of an unfolded protein by a line confocal tracking of single molecule fluorescence Peer-reviewed

Hiroyuki Oikawa, Yuta Suzuki, Masataka Saito, Kiyoto Kamagata, Munehito Arai, Satoshi Takahashi

SCIENTIFIC REPORTS　3　2151　2013/07

DOI： 10.1038/srep02151 　

ISSN： 2045-2322

eISSN： 2045-2322
A New Way to Degrade Heme THE MYCOBACTERIUM TUBERCULOSIS ENZYME MhuD CATALYZES HEME DEGRADATION WITHOUT GENERATING CO Peer-reviewed

Shusuke Nambu, Toshitaka Matsui, Celia W. Goulding, Satoshi Takahashi, Masao Ikeda-Saito

JOURNAL OF BIOLOGICAL CHEMISTRY　288　(14)　10101-10109　2013/04

DOI： 10.1074/jbc.M112.448399 　

ISSN： 0021-9258

eISSN： 1083-351X
Hydration-State Change of Horse Heart Cytochrome c Corresponding to Trifluoroacetic-Acid-Induced Unfolding Peer-reviewed

Yusuke Miyashita, Tetsuichi Wazawa, George Mogami, Satoshi Takahashi, Yoshihiro Sambongi, Makoto Suzuki

BIOPHYSICAL JOURNAL　104　(1)　163-172　2013/01

DOI： 10.1016/j.bpj.2012.11.3825 　

ISSN： 0006-3495

eISSN： 1542-0086
Long-Term Observation of Fluorescence of Free Single Molecules To Explore Protein-Folding Energy Landscapes Peer-reviewed

Kiyoto Kamagata, Toshifumi Kawaguchi, Yoshitomo Iwahashi, Akinori Baba, Kazuya Fujimoto, Tamiki Komatsuzaki, Yoshihiro Sambongi, Yuji Goto, Satoshi Takahashi

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY　134　(28)　11525-11532　2012/07

DOI： 10.1021/ja3020555 　

ISSN： 0002-7863

eISSN： 1520-5126
Staring at a Protein : Ensemble and Single-Molecule Inbestigations on Protein-Folding Dynamics Invited Peer-reviewed

Satoshi Takahashi, Kiyoto Kamagata

Advances in Chemical Physics　146　1-22　2011/11

DOI： 10.1002/9781118131374.ch1 　

ISSN： 0065-2385
Time-Resolved Small-Angle X-ray Scattering Study of the Folding Dynamics of Barnase Peer-reviewed

Tsuyoshi Konuma, Tetsunari Kimura, Shuzo Matsumoto, Yuji Got, Tetsuro Fujisawa, Alan R. Fersht, Satoshi Takahashi

JOURNAL OF MOLECULAR BIOLOGY　405　(5)　1284-1294　2011/02

DOI： 10.1016/j.jmb.2010.11.052 　

ISSN： 0022-2836
Dehydration of main-chain amides in the final folding step of single-chain monellin revealed by time-resolved infrared spectroscopy Peer-reviewed

Tetsunari Kimura, Akio Maeda, Shingo Nishiguchi, Koichiro Ishimori, Isao Morishima, Takashi Konno, Yuji Goto, Satoshi Takahashi

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　105　(36)　13391-13396　2008/09

DOI： 10.1073/pnas.0801316105 　

ISSN： 0027-8424

eISSN： 1091-6490
Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR Peer-reviewed

Takanori Uzawa, Chiaki Nishimura, Shuji Akiyama, Koichiro Ishimori, Satoshi Takahashi, H. Jane Dyson, Peter E. Wright

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　105　(37)　13859-13864　2008/09

DOI： 10.1073/pnas.0804033105 　

ISSN： 0027-8424

eISSN： 1091-6490
Solvation and desolvation dynamics in apomyoglobin folding monitored by time-resolved infrared spectroscopy Peer-reviewed

Shingo Nishiguchi, Yuji Goto, Satoshi Takahashi

JOURNAL OF MOLECULAR BIOLOGY　373　(2)　491-502　2007/10

DOI： 10.1016/j.jmb.2007.08.003 　

ISSN： 0022-2836
Development of a technique for the investigation of folding dynamics of single proteins for extended time periods Peer-reviewed

Masahito Kinoshita, Kiyoto Kamagata, Akio Maeda, Yuji Goto, Tamiki Komatsuzaki, Satoshi Takahashi

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　104　(25)　10453-10458　2007/06

DOI： 10.1073/pnas.0700267104 　

ISSN： 0027-8424
A rapid flow mixer with 11-mu s mixing time microfabricated by a pulsed-laser ablation technique: Observation of a barrier-limited collapse in cytochrome c folding Peer-reviewed

Shuzo Matsumoto, Akira Yane, Satoru Nakashima, Masaki Hashida, Masayuki Fujita, Yuji Goto, Satoshi Takahashi

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY　129　(13)　3840-+　2007/04

DOI： 10.1021/ja0660958 　

ISSN： 0002-7863
Cores and pH-dependent dynamics of ferredoxin-NADP(+) reductase revealed by hydrogen/deuterium exchange Peer-reviewed

Young-Ho Lee, Kosuke Tamura, Masahiro Maeda, Masaru Hoshino, Kazumasa Sakurai, Satoshi Takahashi, Takahisa Ikegami, Toshiharu Hase, Yuji Goto

JOURNAL OF BIOLOGICAL CHEMISTRY　282　(8)　5959-5967　2007/02

DOI： 10.1074/jbc.M608417200 　

ISSN： 0021-9258

eISSN： 1083-351X
3D structure of amyloid protofilaments of beta(2)-microglobulin fragment probed by solid-state NMR Peer-reviewed

Kentaro Iwata, Toshimichi Fujiwara, Yoh Matsuki, Hideo Akutsu, Satoshi Takahashi, Hironobu Naiki, Yuji Goto

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　103　(48)　18119-18124　2006/11

DOI： 10.1073/pnas.0607180103 　

ISSN： 0027-8424
Time-resolved small-angle X-ray scattering investigation of the folding dynamics of heme oxygenase: Implication of the scaling relationship for the submillisecond intermediates of protein folding Peer-reviewed

T Uzawa, T Kimura, K Ishimori, Morishima, I, T Matsui, M Ikeda-Saito, S Takahashi, S Akiyama, T Fujisawa

JOURNAL OF MOLECULAR BIOLOGY　357　(3)　997-1008　2006/03

DOI： 10.1016/j.jmb.2005.12.089 　

ISSN： 0022-2836

eISSN： 1089-8638
Absence of a detectable intermediate in the compound I formation of horseradish peroxidase at ambient temperature Peer-reviewed

M Shintaku, K Matsuura, S Yoshioka, S Takahashi, K Ishimori, Morishima, I

JOURNAL OF BIOLOGICAL CHEMISTRY　280　(49)　40934-40938　2005/12

DOI： 10.1074/jbc.M503472200 　

ISSN： 0021-9258

eISSN： 1083-351X
Specifically collapsed intermediate in the early stage of the folding of ribonuclease A Peer-reviewed

T Kimura, S Akiyama, T Uzawa, K Ishimori, Morishima, I, T Fujisawa, S Takahashi

JOURNAL OF MOLECULAR BIOLOGY　350　(2)　349-362　2005/07

DOI： 10.1016/j.jmb.2005.04.074 　

ISSN： 0022-2836
Nuclear magnetic resonance characterization of the refolding intermediate of beta(2)-microglobulin trapped by non-native prolyl peptide bond Peer-reviewed

A Kameda, M Hoshino, T Higurashi, S Takahashi, H Naiki, Y Goto

JOURNAL OF MOLECULAR BIOLOGY　348　(2)　383-397　2005/04

DOI： 10.1016/j.jmb.2005.02.050 　

ISSN： 0022-2836
Specific collapse followed by slow hydrogen-bond formation of beta-sheet in the folding of single-chain monellin Peer-reviewed

T Kimura, T Uzawa, K Ishimori, Morishima, I, S Takahashi, T Konno, S Akiyama, T Fujisawa

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　102　(8)　2748-2753　2005/02

DOI： 10.1073/pnas.0407982102 　

ISSN： 0027-8424
NO reduction by nitric-oxide reductase from denitrifying bacterium Pseudomonas aeruginosa - Characterization of reaction intermediates that appear in the single turnover cycle Peer-reviewed

H Kumita, K Matsuura, T Hino, S Takahashi, H Hori, Y Fukumori, Morishima, I, Y Shiro

JOURNAL OF BIOLOGICAL CHEMISTRY　279　(53)　55247-55254　2004/12

DOI： 10.1074/jbc.M409996200 　

ISSN： 0021-9258
Steric effects of isoleucine 107 on heme reorientation reaction in human myoglobin Peer-reviewed

H Ishikawa, S Takahashi, K Ishimori, Morishima, I

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　324　(3)　1095-1100　2004/11

DOI： 10.1016/j.bbrc.2004.09.163 　

ISSN： 0006-291X
Direct observation of A beta amyloid fibril growth and inhibition Peer-reviewed

T Ban, M Hoshino, S Takahashi, D Hamada, K Hasegawa, H Naiki, Y Goto

JOURNAL OF MOLECULAR BIOLOGY　344　(3)　757-767　2004/11

DOI： 10.1016/j.jmb.2004.09.078 　

ISSN： 0022-2836
Structural and functional characterization of "laboratory evolved" cytochrome P450cam mutants showing enhanced naphthalene oxygenation activity Peer-reviewed

K Matsuura, T Tosha, S Yoshioka, S Takahashi, K Ishimori, Morishima, I

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　323　(4)　1209-1215　2004/10

DOI： 10.1016/j.bbrc.2004.08.221 　

ISSN： 0006-291X
Refolding processes of cytochrome P450(cam) from ferric and ferrous acid forms to the native conformation - Formations of folding intermediates with non-native heme coordination state Peer-reviewed

T Egawa, T Hishiki, Y Ichikawa, Y Kanamori, H Shimada, S Takahashi, T Kitagawa, Y Ishimura

JOURNAL OF BIOLOGICAL CHEMISTRY　279　(31)　32008-32017　2004/07

DOI： 10.1074/jbc.M310810200 　

ISSN： 0021-9258
Dioxygen reduction by bo-type quinol oxidase from Escherichia coli studied by submillisecond-resolved freeze-quench EPR spectroscopy Peer-reviewed

K Matsuura, S Yoshioka, S Takahashi, K Ishimori, T Mogi, H Hori, Morishima, I

BIOCHEMISTRY　43　(8)　2288-2296　2004/03

DOI： 10.1021/bi0355490 　

ISSN： 0006-2960
Identification of crucial histidines for heme binding in the N-terminal domain of the heme-regulated eIF2 alpha kinase Peer-reviewed

T Inuzuka, BG Yun, H Ishikawa, S Takahashi, H Hori, RL Matts, K Ishimori, Morishima, I

JOURNAL OF BIOLOGICAL CHEMISTRY　279　(8)　6778-6782　2004/02

DOI： 10.1074/jbc.C300464200 　

ISSN： 0021-9258
Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness Peer-reviewed

T Uzawa, S Akiyama, T Kimura, S Takahashi, K Ishimori, Morishima, I, T Fujisawa

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　101　(5)　1171-1176　2004/02

DOI： 10.1073/pnas.0305376101 　

ISSN： 0027-8424
In situ observation on the protein folding dynamics by small angle X-ray scattering Peer-reviewed

Satoshi Takahashi, Takanori Uzawa, Tetsuro Fujisawa

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme　49　(2)　135-140　2004/01

ISSN： 0039-9450
NMR study on the structural changes of cytochrome P450cam upon the complex formation with putidaredoxin - Functional significance of the putidaredoxin-induced structural changes Peer-reviewed

T Tosha, S Yoshioka, S Takahashi, K Ishimori, H Shimada, Morishima, I

JOURNAL OF BIOLOGICAL CHEMISTRY　278　(41)　39809-39821　2003/10

DOI： 10.1074/jbc.M304265200 　

ISSN： 0021-9258
Kinetic and spectroscopic characterization of a hydroperoxy compound in the reaction of native myoglobin with hydrogen peroxide Peer-reviewed

T Egawa, S Yoshioka, S Takahashi, H Hori, S Nagano, H Shimada, K Ishimori, Morishima, I, M Suematsu, Y Ishimura

JOURNAL OF BIOLOGICAL CHEMISTRY　278　(43)　41597-41606　2003/10

DOI： 10.1074/jbc.M210383200 　

ISSN： 0021-9258
The observation of the dioxygen activation of cytochrome bo from Escherichia coli by submillisecond-resolved freeze quench EPR spectroscopy Peer-reviewed

Koji Matsuura, Shiro Yoshioka, Satoshi Takahashi, Koichiro Ishimori, Tatsushi Mogi, Hiroshi Hori, Isao Morishima

Journal of Inorganic Biochemistry　96　(1)　188　2003/07
Publisher: Elsevier {BV}
DOI： 10.1016/s0162-0134(03)80705-5 　
Activation of hydrogen peroxide in horseradish peroxidase occurs within similar to 200 mu s observed by a new freeze-quench device Peer-reviewed

M Tanaka, K Matsuura, S Yoshioka, S Takahashi, K Ishimori, H Hori, Morishima, I

BIOPHYSICAL JOURNAL　84　(3)　1998-2004　2003/03

DOI： 10.1016/S0006-3495(03)75008-5 　

ISSN： 0006-3495
Activation of hydrogen peroxide in horseradish peroxidase occurs within approximately 200 micro s observed by a new freeze-quench device. International-journal Peer-reviewed

Motomasa Tanaka, Koji Matsuura, Shiro Yoshioka, Satoshi Takahashi, Koichiro Ishimori, Hiroshi Hori, Isao Morishima

Biophysical journal　84　(3)　1998-2004　2003/03

ISSN： 0006-3495

More details Close

To observe the formation process of compound I in horseradish peroxidase (HRP), we developed a new freeze-quench device with approximately 200 micro s of the mixing-to-freezing time interval and observed the reaction between HRP and hydrogen peroxide (H(2)O(2)). The developed device consists of a submillisecond solution mixer and rotating copper or silver plates cooled at 77 K; it freezes the small droplets of mixed solution on the surface of the rotating plates. The ultraviolet-visible spectra of the sample quenched at approximately 1 ms after the mixing of HRP and H(2)O(2) suggest the formation of compound I. The electron paramagnetic resonance spectra of the same reaction quenched at approximately 200 micro s show a convex peak at g = 2.00, which is identified as compound I due to its microwave power and temperature dependencies. The absence of ferric signals in the electron paramagnetic resonance spectra of the quenched sample indicates that compound I is formed within approximately 200 micro s after mixing HRP and H(2)O(2). We conclude that the activation of H(2)O(2) in HRP at ambient temperature completes within approximately 200 micro s. The developed device can be generally applied to investigate the electronic structures of short-lived intermediates of metalloenzymes.
Roles of the proximal hydrogen bonding network in cytochrome P450(cam)-catalyzed oxygenation Peer-reviewed

S Yoshioka, T Tosha, S Takahashi, K Ishimori, H Hori, Morishima, I

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY　124　(49)　14571-14579　2002/12

DOI： 10.1021/ja0265409 　

ISSN： 0002-7863
NO-induced activation mechanism of the heme-regulated eIF2 alpha kinase Peer-reviewed

H Ishikawa, BG Yun, S Takahashi, H Hori, RL Matts, K Ishimori, Morishima, I

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY　124　(46)　13696-13697　2002/11

DOI： 10.1021/ja0272336 　

ISSN： 0002-7863
Molecular mechanism of the electron transfer reaction in cytochrome P450(cam)-putidaredoxin: Roles of glutamine 360 at the heme proximal site Peer-reviewed

T Tosha, S Yoshioka, H Hori, S Takahashi, K Ishimori, Morishima, I

BIOCHEMISTRY　41　(47)　13883-13893　2002/11

DOI： 10.1021/bi0261037 　

ISSN： 0006-2960
Direct observation of the multistep helix formation of poly-L-glutamic acids Peer-reviewed

T Kimura, S Takahashi, S Akiyama, T Uzawa, K Ishimori, Morishima, I

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY　124　(39)　11596-11597　2002/10

DOI： 10.1021/ja026639f 　

ISSN： 0002-7863
Conformational landscape of cytochrome c folding studied by microsecond-resolved small-angle x-ray scattering Peer-reviewed

S Akiyama, S Takahashi, T Kimura, K Ishimori, Morishima, I, Y Nishikawa, T Fujisawa

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　99　(3)　1329-1334　2002/02

DOI： 10.1073/pnas.012458999 　

ISSN： 0027-8424
Pressure Effects on the Intramolecular Electron Transfer Reactions in Hemoproteins Peer-reviewed

Yoshiaki Furukawa, Yoichi Sugiyama, Satoshi Takahashi, Koichiro Ishimori, Isao Morishima

187　2002
Publisher: Springer Nature
DOI： 10.1007/978-3-662-04802-3_9 　
Time dependence of the catalytic intermediates in cytochrome c oxidase Peer-reviewed

S Han, S Takahashi, DL Rousseau

JOURNAL OF BIOLOGICAL CHEMISTRY　275　(3)　1910-1919　2000/01

DOI： 10.1074/jbc.275.3.1910 　

ISSN： 0021-9258
CD measurements on the early folding intermediate of cytochrome c using the fast flow mixer Peer-reviewed

S Takahashi, S Akiyama, K Ishimori, Morishima, I

OLD AND NEW VIEWS OF PROTEIN FOLDING　1194　75-84　1999

ISSN： 0531-5131
Detection of a tryptophan radical as an intermediate species in the reaction of horseradish peroxidase mutant (Phe-221 → Trp) and hydrogen peroxide Peer-reviewed

Morimoto A, Tanaka M, Takahashi S, Ishimori K, Hori H, Morishima I, Pond A.E, Dawson J.H

Chemtracts　12　(2)　87-95　1999

ISSN： 1431-9268
Sequence-specific changes in the metal site of ferric bleomycin induced by the binding of DNA Peer-reviewed

JW Sam, S Takahashi, Lippai, I, J Peisach, DL Rousseau

JOURNAL OF BIOLOGICAL CHEMISTRY　273　(26)　16090-16097　1998/06

DOI： 10.1074/jbc.273.26.16090 　

ISSN： 0021-9258
Heme oxygenase: A central enzyme of oxygen-dependent heme catabolism and carbon monoxide synthesis Peer-reviewed

M Ikeda-Saito, H Fujii, KM Matera, S Takahashi, CT Migita, DL Rousseau, T Yoshida

OXYGEN HOMEOSTASIS AND ITS DYNAMICS　1　304-314　1998
Heme degradation mechanism by heme oxygenase: Conversion of alpha-meso-hydroxyheme to verdoheme IX alpha Peer-reviewed

H Fujii, KM Matera, S Takahashi, CT Migita, H Zhou, T Yoshida, M Ikeda-Saito

OXYGEN HOMEOSTASIS AND ITS DYNAMICS　1　315-321　1998
Stereochemistry of the Fe(II)- and Fe(III)-cyanide complexes of the homodimeric Scapharca inaequivalvis hemoglobin. A resonance Raman and FTIR study Peer-reviewed

A Boffi, E Chiancone, S Takahashi, DL Rousseau

BIOCHEMISTRY　36　(15)　4505-4509　1997/04

DOI： 10.1021/bi9618880 　

ISSN： 0006-2960
Submillisecond protein folding kinetics studied by ultrarapid mixing Peer-reviewed

CK Chan, Y Hu, S Takahashi, DL Rousseau, WA Eaton, J Hofrichter

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　94　(5)　1779-1784　1997/03

DOI： 10.1073/pnas.94.5.1779 　

ISSN： 0027-8424
The unique heme-heme interactions of the homodimeric Scapharca inaequivalvis hemoglobin as probed in the protein reconstituted with unnatural 2,4 heme derivatives Peer-reviewed

C Zamparelli, D Verzili, A Boffi, E Chiancone, S Takahashi, DL Rousseau, R Regan, QH Gibson

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS　339　(2)　275-282　1997/03

DOI： 10.1006/abbi.1996.9829 　

ISSN： 0003-9861
Resonance Raman spectroscopic characterization of alpha-hydroxyheme and verdoheme complexes of heme oxygenase Peer-reviewed

S Takahashi, KM Matera, H Fujii, H Zhou, K Ishikawa, T Yoshida, M IkedaSaito, DL Rousseau

BIOCHEMISTRY　36　(6)　1402-1410　1997/02

DOI： 10.1021/bi962361q 　

ISSN： 0006-2960
Folding of cytochrome c initiated by submillisecond mixing Peer-reviewed

S Takahashi, Yeh, SR, TK Das, CK Chan, DS Gottfried, DL Rousseau

NATURE STRUCTURAL BIOLOGY　4　(1)　44-50　1997/01

DOI： 10.1038/nsb0197-44 　

ISSN： 1072-8368
Ligand exchange during cytochrome c folding Peer-reviewed

Yeh, SR, S Takahashi, BC Fan, DL Rousseau

NATURE STRUCTURAL BIOLOGY　4　(1)　51-56　1997/01

DOI： 10.1038/nsb0197-51 　

ISSN： 1072-8368
Oxygen and one reducing equivalent are both required for the conversion of alpha-hydroxyhemin to verdoheme in heme oxygenase Peer-reviewed

KM Matera, S Takahashi, H Fujii, H Zhou, K Ishikawa, T Yoshimura, DL Rousseau, T Yoshida, M IkedaSaito

JOURNAL OF BIOLOGICAL CHEMISTRY　271　(12)　6618-6624　1996/03

DOI： 10.1074/jbc.271.12.6618 　

ISSN： 0021-9258
REACTION OF CYTOCHROME BO3 WITH OXYGEN - EXTRA REDOX CENTER(S) ARE PRESENT IN THE PROTEIN Peer-reviewed

JL WANG, J RUMBLEY, YC CHING, S TAKAHASHI, RB GENNIS, DL ROUSSEAU

BIOCHEMISTRY　34　(47)　15504-15511　1995/11

DOI： 10.1021/bi00047a016 　

ISSN： 0006-2960

eISSN： 1520-4995
IDENTIFICATION OF THE OVERTONE OF THE FE-CO STRETCHING MODE IN HEME-PROTEINS - A PROBE OF THE HEME ACTIVE-SITE Peer-reviewed

JL WANG, S TAKAHASHI, DL ROUSSEAU

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　92　(20)　9402-9406　1995/09

DOI： 10.1073/pnas.92.20.9402 　

ISSN： 0027-8424
2 CONFORMATIONS OF THE CATALYTIC SITE IN THE AA(3)-TYPE CYTOCHROME-C-OXIDASE FROM RHODOBACTER-SPHAEROIDES Peer-reviewed

JL WANG, S TAKAHASHI, JP HOSLER, DM MITCHELL, S FERGUSONMILLER, RB GENNIS, DL ROUSSEAU

BIOCHEMISTRY　34　(31)　9819-9825　1995/08

DOI： 10.1021/bi00031a001 　

ISSN： 0006-2960
MICROSECOND GENERATION OF OXYGEN-BOUND CYTOCHROME-C-OXIDASE BY RAPID SOLUTION MIXING Peer-reviewed

S TAKAHASHI, YC CHING, JL WANG, DL ROUSSEAU

JOURNAL OF BIOLOGICAL CHEMISTRY　270　(15)　8405-8407　1995/04

DOI： 10.1074/jbc.270.15.8405 　

ISSN： 0021-9258
HEME OXYGENASE-2 - PROPERTIES OF THE HEME COMPLEX OF THE PURIFIED TRYPTIC FRAGMENT OF RECOMBINANT HUMAN HEME OXYGENASE-2 Peer-reviewed

K ISHIKAWA, N TAKEUCHI, S TAKAHASHI, KM MATERA, M SATO, S SHIBAHARA, DL ROUSSEAU, M IKEDASAITO, T YOSHIDA

JOURNAL OF BIOLOGICAL CHEMISTRY　270　(11)　6345-6350　1995/03

DOI： 10.1074/jbc.270.11.6345 　

ISSN： 0021-9258
STRUCTURAL CHARACTERIZATION OF OXIDIZED DIMERIC SCAPHARCA-INAEQUIVALVIS HEMOGLOBIN BY RESONANCE RAMAN-SPECTROSCOPY Peer-reviewed

A BOFFI, S TAKAHASHI, C SPAGNUOLO, DL ROUSSEAU, E CHIANCONE

JOURNAL OF BIOLOGICAL CHEMISTRY　269　(32)　20437-20440　1994/08

ISSN： 0021-9258
HEME-HEME OXYGENASE COMPLEX - STRUCTURE AND PROPERTIES OF THE CATALYTIC SITE FROM RESONANCE RAMAN-SCATTERING Peer-reviewed

S TAKAHASHI, JL WANG, DL ROUSSEAU, K ISHIKAWA, T YOSHIDA, N TAKEUCHI, M IKEDASAITO

BIOCHEMISTRY　33　(18)　5531-5538　1994/05

DOI： 10.1021/bi00184a023 　

ISSN： 0006-2960

eISSN： 1520-4995
HEME-HEME OXYGENASE COMPLEX STRUCTURE OF THE CATALYTIC SITE AND ITS IMPLICATION FOR OXYGEN ACTIVATION Peer-reviewed

S TAKAHASHI, JL WANG, DL ROUSSEAU, K ISHIKAWA, T YOSHIDA, HOST, JR, M IKEDASAITO

JOURNAL OF BIOLOGICAL CHEMISTRY　269　(2)　1010-1014　1994/01

ISSN： 0021-9258
TIME-RESOLVED RESONANCE RAMAN ELUCIDATION OF THE PATHWAY FOR DIOXYGEN REDUCTION BY CYTOCHROME-C-OXIDASE Peer-reviewed

T OGURA, S TAKAHASHI, S HIROTA, K SHINZAWAITOH, S YOSHIKAWA, EH APPELMAN, T KITAGAWA

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY　115　(19)　8527-8536　1993/09

DOI： 10.1021/ja00072a002 　

ISSN： 0002-7863

eISSN： 1520-5126
THE MOLECULAR-FEATURES AND CATALYTIC ACTIVITY OF CU(A)-CONTAINING ACO3-TYPE CYTOCHROME-C-OXIDASE FROM A FACULTATIVE ALKALOPHILIC BACILLUS Peer-reviewed

YUMOTO, I, S TAKAHASHI, T KITAGAWA, Y FUKUMORI, T YAMANAKA

JOURNAL OF BIOCHEMISTRY　114　(1)　88-95　1993/07

DOI： 10.1093/oxfordjournals.jbchem.a124145 　

ISSN： 0021-924X
RESONANCE RAMAN STUDIES ON THE CU(A) SITE OF CYTOCHROME-C-OXIDASE USING A MULTICHANNEL SCANNING RAMAN SPECTROMETER WITH A CCD DETECTOR Peer-reviewed

S TAKAHASHI, T OGURA, K SHINZAWAITOH, S YOSHIKAWA, T KITAGAWA

BIOCHEMISTRY　32　(14)　3664-3670　1993/04

DOI： 10.1021/bi00065a020 　

ISSN： 0006-2960

eISSN： 1520-4995
OBSERVATION OF THE FE-4+=O STRETCHING RAMAN BAND FOR CYTOCHROME-OXIDASE COMPOUND-B AT AMBIENT-TEMPERATURE Peer-reviewed

T OGURA, S TAKAHASHI, K SHINZAWAITOH, S YOSHIKAWA, T KITAGAWA

JOURNAL OF BIOLOGICAL CHEMISTRY　265　(25)　14721-14723　1990/09

ISSN： 0021-9258

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Misc. 107

一分子蛍光顕微鏡によるp53変異体の標的配列探索ダイナミクスの観察

伊藤優志, 村田崇人, 坂本清志, 七谷圭, 和田健彦, 高橋聡, 鎌形清人

日本化学会講演予稿集　95th　(3)　869　2015/03/11

ISSN： 0285-7626
Development of the Line Confocal System for the Single Molecule Tracking of Fast Folding Dynamics of Proteins

Hiroyuki Oikawa, Kiyoto Kamagata, Munehito Arai, Atsuhito Fukasawa, Hiroaki Yokota, Toru Ide, Satoshi Takahashi

BIOPHYSICAL JOURNAL　108　(2)　50A-51A　2015/01

DOI： 10.1016/j.bpj.2014.11.309 　

ISSN： 0006-3495

eISSN： 1542-0086
マイクロ秒分解一分子蛍光測定で観るタンパク質の構造変化 Invited Peer-reviewed

小井川浩之, 齊藤雅嵩, 高橋聡

生物物理　54　(5)　276-279　2014/09

DOI： 10.2142/biophys.54.276 　
Practical Techniques : Single-Molecule Fluorescence Tracking at High Time Resolution by Line-Confocal Microscopy Invited Peer-reviewed

Hiroyuki Oikawa, Satoshi Takahashi

Journal of the spectroscopical research of Japan　62　(5)　232-234　2013/10/15
Publisher: 日本分光学会
ISSN： 0038-7002
2P289 Development of hydrodynamic focusing system for single molecule sorting device(26. Measurements,Poster)

Kubota Toshihiko, Oikawa Hiroyuki, Kamagata Kiyoto, Takahashi Satoshi

Seibutsu Butsuri　53　(1)　S206　2013
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.53.S206_6 　
2P061 Dynamics of ubiquitin folding detected by single molecule fluorescence spectroscopy(01C. Protein: Property)

Saito Masataka, Chen Hsin-Liang, Chen Rita, Kamagata Kiyoto, Oikawa Hiroyuki, Takahashi Satoshi

Seibutsu Butsuri　53　(1)　S169　2013
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.53.S169_1 　
2P062 Improvements of the line confocal system for the single molecule tracking of fast folding dynamics of the B domain of protein A(01C. Protein: Property)

Oikawa Hiroyuki, Kamagata Kiyoto, Arai Munehito, Takahashi Satoshi

Seibutsu Butsuri　53　(1)　S169　2013
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.53.S169_2 　
2P026 Direct observation of the multiple sliding modes of a tumor suppressor p53(01B. Protein: Structure & Function,Poster)

Murata Agato, Kashima Risa, Itoh Yuji, Tokino Takashi, Takahashi Satoshi, Kamagata Kiyoto

Seibutsu Butsuri　53　(1)　S163　2013
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.53.S163_2 　
2E1412 Single-molecule structural analysis of tumor suppressor p53 using scanning probe microscopes(Proteins:Property II,Oral Presentation,The 50th Annual Meeting of the Biophysical Society of Japan)

Takahashi Seiya, Sainoo Yasuyuki, Kashima Risa, Tokino Takashi, Komeda Tadahiro, Takahashi Satoshi, Kamagata Kiyoto

Seibutsu Butsuri　52　S45-S46　2012
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.52.S45_5 　
1PT222 Construction of a microfluidic system for fluorescence-based single-molecule sorting(The 50th Annual Meeting of the Biophysical Society of Japan)

Saitoh Sho, Oikawa Hiroyuki, Kamagata Kiyoto, Takahashi Satoshi

Seibutsu Butsuri　52　S106　2012
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.52.S106_2 　
1PT152 Microsecond-resolved traces of protein folding by single-molecule FRET measurements(The 50th Annual Meeting of the Biophysical Society of Japan)

Oikawa Hiroyuki, Suzuki Yuta, Kamagata Kiyoto, Arai Munehito, Takahashi Satoshi

Seibutsu Butsuri　52　S94　2012
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.52.S94_5 　
長時間一分子蛍光測定による緑膿菌由来シトクロムcのフォールディング機構の研究

川口敏史, 鎌形清人, 小井川浩之, 三本木至宏, 馬場昭典, 小松崎民樹, 高橋聡

日本化学会講演予稿集　92nd　(3)　2012

ISSN： 0285-7626
新規一分子蛍光観測法による蛋白質の折り畳みの自由エネルギー地形の探索

鎌形清人, 川口敏史, 馬場昭典, 三本木至宏, 小松崎民樹, 高橋聡

日本蛋白質科学会年会プログラム・要旨集　12th　2012
一分子蛍光観察法を用いたタンパク質フォールディングの解明

小井川浩之, 鎌形清人, 高橋聡

高分子　60　(11)　801-803　2011/11
Publisher: 高分子学会
ISSN： 0454-1138
地域と世界に貢献する東北大学多元物質科学研究所タンパク質ダイナミクスからみた生命現象

高橋聡, 鎌形清人

Mater Integr　24　(4/5)　33-37　2011/06/01

ISSN： 1344-7858
Flow and Stop Protocol for the Long-Time Observation of Fluorescence from Free Single Molecules: Application to the Time-Series Analysis of Protein Folding

Kiyoto Kamagata, Akinori Baba, Tamiki Komatsuzaki, Satoshi Takahashi

BIOPHYSICAL JOURNAL　100　(3)　212-213　2011/02

ISSN： 0006-3495
3H1322 P25 Microsecond-resolved time traces of protein conformational change by single-molcule fluorescence detection(3H Protein: Property 5,The 49th Annual Meeting of the Biophysical Society of Japan)

Oikawa Hiroyuki, Kamagata Kiyoto, Iijima Issei, Hohsaka Takahiro, Sambongi Yoshihiro, Takahashi Satoshi

Seibutsu Butsuri　51　S134　2011
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.51.S134_4 　
3H1124 Imaging and tracking of single BLG molecules in solution(3H Protein: Property 4,The 49th Annual Meeting of the Biophysical Society of Japan)

Ando Shion, Oikawa Hiroyuki, Kamagata Kiyoto, Sakurai Kazumasa, Goto Yuji, Takahashi Satoshi

Seibutsu Butsuri　51　S134　2011
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.51.S134_3 　
2SG-02 Submillisecond dynamics of protein folding and ligand binding detected by the line conforcal microscopy(2SG New approaches for probing biomolecular fluctuations,The 49th Annual Meeting of the Biophysical Society of Japan)

Oikawa Hiroyuki, Kamagata Kiyoto, Iijima Issei, Hohsaka Takahiro, Sambongi Yoshihiro, Takahashi Satoshi

Seibutsu Butsuri　51　S19　2011
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.51.S19_1 　
See how they run : exploring the energy landscape of protein folding using time-series detection of fluorescence of free single molecules

(2011)　25-28　2011
Publisher: [日本分光学会]
新規-分子蛍光観測法による蛋白質の自由エネルギー地形の解析

鎌形清人, 川口敏史, 馬場昭典, 三本木至宏, 小松崎民樹, 高橋聡

日本蛋白質科学会年会プログラム・要旨集　11th　2011
3I1434 Additives inhibiting the bleaching of fluorophores for the long-time fluorescence observation of single proteins(3I Protein: Measurement & Analysis 2,The 49th Annual Meeting of the Biophysical Society of Japan)

Itabashi Hironao, Mano Eriko, Oikawa Hiroyuki, Araki Yasuyuki, Kamagata Kiyoto, Wada Takehiko, Takahashi Satoshi

Seibutsu Butsuri　51　(0)　S139　2011
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.51.S139_1 　

ISSN： 0582-4052
新しいタンパク質科学の可能性-新時代を切り拓くマルチタレントDavid Baker-

鎌形清人, 高橋聡

化学　2011/01
2P081 Single molecule observation of the ligand binding of maltose binding protein doubly labeled by cell free system(The 48th Annual Meeting of the Biophysical Society of Japan)

Yada Tetsuya, Yamamori Akihiro, Iijima Issei, Hohsaka Takahiro, Oikawa Hiroyuki, Kamagata Kiyoto, Takahashi Satoshi

Seibutsu Butsuri　50　(2)　S96　2010
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.50.S96_3 　
3P051 Hydration study of trifluoroacetic acid-induced unfolding of horse heart cytochrome c by dielectric relaxation spectroscopy(Protein: Property,The 48th Annual Meeting of the Biophysical Society of Japan)

Miyashita Yusuke, Wazawa Tetsuichi, Kamagata Kiyoto, Sambongi Yoshihiro, Takahashi Satoshi, Suzuki Makoto

Seibutsu Butsuri　50　(2)　S153-S154　2010
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.50.S153_7 　
3P064 Denatured state dynamics of cytochrome c551 observed by single-molecule measurement with improved time resolution(Protein: Property,The 48th Annual Meeting of the Biophysical Society of Japan)

Kawaguchi Toshifumi, Kamagata Kiyoto, Oikawa Hiroyuki, Sambongi Yoshihiro, Takahashi Satoshi

Seibutsu Butsuri　50　(2)　S156　2010
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.50.S156_2 　
3P062 Microsecond-resolved time traces of protein folding by single-molecule fluorescence detection(Protein: Property,The 48th Annual Meeting of the Biophysical Society of Japan)

Oikawa Hiroyuki, Kamagata Kiyoto, Takahashi Satoshi

Seibutsu Butsuri　50　(2)　S155-S156　2010
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.50.S155_5 　
3P074 Single-molecule observation of the translation diffusion and fluorescence intensity of β-LG(Protein: Property,The 48th Annual Meeting of the Biophysical Society of Japan)

Ando Shion, Kamagata Kiyoto, Oikawa Hiroyuki, Sakurai Kazumasa, Goto Yuji, Takahashi Satoshi

Seibutsu Butsuri　50　(2)　S157-S158　2010
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.50.S157_6 　
2SD0905 Flow and stop protocol for the long-time observation of fluorescence from free single molecules : application to protein folding(2SD Bridging Single Molecule Biophysics and System Biology:New Experimental and Theoretical Challenges,The 48th Annual Meeting of the Biophysical Society of Japan)

Kamagata Kiyoto, Baba Akinori, Komatsuzaki Tamiki, Takahashi Satoshi

Seibutsu Butsuri　50　(2)　S12　2010
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.50.S12_1 　
New Insights into the Mechanism of Protein Folding by Single Molecule Detection Invited Peer-reviewed

KAMAGATA Kiyoto, KINOSHITA Masahito, TAKAHASHI Satoshi

Seibutsu Butsuri　49　(6)　282-286　2009/12
Publisher: The Biophysical Society of Japan
DOI： 10.2142/biophys.49.282 　

ISSN： 0582-4052 1347-4219

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To understand the molecular mechanism of protein folding, it is necessary to reveal the dynamics of single proteins from the unfolded state to the folding transition state. We therefore developed a new system that can detect fluorescence signals from free single proteins for extended time periods. The system enabled us to characterize a slow conformational dynamics for the unfolded state of cytochrome c. Furthermore, we proposed a method to estimate the equilibration time for the intermediate state of protein folding.
Protein, Solution Mixing and Microfabrication : Observation of Protein Folding Dynamics by Rapid Mixing Device

TAKAHASHI Satoshi

rle　37　(7)　515-520　2009/07/15
Publisher: The Laser Society of Japan
DOI： 10.2184/lsj.37.515 　

ISSN： 0387-0200

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device by femtosecond pulsed laser ablation technique. To detect elementary processes involved in
Protein Folding Dynamics by Time-Resolved SAXS and Single Molecule Fluorescence Spectroscopy

TAKAHASHI Satoshi

Journal of the Crystallographic Society of Japan　51　(1)　52-54　2009/02/28
Publisher: 日本結晶学会
ISSN： 0369-4585
Development of single-molecule detection system for the investigation of protein folding

Bio industry　26　(2)　7-14　2009/02
Publisher: シーエムシー出版
ISSN： 0910-6545
1YP1-02 Long-time observation of a single molecule trapped in a capillary cell : application for protein folding(1YP1 Early Research in Biophysics Award Candidate Presentations,The 47th Annual Meeting of the Biophysical Society of Japan)

Kamagata Kiyoto, Goto Yuji, Takahashi Satoshi

Seibutsu Butsuri　49　S1　2009
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.49.S1_2 　
3P-042 Microsecond-resolved single-molecule time traces of protein folding by a line-illuminated confocal microscopy(Protein:Property,The 47th Annual Meeting of the Biophysical Society of Japan)

Oikawa Hiroyuki, Kamagata Kiyoto, Goto Yuji, Takahashi Satoshi

Seibutsu Butsuri　49　S158　2009
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.49.S158_1 　
1P-057 Long-time observation of a single molecule trapped in a capillary cell : application for protein folding(Protein:Property, The 47th Annual Meeting of the Biophysical Society of Japan)

Kamagata Kiyoto, Goto Yuji, Takahashi Satoshi

Seibutsu Butsuri　49　S72　2009
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.49.S72_2 　
1P-064 Single molecule observation of the ligand binding and folding dynamics of maltose binding protein doubly labeled by a cell free system(Protein:Function, The 47th Annual Meeting of the Biophysical Society of Japan)

Yamamori Akihiro, Kamagata Kiyoto, Iijima Issei, Hohsaka Takahiro, Goto Yuji, Takahashi Satoshi

Seibutsu Butsuri　49　S73　2009
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.49.S73_1 　
2P-050 Single molecule observation of the folding of bovine β-lactoglobulin(Protein:Property,The 47th Annual Meeting of the Biophysical Society of Japan)

Kadota Takayuki, Kamagata Kiyoto, Oikawa Hiroyuki, Sakurai Kazumasa, Goto Yuji, Takahashi Satoshi

Seibutsu Butsuri　49　S114　2009
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.49.S114_4 　
「一分子測定法による蛋白質の折り畳み研究の展開」

鎌形清人, 木下雅仁, 高橋聡

生物物理　2009
22pWB-7 Protein folding dynamics detected by flow detection of single molecules

Takahashi Satoshi

Meeting abstracts of the Physical Society of Japan　63　(2)　331-331　2008/08/25
Publisher: The Physical Society of Japan (JPS)
ISSN： 1342-8349
3P-022 Denatured state dynamics of Cytochrome c551 observed by single molecule fluorescence intensity measurements(The 46th Annual Meeting of the Biophysical Society of Japan)

Fujimoto Kazuya, Kinoshita Masahito, Kamagata Kiyoto, Goto Yuji, Sambongi Yoshihiro, Baba Akinori, Komatsuzaki Tamiki, Takahashi Satoshi

Seibutsu Butsuri　48　S131　2008
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.48.S131_2 　
2P-098 Direct observation of single-molecule trajectories of protein folding using a new sheath flow cell(The 46th Annual Meeting of the Biophysical Society of Japan)

Kamagata Kiyoto, Kinoshita Masahito, Goto Yuji, Takahashi Satoshi

Seibutsu Butsuri　48　S90　2008
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.48.S90_3 　
蛋白質の折り畳み運動を一分子レベルで観測するための新手法の開発

木下雅仁, 鎌形清人, 鎌形清人, 前田晃央, 後藤祐児, 後藤祐児, 小松崎民樹, 高橋聡, 高橋聡

日本化学会講演予稿集　88th　(2)　2008

ISSN： 0285-7626
一分子測定法を用いた緑膿菌由来シトクロムcの折り畳み運動の解明

藤本和也, 木下雅仁, 鎌形清人, 鎌形清人, 後藤祐児, 三本木至宏, 三本木至宏, 馬場昭典, 馬場昭典, 小松崎民樹, 小松崎民樹, 高橋聡, 高橋聡

生体分子科学討論会講演要旨集　35th　2008
蛋白質の折り畳み運動を観測するための一分子測定手法の開発(Development of a technique for the investigation of folding dynamics of single proteins for extended time periods)

木下雅仁, 鎌形清人, 前田晃央, 後藤祐児, 小松崎民樹, 高橋聡

生物物理　47　(Suppl.1)　S139-S139　2007/11
Publisher: (一社)日本生物物理学会
ISSN： 0582-4052

eISSN： 1347-4219
Protein folding dynamics detected by time-resolved synchrotron X-ray small-angle scattering technique

Tetsuro Fujisawa, Satoshi Takahashi

AIP Conference Proceedings　902　89-92　2007/05

DOI： 10.1063/1.2723630 　

ISSN： 0094-243X

eISSN： 1551-7616
2P100 Observation of the initial collapse in apomyoglobin folding by ultra-rapid mixing(Proteins-stability, folding, and other physicochemical properties,Poster Presentations)

Tatsumi Tetsuma, Kinoshita Masahito, Goto Yuji, Takahashi Satoshi

Seibutsu Butsuri　47　S138　2007
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.47.S138_1 　
2P094 Direct observation of single-molecule trajectories of protein folding under non-equilibrium condition(Proteins-stability, folding, and other physicochemical properties,Oral Presentations)

Kamagata Kiyoto, Kinoshita Masahito, Goto Yuji, Takahashi Satoshi

Seibutsu Butsuri　47　S136　2007
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.47.S136_3 　
2P078 Time-resolved small angle x-ray scattering study on the burst phase intermediate in the folding of bamase(Proteins-stability, folding, and other physicochemical properties,Poster Presentations)

Konuma Tsuyoshi, Kimura Tetsunari, Matsumoto Shuzo, Goto Yuji, Fujisawa Tetsuro, Takahashi Satoshi

Seibutsu Butsuri　47　S132　2007
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.47.S132_3 　
2P104 Development of a technique for the investigation of folding dynamics of single proteins for extended time periods(Proteins-stability, folding, and other physicochemical properties,Poster Presentations)

Kinoshita Masahito, Kamagata Kiyoto, Maeda Akio, Goto Yuji, Komatsuzaki Tamiki, Takahashi Satoshi

Seibutsu Butsuri　47　(0)　S139　2007
Publisher: 一般社団法人日本生物物理学会
DOI： 10.2142/biophys.47.S139_1 　

ISSN： 0582-4052
サブミリ秒H/D交換法とNMRによって観測されたアポミオグロビンの折り畳み中間体の構造ゆらぎ

鵜澤尊規, 西村千秋, 秋山修志, 石森浩一郎, 高橋聡, DYSON H. Jane, WRIGHT Peter E

Abstracts. Annual Meeting of the NMR Society of Japan　45th　38-39　2006/11/20
S3f1-4 Generality of Initial Collapse Demonstrated by Scaling Relationship for Submillisecond Intermediates of Protein Folding(S3-f1: "Hydration Effects on Structure and Thermodynamics of Proteins,Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

Uzawa Takanori, Kimura Tetsunari, Ishimori Koichiro, Morishima Isao, Matsui Toshitaka, Ikeda-Saito Masao, Takahashi Satoshi, Akiyama Shuji, Fujisawa Tetsuro

Seibutsu Butsuri　46　(2)　S139　2006
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.46.S139_1 　
1P112 Multiple time scales in the folding dynamics of single chain monellin revealed by single molecule measurements(3. Protein folding and misfolding (1),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Maeda Akio, Kinoshita Masahito, Kamagata Kiyoto, Konno Takashi, Goto Yuji, Takahashi Satoshi

Seibutsu Butsuri　46　(2)　S174　2006
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.46.S174_4 　
1P108 Development of sheath-flow system to observe protein folding events at a single-molecule level(3. Protein folding and misfolding (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Kamagata Kiyoto, Kinoshita Masahito, Goto Yuji, Takahashi Satoshi

Seibutsu Butsuri　46　(2)　S173　2006
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.46.S173_4 　
蛋白質の折り畳みダイナミクス : バルク観察からの知見と一分子観測の可能性(複雑な多谷ポテンシャルエネルギー面上で生起する動力学諸問題-タンパク質とその周辺-,研究会報告)

高橋聡, 木下雅仁

物性研究　86　(1)　52-55　2006
Publisher: 物性研究刊行会
ISSN： 0525-2997
2P089 Time-resolved fluorescence and resonance raman measurements of the initial collapse in cytochrome c folding

Matsumoto S., Yane A., Goto Y., Hashida M., Fujita M., Nakashima S., Takahashi S.

Seibutsu Butsuri　45　S142　2005
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.45.S142_1 　
2P085 Development of the fluorescence detection device to observe single molecule dynamics of protein folding for extended periods

Maeda A., Kinoshita M., Konno T., Goto Y., Takahashi S.

Seibutsu Butsuri　45　S141　2005
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.45.S141_1 　
1P087 Iso-1-Cytochrome c Folding Studied by a New Method of Single Molecule Detection

Kinoshita M., Goto Y., Takahashi S.

Seibutsu Butsuri　45　S53　2005
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.45.S53_3 　
3P065 Helix formation in apomyoglobin folding revealed by submillisecond H/D exchange & NMR

Uzawa T, Nishimura C, Akiyama S, Ishimori K, Takahashi S, Dyson Jane, Wright Peter

Seibutsu Butsuri　45　(0)　S220　2005
Publisher: 一般社団法人日本生物物理学会
DOI： 10.2142/biophys.45.S220_1 　

ISSN： 0582-4052
タンパク質フォールディングの昼と夜

後藤祐児, 高橋聡

現代化学　(402)　26-33　2004/09
Publisher: 東京化学同人
ISSN： 0386-961X
3P019 Development of the rapid mixing apparatus by laser processing and elucidation of the initial collapse in the cytochrome c folding

Matsumoto S., Yane A., Goto Y., Takahashi S., Hashida M., Fujita M., Nakashima S.

Seibutsu Butsuri　44　S194　2004
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.44.S194_3 　
3P022 Helix environment in the early folding intermediate of apomyoglobin detected by time-resolved IR spectroscopy

Nishiguchi S., Goto Y., Takahashi S.

Seibutsu Butsuri　44　S195　2004
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.44.S195_2 　
3P023 Significance of oligomerization and rapid collapse in hemeoxygenase folding

Uzawa T., Kimura T., Takahashi S., Ishimori K., Akiyama S., Fujisawa T., Matsui T., Saito M.

Seibutsu Butsuri　44　S195　2004
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.44.S195_3 　
3P020 Iso-1-Cytochrome c Folding Studied by a New Method of Single Molecule Detection

Kinoshita M., Goto Y., Takahashi S.

Seibutsu Butsuri　44　S194　2004
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.44.S194_4 　
高橋聡・鵜澤尊規・秋山修志・藤澤哲郎「サブミリ秒分割X線小角散乱法の開発と蛋白質の折り畳み過程の研究」

放射光（日本放射光学会誌）　17,/,　2004

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2004 予定
高橋聡「赤外吸収およびラマン散乱分光法とそれらの利用」

生物工学ハンドブック　4章2-8節　2004
高橋聡・鵜澤尊規・藤澤哲郎「X線小角散乱を使った蛋白質の折りたたみ運動のリアルタイム観察」

高橋聡, 鵜澤尊規, 藤澤哲郎

蛋白質核酸酵素　49, 135-140/,　(2)　135-140　2004
Publisher: 共立出版
ISSN： 0039-9450
Direct observation of the multistep helix formation of poly-L-glutamic acids using microsecond-resolved FTIR and CD spectroscopies.

T Kimura, S Takahashi, S Akiyama, T Uzawa, K Ishimori, Morishima, I

BIOPHYSICAL JOURNAL　84　(2)　162A-162A　2003/02

ISSN： 0006-3495
Regulation Mechanism of a Novel Myoglobin with a Metal Binding Loop

Taniguchi S, Ihara M, Takahashi S, K. Koichiro, Morishima I

Seibutsu Butsuri　43　(0)　S77　2003
Publisher: 一般社団法人日本生物物理学会
DOI： 10.2142/biophys.43.S77_3 　

ISSN： 0582-4052
Characterization of hydrophobic cluster in early step of cytochrome c folding

Yane A., takahashi S., Ishimori K., Morishima I.

Seibutsu Butsuri　43　S59　2003
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.43.S59_2 　
Main-chain collapse and subsequent tertiary structure formation during apomyoglobin folding

Uzawa T., Kimura T., Takahashi S., Ishimori K., Morishima I., Akiyama S., Fujisawa T.

Seibutsu Butsuri　43　S60　2003
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.43.S60_2 　
Roles of the N-terminal fluctuating domain in folding of coiled coil GCN4-pl'

Kinoshita M., Kimura T., Takahashi S., Ishimori K., Morishima I.

Seibutsu Butsuri　43　S61　2003
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.43.S61_3 　
Folding Mechanism of Single-chain Monellin Based on the Secondary Structure Formation and the Packing of the Hydrophobic Residues

Kimura T., Takahashi S., Konno T., Ishimori K., Morishima I.

Seibutsu Butsuri　43　S59　2003
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.43.S59_3 　
高橋聡「蛋白質の折り畳み研究の展開」

生化学（日本生化学会誌）　75, 54-59/,　2003
「顕微赤外分光法と高速混合装置を使った蛋白質の折り畳み過程の研究」

高橋聡, 木村哲就

「顕微赤外分光法」アイピーシー社（編集西岡利勝・寺前紀夫）　第4章2節(211-224ページ)　2003
Recent progress in protein folding research Peer-reviewed

S Takahashi

SEIKAGAKU　75　(1)　54-59　2003/01

ISSN： 0037-1017
1L1700 Design of Horseradish Peroxidases with the Enhanced Catalase Activity and their Mechanism of Enhancing the Catalase Activity

Hirose S., Tosha T., Shimada H., Takahashi S., Ishimori K., Morishima I.

Seibutsu Butsuri　42　(2)　S72　2002
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.42.S72_4 　
Molecular mechanism of nitric oxide reduction catalyzed by fungal nitric oxide reductase Peer-reviewed

E Obayashi, H Shimizu, S Park, H Nakamura, S Takahashi, H Shoun, Y Shiro

OXYGEN AND LIFE: OXYGENASES, OXIDASE AND LIPID MEDIATORS　1233　(C)　59-62　2002

DOI： 10.1016/S0531-5131(02)00508-3 　

ISSN： 0531-5131
1K0945 Electronic states of the redox centers in dioxygen reduction of cytochrome bo from Escherichia coli by rapid-freezing EPR Method

Matsuura K., Yoshioka S., Takahashi S., Ishimori K., Morishima I., Mogi T., Hori H.

Seibutsu Butsuri　42　(2)　S60　2002
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.42.S60_3 　
3M1030 Observation on the folding process of single-chain monellin studied by microsecond-resolved FTIR spectroscopy

Kimura T., Takahashi S., Konno T., Ishimori K., Morishima I.

Seibutsu Butsuri　42　(2)　S190　2002
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.42.S190_1 　
蛋白質の折りたたみ運動をとらえる (新世紀における蛋白質科学の進展) -- (第2部蛋白質の構造・物性・進化)

高橋聡, 秋山修志

蛋白質核酸酵素　46　(11)　1545-1552　2001/08
Publisher: 共立出版
ISSN： 0039-9450
Apomyoglobin folding studied by submillisecond small-angle X-ray scattering

Uzawa T, Akiyama S, Kimura T, Takahashi S, Ishimori K, Morishima I, Nishikawa Y, Fujisawa T

Seibutsu Butsuri　41　S166　2001
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.41.S166_1 　
Factors Regulating the Radical Locations in Compound I of Peroxidase

Hirose S., Shintaku M., Yoshioka S., Takahashi S., Ishimori K., Morishima I., Hori H.

Seibutsu Butsuri　41　S105　2001
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.41.S105_4 　
高橋聡・秋山修志「たんぱく質の折りたたみ運動をとらえる」

蛋白質核酸酵素増刊：「新世紀における蛋白質科学の進展」　46, 1514-1552/,　2001
The study on the burst phase of ribonuclease A folding using a rapid mixing device

Kimura T., Akiyama S., Takahashi S., Harada Y., Ishimori K., Morishima I.

Seibutsu Butsuri　40　S161　2000
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.40.S161_1 　

ISSN： 0582-4052
Study on control mechanism of the oxgen affinity in the isolated betachain of hemoglobin by using Leucine 28 mutants

Konishi K., Ishikawa S., Takahashi S., Ishimori K., Morishima I.

Seibutsu Butsuri　40　S45　2000
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.40.S45_3 　

ISSN： 0582-4052
Studies for the design of artificial proteins by the module substitution between heterogenous proteins

Ihara M., Takahashi S., Ishimori K., Morisima I.

Seibutsu Butsuri　40　S187　2000
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.40.S187_3 　

ISSN： 0582-4052
Design of a New Myoglobin by Introducing a Metal Binding Loop

Nishiyama A., Ihara M., Takahashi S., Ishimori K., Morishima I.

Seibutsu Butsuri　40　S187　2000
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.40.S187_1 　

ISSN： 0582-4052
CD Measurements on the Early Folding Intermediates of Cytochrome c Using Fast Flow Mixer

Akiyama S., Takahashi S., Ishimori K., Morishima I.

Seibutsu Butsuri　39　S149　1999
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.39.S149_2 　

ISSN： 0582-4052
2PA087 ヘモグロビンβ鎖の疎水性残基Leu28による配位子結合反応の制御に関する研究

小西一誠, 石川春人, 近藤詩乃, 高橋聡, 石森浩一郎, 森島績

生物物理　39　S122　1999
Publisher: 一般社団法人日本生物物理学会
DOI： 10.2142/biophys.39.S122_3 　

ISSN： 0582-4052
2PA094 転写調節因子CooAの機能発現に関与するヘム軸配位子の研究

山本克彦, 石川春人, 高橋聡, 石森浩一郎, 森島績, 中島洋, 青野重利

生物物理　39　S124　1999
Publisher: 一般社団法人日本生物物理学会
DOI： 10.2142/biophys.39.S124_2 　

ISSN： 0582-4052
Dynamics of Protein Folding Studied Using the Fast Flow Mixer

Takahashi Satoshi, Akiyama Shuji, Ishimori Koichiro, Morishima Isao

Seibutsu Butsuri　39　S13　1999
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.39.S13_2 　

ISSN： 0582-4052
Recent Progress in the Experimental Studies of Protein Folding Dynamics

TAKAHASHI Satoshi

Seibutsu Butsuri　39　(2)　70-75　1999
Publisher: The Biophysical Society of Japan General Incorporated Association
DOI： 10.2142/biophys.39.70 　

ISSN： 0582-4052 1347-4219

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The experimental studies of protein folding dynamics have made remarkable advances lately. This is attributed to the development of new experimental methods that enable to detect submillisecond dynamics of protein folding as well as to the characterization of folding transition states of small single domain proteins. The characterization of folding intermediates, the elucidation of the processes involved before the formation of folding nucleus and the experimental confirmation of the energy landscape theory are several of important problems that are currently discussed.
高橋聡「コンピュータが畳むたんぱく質」

化学　54, 66-67/,　1999
Structure of the folding intermediate of apomyolobin studied by fluorescence quenching.

Tanioka H., Akiyama S., Inaba K., Takahashi S., Ishimori K., Morishima I.

Biophysics　38　(2)　S161　1998/09/07
Publisher: The Biophysical Society of Japan General Incorporated Association
ISSN： 0582-4052
高速液体混合装置を使った生体反応研究の新展開

化学と工業　51　(2)　155　1998
Developments in the Study of Biological Reaction Mechanisms Using Rapid Solution Mixing Devices

TAKAHASHI Satoshi

Chemistry and Chemical Industry　51　(2)　155-162　1998
Publisher: 日本化学会
ISSN： 0022-7684
Heme environmental structure and CO binding reaction of CO-dependent transcriptional regulator under presence and absence of DNA

UCHIDA T., ISHIKAWA H., TAKAHASHI S., ISHIMORI K., MORISHIMA I., OHKUBO K., NAKAJIMA H., SHIMONO T., AONO S.

Biophysics　37　S175　1997/10
Publisher: The Biophysical Society of Japan General Incorporated Association
ISSN： 0582-4052
The structure and function of Cytochrome b_5 mutant H39L

IHARA M., TAKAHASI S., ISIMORI K., MORISHIMA I.

Biophysics　37　S181　1997/10
Publisher: The Biophysical Society of Japan General Incorporated Association
ISSN： 0582-4052
The evolution of allostericity in vertevrate Hemoglobin

KATAOKA H., TAKAHASI S., ISIMORI K., MORISIMA I.

Biophysics　37　S177　1997/10
Publisher: The Biophysical Society of Japan General Incorporated Association
ISSN： 0582-4052
Characterization of iron coordination structure of nitric oxide reductase, cytochrome P450nor

E Obayashi, Y Shiro, S Takahashi, T Iizuka, H Shoun

FASEB JOURNAL　11　(9)　A807-A807　1997/07

ISSN： 0892-6638
Cytochrome c folding initiated by submillisecond mixing and probed by resonance Raman scattering

Yeh, SR, S Takahashi, TK Das, BC Fan, DS Gottfried, DL Rousseau

BIOPHYSICAL JOURNAL　72　(2)　MPMC4-MPMC4　1997/02

ISSN： 0006-3495

eISSN： 1542-0086
Submillisecond protein folding kinetics studied by ultrafast mixing

CK Chan, Y Hu, S Takahashi, DL Rousseau, WA Eaton, J Hofrichter

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY　212　149-PHYS　1996/08

ISSN： 0065-7727
Conversion of alpha-hydroxyhemin to verdoheme in heme oxygenase.

KM Matera, S Takahashi, H Fujii, H Zhou, K Ishikawa, T Yoshimura, DL Rousseau, T Yoshida, M IkedaSaito

BIOPHYSICAL JOURNAL　70　(2)　WP342-WP342　1996/02

ISSN： 0006-3495

eISSN： 1542-0086
Protein folding kinetics studied by ultrafast mixing.

CK Chang, Y Hu, S Takahashi, DL Rousseau, WA Eaton, J Hofrichter

BIOPHYSICAL JOURNAL　70　(2)　MP140-MP140　1996/02

ISSN： 0006-3495

eISSN： 1542-0086
高橋聡・Denis L. Rousseau 「フローミキサーとラマン散乱法を使った、マイクロ秒領域のヘム蛋白反応の測定」

高橋聡, Rousseau Denis L.

生物物理　35, 81-82/,　(2)　31-32　1995
Publisher: 一般社団法人日本生物物理学会
ISSN： 0582-4052
STRUCTURAL CHARACTERIZATION OF IRON-BLEOMYCIN BY RESONANCE RAMAN-SPECTROSCOPY

S TAKAHASHI, JW SAM, J PEISACH, DL ROUSSEAU

BIOPHYSICAL JOURNAL　66　(2)　A374-A374　1994/02

ISSN： 0006-3495

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Books and Other Publications 5

相分離生物学の全貌

髙橋聡, 小井川浩之

東京化学同人　2020/11

ISBN: 9784807913466
揺らぎ・ダイナミクスと生体機能～物理化学的視点から見た生体分子～

小井川浩之, 鎌形清人, 高橋聡

化学同人　2013/09
Protein Folding and Misfolding Shining Light by Infrared Spectroscpy

Satoshi Takahashi, Tetsunari KImura

2011/09
タンパク質の立体構造入門：基礎から構造バイオインフォマティクスへ

高橋聡

2010/12
蛋白質のはたらきを知る一分子機能と生体作用

高橋聡

2009

Presentations 39

単一分子計測から見た分子システム化学：タンパク質とRNAの構造形成 Invited

高橋聡

日本化学会春季年会：オープンシンポジウム「次世代分子システムが拓く未来の化学」　2026/03/17
蛍光相関分光法と⼀分⼦FRET分光法によるSARS-CoV-2のNタンパク質リンカー領域のRNAへの結合における役割の解明

石川史恩, 木村貴洋, 齋藤真之介, 伊藤優志, 高橋聡

2025年度生物物理学会北海道支部－東北支部合同例会　2026/03/06
The characteristic RNA structure of SARS-CoV-2 is modulated by nucleocapsid proteins

T. Kimura, Y. Itoh, S. Ishikawa, Y. Yamano, K. Onizuka, F. Nagatsugi, S. Takahashi

2026/02/24
Investigation of the structure and dynamics of short and long RNA sequences using single-molecule FRET and fluorescence correlation spectroscopies Invited

Satoshi Takahashi

The East Asian Single- Molecule Biophysics Symposium 2025　2025/11/24
Development of site specific fluorophore labeling to target protein by using enzymatic reaction

Ai Hanano, Saori Kanbayashi, Cassidy Schmi, Hiroyuki Oikawa, Tateki Suzuki, Takao Hashiguchi, Yuji Itoh, Satoshi Takahashi

2025/09/25
Ultrafast dynamics of protein and RNA investigated by nanosecond fluorescence correlation spectroscopy

Yuji Itoh, Yutaka Sano, Ibuki Soshino, Shrutarshi Mitra, Divya Rajendran, Athi N. Naganathan, Satoshi Takahashi

2025/09/25
Roles of the linker region of the N protein of SARS-CoV-2 in the association to RNA by sm-FRET and fluorescence correlation spectroscopies

Shion Ishikawa, Takahiro Kimura, Takuya Katayama, Syamil MA Husna, Yuji Itoh, Satoshi Takahashi

2025/09/24
Single-molecule FRET spectroscopy reveals the structure of genomic RNA of SARS-CoV2 virus

T. Kimura, Y. Itoh, T. Katayama, Y. Yamano, K. Onizuka, F. Nagatsugi, S. Takahashi

2025/09/24
Investigation of the structure and dynamics of short and long RNA sequences using single-molecule FRET and fluorescence correlation spectroscopies Invited

Satoshi Takahashi

Telluride workshop Protein Dynamics 2025　2025/07/30
一分子FRET分光法と蛍光相関分光法によるSARS-CoV-2 Nタンパク質のG215C変異とリン酸化模倣変異のRNA結合性および構造の解析

石川史恩, 片山拓也, Syamil MA Husna, 伊藤優志, 高橋聡

第25回日本蛋白質科学会年会　2025/06/19
SARS-CoV-2のヌクレオカプシドタンパク質によるゲノムRNAの折り畳みの解明

伊藤優志, 木村貴洋, 片山拓也, 石川史恩, 金田直也, 高橋聡

第25回日本蛋白質科学会年会　2025/06/18
Local interaction and non-local interaction: Folding of proteins, RNAs and a scientist. Invited

Satoshi Takahashi

IISER Mohali Chemistry Day　2025/03/10
Single-molecule FRET investigation on the long-range contact formation in the folding of a gRNA granule of SARS-CoV-2

T. KimuraY., ItohT. KatayamaY., YamanoK. OnizukaF., NagatsugiS. Takahashi

Indian Biophysical Society Meeting　2025/03/07
Single-molecule FRET and FCS investigation of the interaction between SARS-CoV2 N protein and RNA Invited

Satoshi Takahashi

47th Indian Biophysical Society Meeting　2025/03/07
近い相互作用と遠い相互作用：タンパク質とRNAの構造形成 Invited

高橋聡

第64回生物物理若手の会夏の学校 in 北海道　2024/08/26
Conformational dynamics of SARS-CoV-2 spike protein investigated by single molecule fluorescence spectroscopy

Yuji Itoh, Taisei Mori, Tateki Suzuki, Takao Hashiguchi, Satoshi Takahashi

21st IUPAB and 62nd BSJ Joint Congress 2024　2024/06/27
Elucidating fusion dynamics of FUS protein droplets using fluorescence microscopy and optical tweezers

Syamil Muharror Ahsanul Husna, Atsumi Hando, Saori Kanbayashi, Satoshi Takahashi, Kiyoto Kamagata

2024/06/27
Observation of the compaction process ribonucleoprotein complex formed by SARS-CoV-2 genome RNA and N protein by using fluorescence correlation spectroscopy

Takuya Katayama, Yuji Itoh, Naoya Kaneda, Satoshi Takahashi

21st IUPAB and 62nd BSJ Joint Congress 2024　2024/06/26
Exploring the design rules for artificial phase-separating peptides based on natural phase-separating protein sequences

Joe Mori, Atsumi Hando, Satoshi Takahashi, Keisuke Ikeda, Kiyoto Kamagata

21st IUPAB and 62nd BSJ Joint Congress 2024　2024/06/26
Simple and Efficient Detection Scheme of Two-Color Fluorescence Correlation Spectroscopy for Protein Dynamics Investigation from Nanoseconds to Milliseconds

Yutaka Sano, Yuji Itoh, Atsuhito Fukasawa, Hiroyuki Oikawa, Satoshi Takahashi

21st IUPAB and 62nd BSJ Joint Congress 2024　2024/06/26
Single-molecule FRET and FCS investigation of the interaction between SARS-CoV2 N protein and RNA Invited

Satoshi Takahashi

DIJON COMPLEXBIODYN　2024/06/12
蛍光分光法によるSARS-COV2のNタンパク質とRNAの相互作用解析

伊藤優志, Shrutarshi Mitra, 金田直也, 片山拓也, 高橋聡

第24回日本蛋白質科学会　2024/06/11
Single-molecule FRET and FCS investigation of the interaction between SARS-CoV2 N protein and RNA Invited

Satoshi Takahashi

Protein Society Zoom Webinar　2024/04/25
Conformational elucidation of SARS-CoV2 genomic RNA elements by single-molecule FRET measurements

Shrutarshi Mitra, Yuji Itoh, Takuya Katayama, Satoshi Takahashi

2023/11/15
Investigation and prediction of RNA structure towards the understanding of viral RNA structure

Yuji Itoh, Takuya Katayama, Satoshi Takahashi

The 61st Annual Meeting of the Biophysical Society of Japan　2023/11/15
Lossless photon recording of two-color fluorescence correlation spectroscopy for protein dynamics investigations from nanoseconds to milliseconds

Yutaka Sano, Yuji Itoh, Atsuhito Fukasawa, Hiroyuki Oikawa, Satoshi Takahashi

The 61st Annual Meeting of the Biophysical Society of Japan　2023/11/14
Double fluorophore labeling of SARS-CoV-2 spike protein and its structural dynamics revealed by single molecule fluorescence spectroscopy

Taisei MORI, Yuji ITOH, Tateki SUZUKI, Takao HASHIGUCHI, Satoshi TAKAHASHI

2023/11
Structural characterization of RNA upon the binding with SARS-CoV-2 N protein by single molecule fluorescence measurements

Naoya Kaneda, Shun Endo, Leo Suzuki, Yuji Itoh, Hiroyuki Oikawa, Satoshi Takahashi

2023/11
Single-molecule FRET and FCS investigation of the interaction between SARS-CoV2 N protein and RNA: Does the formation of ribonucleoprotein granule resemble protein folding? Invited

Satoshi Takahashi

8th Asian Spectroscopy Conference 2023　2023/09/04
タイムタグ光子測定方式によるナノ秒蛍光相関分光測定システムの開発

佐野豊, 伊藤優志, 小井川浩之, 高橋聡

日本生物物理学会東北支部会　2023/03/04
ポストAlphaFold時代のフォールディング研究 Invited

髙橋聡

日本生物物理学会東北支部会　2023/03/04
Purification and fluorophore labeling of SARS-CoV2 N protein aiming at single molecule fluorescence measurements

Shun Endo, Leo Suzuki, Yuji Itoh, Hiroyuki Oikawa, Satoshi Takahashi

The 60th Annual Meeting of the Biophysical Society of Japan　2022/09/29
Development of the time-tag photon detection method of nanosecond fluorescence correlation spectroscopy

Yutaka Sano, Hiroyuki Oikawa, Satoshi Takahashi

The 60th Annual Meeting of the Biophysical Society of Japan　2022/09/28
Basics of spectroscopy for protein researches at AlphaFold era: Introduction Invited

Satoshi Takahashi

2022/06/07
Basics of single molecule and ensemble fluorescence spectroscopy for protein researches at AlphaFold era Invited

Satoshi Takahashi

2022/06/08
Single molecule fluorescence investigations on the structure transitions of LAF1- RGG upon the RNA binding and the droplet formation

Kanna Fujita, Michiko Kimura, Hiroto Takahashi, Satoshi Takahashi, Hiroyuki Oikawa

The 59th Annual Meeting of the Biophysical Society　2021/11/27
Structural Characterization of RNA upon the binding with SARS-CoV-2 N Protein by Single Molecule Fluorescence Measurements

Leo Suzuki, Hiroyuki Oikawa, Satoshi Takahashi

The 59th Annual Meeting of the Biophysical Society　2021/11/26
Conformational dynamics of E. coil Cytidine Repressor DNA Binding domain studied by Single-molecule Fluorescence Spectroscopy

Shrutarshi Mitra, Hiroyuki Oikawa, Divya Rajendran, Athi N.Naganathan, Satoshi Takahashi

The 59th Annual Meeting of the Biophysical Society of Japan　2021/11/26
Protein Folding Problem: Efforts to solve the simple but notoriously difficult puzzle are now revolutionizing life sciences Invited

Satoshi Takahashi

Tohoku University STEM Summer Program　2023/07/10

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Research Projects 28

新型コロナウィルスgRNAの構造形成：gRNAのフォールディング仮説の証明と展開

高橋聡

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 基盤研究(B)

Institution: 東北大学

2024/04/01 - 2027/03/31
SARS-CoV2のタンパク質Nによる液滴とRNAタンパク質複合体の形成機構

高橋聡

Offer Organization: 日本学術振興会

System: 科学研究費助成事業基盤研究(B)

Category: 基盤研究(B)

Institution: 東北大学

2021/04 - 2024/03

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新型コロナウイルスは約3万塩基もの長さを持つRNAをウイルス内にコンパクトにパッキングし、遺伝情報として維持している。ウイルスにおいて、本来不安定なRNAを安定化しコンパクトに収縮させる役割を持つと考えられるのが、Nタンパク質である。今年度も、Nタンパク質と短いRNAが形成する複合体の構造を、一分子蛍光分光法と蛍光相関分光法を用いて調べる研究を継続した。用いた試料は、一本鎖構造を持ち、長さが20塩基、30塩基、40塩基のポリアデニル酸と、ステムループ構造を作る複数のRNA配列である。これらの試料の5'および3'末端を蛍光色素でラベル化し、Nタンパク質を滴定し、試料に引き起こされた構造変化を一分子蛍光分光法により観察した。その結果、これらの試料は、RNAが二次構造を持つかどうかに関わらず、Nタンパク質がRNAに対して高い親和性を持ち、不均一な構造を形成することを見出した。ステムループ構造を作る試料の場合、ステムループがある程度引き伸ばされた構造が形成された。これらの結果は、Nタンパク質がRNAと結合することで非特異的な構造変化を引き起こすことを示している。同じ試料を蛍光色素一つでラベル化し、蛍光相関分光法を用いて解析した結果も、RNAとNの高い親和性を示した。数百ベースを超える長鎖RNAをin vitro転写により合成する方法を確立し、さらに、合成した長鎖RNAに蛍光色素をラベル化する方法を開発した。この試料を用いて、RNAのコンパクト化を調べることを可能とした。
Development of all photon time-tag detection method for the fluorescence investigations of single molecules

Takahashi Satoshi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)

Category: Grant-in-Aid for Challenging Research (Exploratory)

Institution: Tohoku University

2020/07/30 - 2022/03/31

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Fluorescence correlation spectroscopy (FCS) is a powerful method for the elucidation of fast protein dynamics. In this investigation, we aim to develop "all photon time-tag detection strategy" for FCS by combining hybrid photodetector (HPD) and digitized fast photon counting board. We noticed that the correlation data obtained by the newly constructed system were affected by the spike shaped noises due to the afterpulsing effect of HPD. We found a way to eliminate the noises in the correlation data, and were successful in observing the FCS data down to the 10 ns time region within an hour, which was a significant reduction in the observation time compared to the conventional system that required more than 10 hours of data accumulation.
Microsecond resolved single molecule fluorescence measurements triggered by solution mixing

Takahashi Satoshi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

Category: Grant-in-Aid for Scientific Research (B)

Institution: Tohoku University

2018/04/01 - 2021/03/31

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We used single molecule fluorescence spectroscopy and revealed the structures and dynamics of biological macromolecules. We developed rapid solution mixing device that can mix two solutions quickly, and enabled the system to be used in combination with the ultrafast single molecule fluorescence detection techniques. We also developed the nano-second region fluorescence correlation spectroscopic method. Using the systems, we investigated the dynamics of various proteins including the wild type of F1-ATPase, the fluorescence intensity fluctuation in the poly-alanine peptide, and the structures of a droplet forming protein, the RGG domain of LAF1.
Tracking of protein folding transition path by single-molecule fluorescence measurements

Oikawa Hiroyuki, Takahashi Satoshi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

Category: Grant-in-Aid for Young Scientists (B)

Institution: Tohoku University

2017/04/01 - 2019/03/31

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We aimed to track transition paths on protein folding events by single-molecule fluorescence resonance energy transfer (FRET) measurements. The time resolution of our existing line-confocal microscope combined with microfluidic chip was insufficient for tracking the transition events. To improve the time resolution, we built a new system based on hybrid photo detectors (HPD). By introducing HPD to the line-confocal microscope, we could obtain the single-molecule FRET traces with the time resolution of ten microsecond and the observation time of more than ten millisecond. As the target for the tracking of the transition path by using the new system, we tried to observe the fast conformational changes of F1-ATPase induced by the ATP hydrolysis. Unfortunately, although the conformational changes of F1-ATPase could not be observed, the method of single-molecule fluorescence measurement for tracking transition paths of biopolymer conformational changes was established.
Development of micro second measurement for functional analysis of DNA binding proteins

Kamagata Kiyoto, TAKAHASHI SATOSHI

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

Category: Grant-in-Aid for Scientific Research (C)

Institution: Tohoku University

2016/04/01 - 2019/03/31

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DNA-binding proteins can search for and bind to the target DNA sequence among a huge non-target sequence. We hypothesized that the proteins use “hopping” and “intersegmental transfer”, which is difficult to measure, during the target search. In this study, we developed sub-millisecond time resolved single-molecule fluorescence microscopy for DNA-binding proteins and aligned DNA array “DNA garden”, and investigated the target search mechanism of the proteins by visualizing the motion at single molecule level. We found that a tumor suppressor p53 searches for the target DNA by hopping and intersegmental transfer. Furthermore, we revealed the accuracy for recognizing the target DNA and proposed the molecular mechanism for bypassing the obstacles on DNA.
Support for International Activities of Studying the Function of Soft Molecular Systems by Concerted Use of Theory and Experiment

TAHARA Tahei, MORITA Akihiro, HAYASHI Shigehiko, TAKAHASHI Satoshi, MURAHASHI Tetsuro, NAKANISHI Takashi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

Institution: Institute of Physical and Chemical Research

2015/11/06 - 2018/03/31

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In the "soft molecular systems" project, we organized a group that supports international activities of members, and conducted two programs. The first program aims at sending young researchers abroad. We started this program at the beginning of the "soft molecular systems" project, 2 years before this grant project (15K21740) started. We requested young researchers not only to participate in the conference abroad, but also to visit laboratories for discussion. They are requested to report their activities in Newsletter. Using this grant, we sent 20 young researchers abroad, whose active communications with foreign researchers can be seen in Newsletter. The second program supported international collaboration of members, for which we sent 6 researchers abroad, and invited 7 researchers from abroad. As a result, 6 papers have been published in high-impact journals, including Nat. Commun. and PNAS. The two programs thus contributed the success of the "soft molecular systems" project.
Investigation on the dynamics of protein folding by single molecule fluorescence spectroscopy

Takahashi Satoshi, KAMAGATA Kiyoto, OIKAWA Hiroyuki, MANO Eriko

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

Institution: Tohoku University

2013/06/28 - 2018/03/31

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We developed a new method of single molecule fluorescence detection that can track single-molecule fluorescence and FRET efficiency at the time resolution of 10 microsecond. We used the method for the investigation of the folding dynamics of proteins, and revealed the unexpected heterogeneity in the unfolded state of BdpA and ubiquitin. In addition, we constructed a fluorescence microscope that can image the sliding of a tumor suppressor p53 along the stretched DNA, and revealed its target search dynamics. We clarified various properties of p53 including the dependency of the target search on the divalent cations, the behavior of p53 near the target sequence and the roles of disordered linker in p53. The current results might open a new research field of investigating proteins based on the assimilation of data obtained by single molecule method and MD calculation. In addition, investigations of the dynamics of proteins in the environment mimicking nucleus might become possible.
Development of New Strategy of Protein Design Based on Single Phage Sorting Technique

TAKAHASHI Satoshi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

Category: Grant-in-Aid for Scientific Research (B)

Institution: Tohoku University

2014/04/01 - 2017/03/31

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We planned to develop a new method of protein design by displaying a library of mutated fluorescent proteins on the surface of phages, and selecting phages displaying desired proteins by monitoring their emission properties. We constructed M13 phages displaying C-terminal fragment of GFP on g8p, and reconstituted GFP on the phage by adding the N-terminal fragment. We could create strongly emitting phages by displaying GFP on g8p proteins. We improved the sensitivity of the single phage detecting system. We were successful in selecting the fluorescent phages and examining the number of the collected phages by colony assay method.
Development of ultrafast detection system for the single molecule fluorescence based on hybrid photo detector

Takahashi Satoshi, KAMAGATA KIYOTO

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

Category: Grant-in-Aid for Challenging Exploratory Research

Institution: Tohoku University

2014/04/01 - 2016/03/31

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Single molecule fluorescence spectroscopy (SMFS) is a powerful method for the characterization of protein dynamics; however, the past methods of SMFS has the limit in the time resolution of the fluorescence detection and can not resolve events occurring within 1 ms. In this investigation, we used line confocal microscopy and combined it with the new photodetector, hybrid photodetector (HPD), and enabled to detect fluorescence signals from single molecules at the time resolution of 10 microsecond and for the duration of 10 millisecond. We further characterized the dynamics of a protein, BdpA, and observed that the unfolded BdpA shows various dynamics that should be important for the understanding of protein folding. The developed method can be applied to various proteins and their dynamics at the single molecule level.
タンパク質の高速揺らぎ運動と細胞内における安定性の一分子観測

高橋聡

Offer Organization: 日本学術振興会

System: 科学研究費助成事業新学術領域研究(研究領域提案型)

Category: 新学術領域研究(研究領域提案型)

Institution: 東北大学

2011/04/01 - 2013/03/31

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本研究は、タンパク質の示す運動を一分子レベルで高速観察すること、さらに、細胞内でのタンパク質の一分子観察を可能にすることを目的として計画した。第一のタンパク質運動を高速測定する装置に関しては、二色の蛍光の同時観察が可能なラインフォーカス型の共焦点顕微鏡を開発し、一分子継続蛍光観察の時間分解能を数十マイクロ秒にまで短縮した。B domain of protein A（BdpA）と呼ばれるタンパク質について、変性過程の一分子観察を行った。その結果、変性状態においてさまざまな構造と運動性を持つ状態が存在することを見いだした。特に、装置の観察時間の5ms程度の間、大きく構造を変えない複数の状態の存在が示唆された。この成果を、Nature系のオンライン誌であるScientific Reportsに投稿し、現在リバイスの過程である。さらに、この装置を用いて、ユビキチンの変性過程についての高速一分子測定を行った。驚くことに、BdpAで観察された複数の構造状態がユビキチンの変性状態にも存在することを見いだした。また、マルトース結合タンパク質について、試料流路と試料との相互作用が、観測に影響を与えることを観察し、装置を使った観測のためには、流路内面の親水的なコーティングが重要であることを見いだした。そのため、流路内面をBSAなどを用いて親水コーティングする手法を開発した。以上のように、本年度の努力により、ラインフォーカス型高速一分子観察装置は、蛍光色素をラベル化した試料さえあれば誰でも簡単に一分子観察が可能な装置として、実用的な装置として完成させることができた。第二の細胞内のタンパク質の観察については、共焦点顕微鏡を開発し、ドナー蛍光とアクセプター蛍光の両方を観察しながら基板においた蛍光試料のイメージングを可能にした。この装置を用いることで、細胞のイメージングが可能になった。
Development of microfluidic cell based on light guide for the single molecule fluorescence measurements

TAKAHASHI Satoshi, KAMAGATA Kiyoto

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

Category: Grant-in-Aid for Challenging Exploratory Research

Institution: Tohoku University

2011 - 2012

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We developed several flow cells for the single molecule fluorescence measurements. The first can maintain stable sheath flow. The second can mix two solutions for the kinetic measurements.
一分子観察法による生体分子の並進拡散運動と分子内構造変化の相関の解明

高橋聡

Offer Organization: 日本学術振興会

System: 科学研究費助成事業新学術領域研究(研究領域提案型)

Category: 新学術領域研究(研究領域提案型)

Institution: 東北大学

2009 - 2011

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本研究では、タンパク質が一分子レベルで溶液中を拡散する過程を直接観察し、タンパク質の流体力学的半径を計測する方法を開発すること、さらに、タンパク質の構造変化を同時観測することで分子内の構造変化と溶媒との衝突によって引き起こされる並進運動の関わりを計測することを目的とした。研究実施項目として、タンパク質の拡散運動を計測するために、セルの前後に取り付けたバルブをコントロールすることで溶液をセル中で完全に止める実験方法を確立した。次に、蛍光色素の位置の時間変化を確定し、位置の時間変化から拡散定数を求めるための方法を検討した。その結果、一般に行われている平均二乗変位の時間変化をプロットする方法よりも、拡散幅の分布関数をガウスフィットして幅を求め、その時間変化をプロットする方法の方がより正確な拡散定数を求められることを見いだした。以上の基礎データを積み重ねた後に、蛍光色素、蛍光ピース、βラクトグロブリンなどについて、一分子レベルでの拡散運動観察を行った。まず、蛍光色素単体と蛍光ビーズの拡散運動を観察したところ、それぞれに期待される分子量に応じた拡散運動の違いが確認された。次に、βラクトグロブリンを蛍光色素でラベルし、さまざまな変性状態において拡散運動を観察したところ、拡散運動を早く示す成分と遅く示す成分があり、それぞれ蛍光強度が異なることを見いだした。これは、一分子レベルで拡散運動を観察することで、試料の不均一性を見いだしたはじめての例である。
Investigation of the folding dynamics of proteins based on the advanced method of single molecule detection

TAKAHASHI Satoshi, KAMAGATA Kiyoto, OIKAWA Hiroyuki

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

Category: Grant-in-Aid for Scientific Research (B)

Institution: Tohoku University

2009 - 2011

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In this project, we first developed spherical mirror system for the detection of fluorescence from single molecules for long periods. Based on the developed system, we successfully determined the energy landscape for cytochrome c. We next developed line conforcal microscopy for the fast time resolved measurements of single molecule fluorescence time series. The method was used to investigate the unfolded state dynamics of the B domain of protein A, and revealed the presence of substates in the unfolded ensemble. Further applications of the developed methods for other protein systems are currently underway.
蛋白質翻訳過程の一分子観察法の確立

高橋聡

Offer Organization: 日本学術振興会

System: 科学研究費助成事業挑戦的萌芽研究

Category: 挑戦的萌芽研究

2008 - 2009

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蛋白質は、細胞の中においてリボソームにより合成される。この過程は、構造を持たないポリペプチド鎖が特定の形に折り畳む過程でもある。しかし、この過程で生合成途中のポリペプチド鎖がどのような構造と運動特性を持つのかを調べる研究はほとんどなされていない。本申請研究は、申請者が開発してきた一分子観察法を用いることで、合成途中の蛋白質の性質を調べ、真の折り畳み過程を解明することを目的とする。特に、申請者が開発した蛋白質の一分子運動の観察装置を用いて、研究の難しかった蛋白質合成過程の観察を行う。この実験のために、北陸先端大の芳坂博士が開発された蛋白質のN末端残基を蛍光ラベル化する技術を用いる。本年度は,N末端に蛍光色素をラベルしたマルトース結合蛋白質の一分子観察を行い,全長を合成した状態における揺らぎ運動の解析を行った。本申請研究の終了後も,マルトース結合蛋白質を用いたタンパク質合成過程の観察を続ける予定である。
一分子計測法による蛋白質の折り畳み運動の観測

高橋聡

Offer Organization: 日本学術振興会

System: 科学研究費助成事業特定領域研究

Category: 特定領域研究

Institution: 大阪大学

2006 - 2007

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本研究では、新しい一分子観察法を提案し、実際の蛋白質を対象に手法の有用性を示した。この手法では、蛍光色素でラベルした蛋白質を、レーザー照射したキャピラリーにー分子ごとに流し、分子が発する蛍光を輝線として検出する。この手法を使い、シトクロムcが折り畳み転移する様子の観察を行った。得られた-分子の蛍光強度の頻度分布には、中間状態と変性状態に対応する二つのピークが見られた。さらに、時系列データの自己相関関数を計算したところ、中間状態はミリ秒以内に減衰を示した一方で、変性状態の減衰は15ミリ秒程度だった。この結果は、変性蛋白質の運動が、従来考えられていたよりも遅いことを示唆する(発表論文1)。新しい装置開発として、二重のキャピラリーを持つ「鞘流セル」を制作した。このセルでは、蛋白質資料の位置を流路の中心部のみに制限することで、データのS/N比の向上と試料の吸着を減らした観測が可能になった。さらに、溶液混合を用いた非平衡状態での観測が可能となった。このセルを用いることで、変性状態からpHジャンプを起こした後のシトクロムcの運動について、予備的な観測を行った。アポミオグロビンの折り畳み過程について、時間分解赤外吸収観測を行った。蛋白質のアミドI領域を観察することで、さまざまな二次構造要素の分別が可能である。特に、疎水的環境に埋もれたヘリックス構造と、水に接触した水和ヘリックスの区別が可能である。折り畳み開始直後から水和したヘリックスの含量が大きく増え、折り畳みの律速段階で量を減らした。アポミオグロビンが水を巻き込んだ形で折り畳み中間体を作り、その水が脱水和することで折り畳み構造が作られることを示している(発表論文2)。
新しい一分子計測法による蛋白質の折り畳みダイナミクス

高橋聡

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 特定領域研究

Institution: 大阪大学

2004 - 2005

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蛋白質の折り畳みダイナミクスを一分子レベルで観察する新しい手法を開発し、シトクロムcなどの折り畳み過程を解析することが本研究の目的である。本年度は、実験装置の開発とデータ取得条件の最適化を検討した。 1)一分子蛍光観測用光学システムの開発:一分子観測のためのシステムとして、レーザーとイメージインテンシファイアー、CCDカメラによる系を構築した。また、レーザー光を変調することでデータに時間マーカーを入れるためのEO変調素子を導入した。以上の開発により、一分子の蛍光トレースを観測することが可能になった。2)一分子の蛍光観察のための「さや流セル」の開発:一分子レベルの試料を扱うためには、フローセル内面に試料が吸着を起こさずに、一定流速で流れることが必要だった。そのために、セルの中心部分にのみ試料を流すためのさや流セルを開発した。今年度は、ベイバイオサイエンス社(神戸)に依頼し、セルソーターに使われるさや流セルを、特別に我々の実験目的に合うようにデザインした。このセルを用いることで、試料の吸着などの問題をある程度回避できるようになった。3)平衡条件下における折り畳み過程の一分子観測:蛍光ラベルしたシトクロムcを用いて、平衡条件下で折り畳み転移の観察を行った。得られた一分子の蛍光トレースは、様々な強度の間を比較的ゆっくりと行き来した。蛍光強度の頻度分布を計算したところ、集団変性観察から推定されたN、I、U状態に対応する三つのピークが確認された。ピーク分布のグアニジン濃度依存性も、分子集団観察の結果と一致した。従って、今回観察された蛍光強度トレースは、ラベル化試料の特性を正しく反映すると推論できた。以上のように、本研究を行うことで、我々は一分子の蛍光観察にはじめて成功したと考えている。
Experimental investigation on the folding dynamics of proteins

TAKAHASHI Satoshi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (B)

Institution: Osaka University

2003 - 2005

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Proteins can fold from extended conformations of unfolded polypeptides to tightly packed and functional structures. Therefore, the mechanism of protein folding is important for the understanding of the principles of protein structures and functions. In this study, we investigated the structural events involved in the dynamics of protein folding based on the time-resolved technique combined with various spectroscopic techniques. We utilized small angle X-ray scattering for the characterization of protein compactness and circular dichroism, infrared absorption, fluorescence and resonance Raman spectroscopies for the detection of secondary and tertiary structure contents. The rapid mixing method was developed to increase the time resolution of the dynamics. We investigated the folding process of apomyogrobin, single chain monnelin, cytochrome c, heme oxygenase and ribonuclease A, and observed that the folding transitions of proteins with more than 100 residues generally follow the collapse and search mechanism, in which the rapid collapse and the subsequent search in the collapsed conformations are the characteristic events. Furthermore, we presented evidence suggesting that the initial collapse is caused by the coil-globule transition. Interestingly, it was observed that proteins of less than 100 residues usually do not accumulate kinetic intermediates, and possess the expanded R_g after the refolding jump. We propose that T_θ for small proteins is lower than the experimental temperatures causing small proteins to remain expanded until the moment of folding. Thus, the coil-globule transition offers a general explanation for the initial folding mechanisms of small and larger proteins. A further understanding on the molecular mechanism of the collapse will likely give an important insight that might help improve the structure prediction.
Structure and Function of Heme-regulated Proteins and Their Molecular Mechanisms

ISHIMORI Koichiro, TAKAHASHI Satoshi, WAKASUGI Keisuke

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

Category: Grant-in-Aid for Scientific Research (B)

2003 - 2005

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The major results we have obtained in this research project are as follows: 1. Ligation of Cys to Ferric Heme in Irr and IRP2. Based on the resonance Raman spectra, we successfully identified the Fe-Cys stretching modes in ferric heme-bound Irr and IRP2. These Fe-Cys stretching modes were downshifted, compared with that in conventional Cys-ligated hemoproteins such as P450cam. The downshifted Fe-Cys stretching modes correspond to the lower affinities of these proteins to ferric heme, which is also supported by fluorescence heme titration in Irr. Such weak affinities of these proteins to heme would be one of the characteristics of heme-regulated proteins having "Heme Regulatory Motif' 2. Redox-dependent Replacement of Axial Ligands. We confirmed the ligation of Cys to ferric heme in Irr and IRP2. By reduction of the heme iron, however, the absorption spectra of heme-bound Irr and IRP2 were drastically changed and the resultant spectra were quite different from ferrous P450cam, which were rather similar to bis-His ligated hemoproteins like cytochrome b_5. The spectral similarity to His-ligated hemoprotein was more evident in the CO adducts of heme-bound Irr and IRP2. The resonance Raman measurements. clearly showed the Fe-His stretching modes in ferrous heme bound lrr and IRP2 and the Fe-C and FeC-O stretching modes in the CO adducts, confirming that axial Cys is replaced with His by reduction of the heme iron. Considering that molecular oxygen can bind to ferrous heme, not ferric heme, the His ligated species in these proteins would be active species to generate reactive oxygen species, which leads to the oxidative modification of the peptide and protein degradation.
急速凍結分光法を使った金属酵素およびセンサー蛋白質の反応機構の研究

石森浩一郎, 高橋聡, 堀洋, 若杉桂輔

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 萌芽研究

Institution: 京都大学

2003 - 2004

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本研究課題で得られた成果は以下のとおりである. 1.P450酵素における活性阻害剤の結合様式の検討ヘム酵素であるP450はバクテリアから高等動物まで広く分布し,種々の薬物の代謝において重要な役割を担っている.したがって,感染症の原因となる真菌類のP450の機能を選択的に阻害できれば抗菌剤として有望な薬剤の開発が期待できる.そこで本研究課題では結核菌とヒトのP450(CYP51)に対する活性阻害剤であるいくつかのアゾール化合物について,その活性阻害様式を共鳴ラマンスペクトルやEPRを用いることで,構造化学的に検討した.その結果,アゾール環の置換基の立体障害とその疎水性度を制御することによって結核菌のP450のみ選択的に結合するアゾール化合物の分子設計が可能であることを示した. 2.ヘム酸化酵素中間体におけるラジカル位置の制御ヘムを含む酸化酵素であるペルオキシターゼ類は多くの動植物に存在し,種々の酸化反応を触媒している.これらの酵素はその反応中間体としてラジカル種を形成するが,そのラジカルの位置については基質の大きさに依存して異なることが知られている.つまり,小さな基質の場合は蛋白質に埋め込まれたポルフィリン環上に,大きな基質の場合は蛋白質表面に露出したアミノ酸上に形成される.われわれは既に小さな基質に対する酸化酵素である西洋わさびペルオキシターゼ(HRP)について,蛋白質表面に芳香族アミノ酸を導入することで、ラジカル位置をポルフィリン環上から導入した芳香族アミノ酸に移動できることを報告してきた.今回,その移動したラジカル種による活性を検証するために,野生型のHRPでは反応性が低い立体障害の大きな基質を用いて検討したところ,蛋白質表面にラジカル種を有する変異体ではその活性が数十倍に増大し,ラジカル位置の制御によってヘム酵素の基質特異性が制御できることが示された.
構造及び機能単位としてのモジュールを組み合わせた新規蛋白質の分子設計と創製

森島績, 若杉桂輔, 高橋聡, 石森浩一郎

Offer Organization: 日本学術振興会

System: 科学研究費助成事業特別推進研究

Category: 特別推進研究

Institution: 京都大学

2000 - 2003

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1.モジュール間相互作用の再生を目指した方法論の開発ヘモグロビンα鎖のヘム結合モジュールをシトクロムb_5の対応する領域に導入したb_5αb_5を作製し、ランダム変異導入後、ヘム親和性を指標に構造力安定化している蛋白質のスクリーニングを行ったところ、ヘム結合モジュールに近接する特定の部位に変異が集中していた。そこで、構造上の歪みがかかっていると考えられるこれら残基に範囲をしぼってランダム変異を導入した変異体集団を作製し、さらに、スクリーニング過程に、崩れた構造の変異蛋白質を蛋白質分解酵素により除去するプロセスを新たに加え方法論の改良を行ったところ、モジュール置換蛋白質の安定性の向上に成功した。 2.新規機能性蛋白質の創製に成功ヒトのトリプトファニルtRNA合成酵素(TrpRS)と相互作用する蛋白質を探索したところ、解糖系の酵素であるグリセルアルデヒド3-リン酸デヒドロゲナーゼ(GapDH)がTrpRSと結合することが明らかになった。TrpRSと結合するGapDHの結合部位の特定を試みるために、様々なキメラ蛋白質を作製した。ミオグロビン(Mb)のN末端側にGapDHのあるモジュールを融合したキメラ蛋白質は、野生型Mb同様、酸素を可逆的に配位でき、またGAPDH同様にTrpRSと会合する安定な新規蛋白質であることが明らかになった。 3.酸化ストレス応答性新規グロビン蛋白質の分子機構の解明とその知見に基づく人工蛋白質の設計「ニューログロビン(Ngb)」には酸化ストレスに伴う神経細胞死を抑制する働きがあると指摘されている。今回、このNgbが脳神経系においてシグナル伝達系を制御する分子として機能しているという仮説を立て、Ngbが関与する脳神経シグナル伝達系を明らかにすることを目指した。その結果、酸化ストレス下で生成する鉄3価Ngbが細胞内シグナル伝達蛋白質であるGαと特異的に結合すること、他方、通常の酸素正常状態の鉄2価NgbはGαとは相互作用しないことを発見した。また、Ngbは酸化ストレス応答性のセンサー蛋白質として働き、酸化ストレスを受けた時のみGαと結合し、GαのGDP/GTP交換反応抑制蛋白質として機能することにより、神経細胞死を抑制することを明らかにした。さらに、Ngbに関しモジュール置換した種々のキメラ蛋白質を作製し、それらの解析を行うことにより、制御メカニズムを分子レベルで明らかにすることに成功した。
αヘリックスの形成過程に注目した蛋白質の折れ畳み機構の実験的研究

高橋聡

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 奨励研究(A)

Institution: 京都大学

2000 - 2001

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蛋白質の折り畳み過程を詳しく観察することで、蛋白質のアミノ酸配列から折り畳み構造を予測するための手がかりが得られると期待できる。本研究は、蛋白質を構成する基本構造であるαヘリックスが作られる動的過程を、実験的に観察することを目的とした。具体的には以下の三つのテーマの実験を行い、成果を挙げることができた。 1)シトクロムcの折り畳み過程の測定。独自に開発した溶液混合セルを用いて、シトクロムcの折り畳み過程の時分割測定を、100μsの時間分解能で行った。シトクロムcが、二次構造とコンパクトさを同期させて折り畳みを進めることを明らかにした。本研究の成果は、Nature Stuct. Biol.誌や、PNAS誌に受理された。 2)ポリグルタミン酸のコイルヘリックス転移過程の観察。比較的短いポリペプチドが、αヘリックスが形成する過程を、溶液混合装置を使って追跡した。測定の結果、ポリグルタミン酸のコイルヘリックス転移には、いったん形成した短いヘリックス構造が連結して、長いヘリックスを作る運動の相があることを明らかにした。本研究の成果を、JACS誌に投稿すべく現在準備中である。 3)アポミオグロビンの折り畳み過程の時分割測定。アポミオグロビンは7本のヘリックスを持つ蛋白質である。これらのヘリックスが段階的に形成する様子を、時分割測定により明らかにした。特に、折り畳み開始後の100μs以内に超高速のヘリックス形成過程があることを実証した。
Development of time-resolved spectroscopic observation systems for the fast dynamics of protein folding

TAKAHASHI Satoshi, ISHIMORI Koichiro, HARADA Yoshiyuki

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (B)

Institution: KYOTO UNIVERSITY

2000 - 2001

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We developed the following original instruments to observe the early dynamics of protein folding. 1) The rapid mixing device that can initiate protein folding within 50 μs. The previous mixing devices has the mixing time of 300〜400 μs. Thus, we achieved to reduce the mixing times to nearly an order shorter. 2) We constructed to the observation system for the CD spectroscopy based on the prism polychrometer and a CCD detector. We resolved several problems associated with the new ideas of spectroscopic observation system, such as the effect of optical retardation. The developed system enabled us to observe the circular dichroism spectra with a high S/N ratio based on the multichannel detection. 3) We tried to combine the developped flow cell system and the spectroscopic observation system ; however, faild to observe reliable data for the repid-mixing cell. We are currently working to reduce the optical retardations of the cells. Using the developed flow cell systems, we obtained the following results that were already reported in scientific journals. 1) We observed time-resolved small-angle X-ray scattering profiles for the protein folding dynamics of cytochrome c. The developed cell enabled us to obtain information on the early submillisecond phase of cytohrome c folding that were inaccessible by the conventional devices. We conclude that the protein compaction and the secondary structure formations proceed simultaneously in the folding process of cytochrome c. 2) We observed the helix formation processes of polyglutamic acid using the developed rapid-mixing cell. We demonstrated that a short helix formation precedes the longer helix formation process in the PGA helix formation. This is a new dynamical event observed in the helix formation of polypeptides. 3) The helix-formation and the collapse process of apomyoglobin were observed using the developed rapid mixing systems in the submillisecond time domain. We clarified that the apomyoglobin folding proceed as a stepeise process that is similar to the observation for cytochrome c.
金属酵素の不安定中間体を観測するための高速溶液混合実験システムの開発

森島績, 高橋聡, 石森浩一郎

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 基盤研究(B)

Institution: 京都大学

1999 - 2000

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平成12年度はペルオキシターゼの活性中間体であるCompound Iの生成機構を検討するため,独自に開発した高速混合装置を用いて,西洋ワサビペルオキシターゼ(HRP)と過酸化水素との反応を追跡した。装置としては,本研究者らが開発した高速混合装置を改良することにより,時間分解能50マイクロ秒で可視紫外吸収スペクトルことに成功した。この装置を用いて,上記反応を追跡したところ,HRPの休止状態からCompound Iへのスペクトル変化が,ほぼ等吸収点をとりながら観測できた。このことは休止状態からCompound Iの変化において中間体が存在しないことを意味しているが,過酸化水素の濃度に対するCompound I生成速度の依存性は,速度論的な中間体が存在する明らかに飽和現象を示し,Michaelis-Menten型の解析をすることで,その寿命(半減期)が約10μ秒であることが明らかとなった。さらにこの値をもとに,このような寿命を持つ中間体を仮定したモデルを考え,反応後50マイクロ秒後の中間体の存在割合を求めたところ,約15%と見積もられ,その推定される紫外可視スペクトルは休止状態に類似した鉄3価高スピン型であった。このことは,Compound I生成の中間体として,従来想定されていた3価のヘム鉄にアニオン化した過酸化水素(HOO^-)が結合した鉄3価低スピン型ではなく,中性の過酸化水素(HOOH)が結合した状態であると推定できた。このような中間体を形成する反応機構として,中性の過酸化水素がヘム鉄に結合できるように,ヘム近傍の遠位ヒスチジンと遠位アルギニンが同時に過酸化水素と相互作用しているモデルを新たに提出した。
蛋白質構造の「揺らぎ」による電子移動過程の制御

森島績, 若杉桂輔, 高橋聡, 石森浩一郎

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 基盤研究(B)

Institution: 京都大学

1999 - 2000

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平成12年度は電子伝達蛋白質のモデル系として,亜鉛置換ミオグロビンとチトクロムb_5の系を構築し,レーザー光の照射による光誘起電子移動反応を検討することで,この電子移動反応における電子移動複合体に構造について考察を行った。従来,この系は電子移動反応を元にした会合定数が,等温適定実験からの会合定数よりはるかに小さいことが予想されており,電子移動を行える会合体は全会合体の一部であると考えられてきた。しかし,この系では,その会合が弱いことから電子移動反応を元にした正確な会合定数は,報告されておらず,実際に一部の会合体しか電子移動を行えないのかどうかは明確ではない。そこで,本研究ではこの系の会合における相互作用が静電的相互作用であることに注目し,イオン強度を下げることにより,Michaelis-Menten型の解析から,その会合定数を8.0x10^4M^<-1>と決定できた。一方,すべての会合体に対する結合定数を求めるために,亜鉛ミオグロビンの蛍光がチトクロムb_5により消光されることに注目した。その結果,会合定数は4.0x10^4M^<-1>と求めることができ,この値は,電子移動反応から求められた値とほぼ同程度であった。以上の結果は,亜鉛置換ミオグロビンとチトクロムb_5における電子移動はほとんどすべての会合体で起こっていることになり,従来の予想を否定する結果となった。さらに,電子移動反応のイオン強度依存性の結果から,この会合体は両蛋白質のヘム面が向かい合った状態であることが示唆され,これはマーカスの式から予想される電子移動反応の距離ともほぼ一致した。
二量体アポミオグロビンをモデルに使った蛋白質の折れ畳み初期過程の研究

高橋聡

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 奨励研究(A)

Institution: 京都大学

1997 - 1998

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本研究では,蛋白質の折れ畳み初期過程で働く相互作用の種類を知るために,アポミオグロビンの二量体を遺伝子工学の方法で合成し,単量体との性質の違いを調べることを目的とした.以下の成果を得た. 1. 二量体アポミオグロビンの合成.二つのミオグロビンのNとC末端を遺伝子工学の方法を使ってつなぎ,二量体化した.遺伝子の作成に手間がかかったが,二量体蛋白質の合成に成功し,性質の決定を行っている. これまでの結果は,ミオグロビンは二量体化しても,安定性が単量体と比べてあまり落ちていない.すなわち,蛋白質が折れ畳む速度は,単量体と二量体であまり変わらないようにみえる.今後,実際の折れ畳み速度の測定を行い観察を確かめた上で,学会発表する予定である. 2. 高速CD測定装置の開発.二量体ミオグロビンの折れ畳み過程測定の手段として,サブミリ秒の時間分解能で折れ畳み中間体のCDスペクトルを測定する装置の開発を行った.いくつかの問題点を解決することで,約500μsの時間分解能を持つ装置を作ることに成功した. 二量体アポミオグロビンの合成が遅れたため,チトクロムcを使い,折れ畳み過程のCD測定を行った.得られた結果は,チトクロムcが折れ畳む際に,いったん蛋白質全体が収縮した後に,二次構造が増加することを示している.この結果は,本年度の谷口シンポジウムで発表した. 本研究を通じ,蛋白質の折れ畳み初期過程について,いくつかの重要な知見を得ることに成功した.ミオグロビンの結果は局所的な相互作用が働くこと,チトクロムcの結果は非局所的な相互作用が働くことを示している.今後,実験を重ねることで,総合的な知見を得ることが必要である.結果の論文化が遅れているが,半年以内に公表する予定である.
Protein Engineering for New Functional Hemoproteins Based on Module Substitution

MORISHIMA Isao, TAKAHASHI Satoshi, ISHIMORI Koichiro

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (A)

Institution: KYOTO UNIVERSITY

1995 - 1997

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The primary results in this research project are as followa : (1)Module Substitution in Globins and Peroxidases. The structural and functional properties of the module-substituted globins and peroxidases have been characterized. Most of the module-substituted proteins exhibited highly destabilized protein structure, probably due to the missing or severe perturbation in the key interactions between the modules. The destabilized protein structure also interfered the formation of the specific dimers or tetramers in globins and enzymatic activity in peroxidases. It can be, therefore, concluded that the module-module interactions are also essential to design new stable and functional proteins as well as the module substitution. (2)Identification of New Module Structure in Globins. The amino acid substituteions based on the structural unit of wchic boundaries are located at the middle of the conventional modules have revealed that the module previously proposed can be divided into new and smaller structural units. In this research project, I focused upon one of the newly identified structural units, which includes the iron-liganded histidine and about 20 amino acid residues in the proximal site, "sub-module m6". The substitution of the sub-module m6 in globins drastically affect the electronic state of heme and configuration of the iron-liganded histidine, indicating that the sub-module m6 would be a "heme binding modules" respondible for the heme binding structure.
Developement and Application of High Pressure Multi-Dimensional NMR Spectroscopy

MORISHIMA Isao, TAKAHASHI Satoshi, ISHIMORI Koichiro

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)

Category: Grant-in-Aid for Scientific Research (A)

Institution: KYOTO UNIVERSITY

1995 - 1997

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The primary results in this research project are as follows : (1)Development of High Pressure Multi-Dimensional NMR Spectroscopy. We have tried to measure 2-dimensional proton NMR spectrum of hemoprotein under high pressure (up to 2000 bar). Our preliminary measurement in normal pressure has revealed that the very high sample concentration (more than 10 mM) and 40-60 hours accumulation would be required to obtain the 2D NMR spectrum by using a glass capillary as sample tube. Although the glass capillary needs enough space below the detection coil in the NMR probe, the bottom part of the our probe was used for the temperature control unit, which severely limits the size of the glass capillary. We final concluded that our NMR probe must be modified to use the glass capillary. (2)Application of High Pressure Laser Flash Photolysis to Dynamic Properties of Hemoproteins. Since pressure has been considered to perturb thermal fluctuation in protein structure, we examined the effects of the thermal fluctuation on dynamic properties of hemoprotein. One of the dynamic properties we focused on in this research project was the ligand binding in hemoproteins. Systematic combination of mutant myoglobins and kinetic measurement under high pressure has clearly shown that the some of the hydrophobic amino acid residues play a key role in controlling the ligand binding by maintaining the high hydrophobic environments in the heme pocket. Another dynamic property is the electron transfer reaction in hemoproteins. Basied on the measurements of the reaction rates for electron transfer in hemoprotein (myoglobin) under high pressure, we can pointed out that the electron transfer pathway and free energy difference for the reaction would highly depend on the thermal fluctuation in protein structure, and the thermal fluctuation is one of the critical factors of the molecular mechanism for the electron transfer in proteins.

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