Summary of Research Project Results Under the JSPS FY2000
"Research for the future Program"



1.Research Institution Kyoto University
 
2.Research Area Life Sciences
 
3.Research Field Structure and Functional Control mechanism of Biomolecules
 
4.Term of Project FY1996〜FY2000
 
5.Project Number 96L00502
 
6.Title of Project Structural Study of Molecular Interaction in Living Organism

7.Projetct Leader
Name Institution,Department Title of Position
Yoshinori Fujiyoshi Graduate School of Science, Kyoto University Professor

8.Core Members

Names Institution,Department Title of Position
Hiromichi Sato Graduate School of Pharmaceutical Science, Kyoto University Professor

9.Cooperating Researchers

Names Institution,Department Title of Position
Kaoru Mitsuoka Graduate School of Science, Kyoto University Assistant

10.Summary of Research Results

The project was executed by focusing on structural study of membrane proteins including channels through technical and methodological development for structure analysis of membrane proteins which were involved in important biological functions such as signal transduction. The following are the main results in this project.
1) A structure of water channel, aquaporin-1 (AQP-1) was analysed at a resolution of 6Å by electron crystallography (Nature, 387, 624-627 (1997).). It was suggested that charge shifts on bacteriorhodopsin molecule were discriminated based on the structure analysis at 3Å resolution by electron crystallography (Nature, 389, 206-211 (1997). & J. Mol. Biol., 286, 861-882 (1999).). An atomic structure of AQP-1 was analysed and the model provides a possible molecular explanation to longstanding puzzles in physiology; i) How does mercurial ion inhibit the water permeability through the channel? ii) How does the channel achieve marked selectivity? iii) How does the channel achieve fast water transport? iv) How does the molecule block the proton transport despite the high-speed water flow? (Nature, 405, 599-605 (2000).).
2) The structure of Nicotinic acetylcholine receptor, which was found in the postsynaptic membranes of muscle cells, was analysed at
4.6Å resolution (J. Mol. Biol., 288, 765-786 (1999).).
3) Three-dimensional structure of voltage sensitive Na+-channel, which was purified from electric eel, was analysed at 19Å resolution based on single particle analysis using an electron cryo-microscope (Nature, 409, 1047-1051 (2001).).
4) An electron microscope with helium cooled stage was improved by modifying the microscopic system including the stage which was developed by Fujiyoshi et al. in 1986.
5) Functional studies of G-protein coupled receptors were proceeded through developing a system for over-expression and purification (for example, Biochemistry, 39, 686-692 (2000).).

11.Key Words

(1)membrane proteins、(2)three-dimensional structure、(3)water channel
(4)Nicotinic acetylcholine receptor、(5)bacteriorhodopsin、(6)voltage sensitive Na+-channel
(7)G-protein coupled receptors、(8)electron crystallography、(9)electron cryo-microscope

12.References

[Reference Articles]
Author Title of Article
A. H. Iwane, I. Ikeda, Y. Kimura, Y. Fujiyoshi, K. Aitendorf and W. Epstein Two-Dimensional Crystal of the Kdp-ATPase of Escherichia coli.
Journal Volume Year Pages Concerned
FEBS Letters 396 1996 172-176

Author Title of Article
Y. M. Kiyosue, F. Vonderviszt, I. Yamashita, Y. Fujiyoshi and K. Namba. Direct Interaction of Flagellin Termini Essential for polymorphic Ability of Flagellar Filament.
Journal Volume Year Pages Concerned
Proc. Natl. Acad. Sci. USA 93 1996 15108-15113

Author Title of Article
T. Walz, T. Hirai, K. Murata, B. L. Smith, J. B. Heymann, K. Mitsuoka, Y. Fujiyoshi, P. Agre and A. Engel Theree-Dimensional Structure of Aquaporin 1.
Journal Volume Year Pages Concerned
Nature 387 1997 624-627

Author Title of Article
Y. Kimura, D. G. Vassylyev, A. Miyazawa, A. Kidera, M. Matsushima, K. Mitsuoka, K. Murata, T. Hirai and Y. Fujiyoshi Surface of bacteriorhodopsin revealed by high resolution electron crystallography.
Journal Volume Year Pages Concerned
Nature 389 1997 206-211

Author Title of Article
H. Hebert, I. Schmidt-Krey, R. Morgenstem, K. Murata, T. Hirai, K. Mitsuoka and Y. Fujiyoshi The 3.0Å projection structure of microsomal glutathione transferase as determind by electron crystallography of p21212 two-dimenstional crystals.
Journal Volume Year Pages Concerned
J. Mol. Biol. 271 1997 751-758

Author Title of Article
Y. Kimura, D. G. Vassylyev, A. Miyazawa, A. Kidera, M. Matsushima, K. Mitsuoka, K. Murata, T. Hirai and Y. Fujiyoshi High resolution structure of bacteriorhodopsin determined by electron crystallography.
Journal Volume Year Pages Concerned
Photochemistry and Photobiology 66 1997 764-767

Author Title of Article
T. Doi, Y. Hioaki, I. Arimoto, Y. Fujiyoshi, T. Okamoto, M. Satoh and Y. Furichi Characterization of human endothelin ETB receptor and mutants expressed in insect cells.
Journal Volume Year Pages Concerned
Eur. J. Biochem. 248 1997 139-148

Author Title of Article
Y. Zhu, A. M. Hemmings, K. Iwasaki, Y. Fujiyoshi, B. Zhong, J. Yan, M. Isogai and T. Omura Details of the arrangement of the outer capsid of rice dwarf phytoreovirue as visualized by two-dimensional crystallograohy.
Journal Volume Year Pages Concerned
J. Virol. 71 1997 8899-8901

Author Title of Article
K. Mitsuoka, K. Murata, Y. Kimura, K. Namba and Y. Fujiyoshi Examination of the leafscan 45, a line-illuminating micro-densitometer for its use in electron crystallography.
Journal Volume Year Pages Concerned
Uitramicroscopy 68 1997 109-121

Author Title of Article
T. Ohtaki, K. Ogi, Y. Masudall, K. Mitsuoka, Y. Fujiyoshi, C. Kitada, H. Sawada, H. Oda and M. Fujino. Expression, purification and reconstitution of receptor for pituitary adenylate cyclase-activating polypeptide.
Journal Volume Year Pages Concerned
J. Bipl. Chem. 273 1998 15464-15473

Author Title of Article
Y. M. Kiyosue, I. Yamashita, Y. Fujiyoshi, S. Yamaguchi and K. Namba Role of the outermost subdomain of salmonella flagelline in the filament structure revealed by electron cryomicroscopy.
Journal Volume Year Pages Concerned
J. Mol. Biol. 284 1998 521-530

Author Title of Article
Y. Fujiyoshi The structural study of membrance proteins by electron crystallography.
Journal Volume Year Pages Concerned
Adv. Biophys. 35 1998 25-80

Author Title of Article
I. Ikeda, T. Odahara, K. Mitsuoka, Y. Fujiyoshi and K. Murata Projection map of the reaction center-light harvesting complex from rhodopseudomanas viridis at 10 A resolution.
Journal Volume Year Pages Concerned
FEBS Letters. 425 1998 505-508

Author Title of Article
K. Mitsuoka, T. Hirai, K. Murata, A. Miyazawa, A. Kidera, Y. Kimura and Y. Fujiyoshi The structure of bacteriorhodopsin at 3.0 Å rsolution based on electron crystallography: Implication of the charge distribution.
Journal Volume Year Pages Concerned
J. Mol. Biol. 286 1999 861-882

Author Title of Article
Y. Hiroaki, K. Mitsuoka, Y. Cheng H. Hiroaki and Y. Fujiyoshi Influence of various nucleotides on the in situ crystallization of Ca2+ -ATPase.
Journal Volume Year Pages Concerned
Biochemica et Biophysica Acta 1415 1999 361-368

Author Title of Article
T. Doi, H. Sugimoto, I. Arimoto, Y. Hiroaki and Y. Fujiyoshi Interactions of endothelin receptor subtypes A and B with Gi, Go and Gq in reconstituted phospholipid vesicles.
Journal Volume Year Pages Concerned
Biochemistry 38 1999 3090-3099

Author Title of Article
Y. Fujiyoshi Electron crystallography of membrance proteins.
Journal Volume Year Pages Concerned
G. protein-coupled receptors(CRC Press: Edited by T. Haga and G. Berstein)   1999 323-354

Author Title of Article
A. Miyazawa, Y. Fujiyoshi, M. Stowell and N. Unwin Nicotinic Acetylcholine Receptor at 4.6Å Resolution: Transverse Tunnels in the Channel Wall.
Journal Volume Year Pages Concerned
J. Mol. Biol. 288 1999 765-786

Author Title of Article
T. Hirai, K. Murata, K. Mitsuoka, Y. Kimura and Y. Fujiyoshi Trehalose embedding technique for high-resolution electron crystallography.
Journal Volume Year Pages Concerned
J. Electron Microsc. 48 1999 653-658

Author Title of Article
Y. Fujiyoshi Molecular structure of proton pump revealed with electron crystallography.
Journal Volume Year Pages Concerned
The FASEB Journal.   1999 S191-s194

Author Title of Article
K. Mitsuoka, K. Murata, T. Walz, T. Hirai, P. Agre, J. B. Heymann, A. Engel and Y. Fujiyoshi. The Structure of Aquaporin-1 4.5Å Resolution Reveals Short α-Helices in the Center of the Monomer.
Journal Volume Year Pages Concerned
J. Structure Biol. 128 1999 34-43

Author Title of Article
F. Imamura, I. Arimoto, Y. Fujiyoshi and T. Doi W276 Mutation in the Endothelin Receptor subtype B Imaparis Gq Coupling but not Gi or Go Coupling.
Journal Volume Year Pages Concerned
Biochemistry. 39 2000 686-692

Author Title of Article
Y. Tahara, A. Oshima, T. Hirai, K. Mitsuoka, Y. fujiyoshi and Y. Hayashi. The 11Å resolution projection map of Na+/K+-ATPase calculated by application pf single particle anaysis to two-dimensional crystal images.
Journal Volume Year Pages Concerned
J. Electron Microsc. 49 2000 583-587

Author Title of Article
B. L. de Groot, J. B. Heymann, A. Engel, K. Mitsuoka, Y. Fujioshi and H. Grubmueller The fold of human aquaporinl
Journal Volume Year Pages Concerned
J. Mol. Biol. 300 2000 987-994

Author Title of Article
K. Murata, K. Mitsuoka, T. Hirai, T. Walz, P. Agre, J. B. Heymann, A. Engel and Y. Fujiyoshi Structure determinants of water permeation through aqaporin-1.
Journal Volume Year Pages Concerned
Nature. 407 2000 599-605

Author Title of Article
I. Schmidt-Krey, K. Mitsuoka, T. Hirai, K. Murata, Y. Cheng, Y. Fujiyoshi, R. Morgenstern and H. Hebert The three-dimensional map of microsomal glutathione transferase 1 at 6 Å resolution
Journal Volume Year Pages Concerned
EMBO Journal. 19 2000 6311-6316

Author Title of Article
C. Sato, Y. Ueno, K. Asai, K. Takahashi, M. Sato, A. Engel and Y. Fujiyoshi The voltage-sensitive sodium channel is a bell-shaped molecule with several cavities.
Journal Volume Year Pages Concerned
Neture. 409 2001 1047-1051

Author Title of Article
A. Engel, Y. Fujiyoshi and P. Age The Importance of Aquaporin Water Channel Protein structures
Journal Volume Year Pages Concerned
EMBO J 19 2000 800-806


back