Laboratory of Protein Organic Chemistry

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Professor
Saburo AIMOTO
mail aimoto@protein.osaka-u.ac.jp
Associate Professor
Toru KAWAKAMI
mail kawa@protein.osaka-u.ac.jp
Assistant Professor
Takeshi SATO
mail takeshi@protein.osaka-u.ac.jp
Fields

Chemical Biology

Belong

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Location

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Research Theme

One of our research objectives is to establish a general method that permits the synthesis of a wide variety of proteins including membrane and highly modified proteins like histone. Currently our efforts on this line are focused on 1) methodological studies for protein synthesis, 2) development of methods for peptide thioester preparation, 3) synthetic studies of membrane proteins, 4) synthetic studies of post-translationally modified histone H3. The other is structural and functional studies of proteins, such as the activation mechanism of cell surface receptors, which are prepared based on the developed methods.

Methodological Studies for Protein Synthesis

Currently the thioester method and the native chemical ligation method are used for protein synthesis. The thioester method permits peptide segment coupling at any given site in the presence of silver ions, however, this method requires protecting groups at amino and thiol groups. On the other hand, the native chemical ligation method allows unprotected peptide chains to be condensed in an aqueous neutral buffer, however, a cysteine residue is always required at the coupling site. To overcome the weak points involved in the both methods, a new ligation auxiliary group is under development.

Development of a Method for Peptide Thioester Preparation via an N-S Acyl Shift Reaction

Peptide thioester is a common intermediate in contemporary methods for protein synthesis. Therefore, one of the key steps in protein synthesis is to prepare peptide thioesters in a good yield without epimerization. A new method for peptide thioester preparation under development is based on an N-S acyl shift reaction after accomplishment of peptide chain elongation cycles. This alternative for thioester production is amenable to standard Fmoc chemistry. This newly developed strategy will provide partially protected peptide thioesters, which are necessary for the synthesis of polypeptides via the thioester method, as well as side-chain unprotected peptide thioester used for the native chemical ligation.

Synthetic Studies of Membrane Proteins

About 30% of the human genome encodes membrane proteins. Much of the information concerns the structure and function of these membrane proteins, however, remains to be uncovered because of the difficulties associated with biochemical sample preparation. As an alternative approach to obtain membrane proteins, chemical synthesis represents a viable candidate. In order to examine the utility of a developing method, we are applying it to the synthesis of membrane proteins. One of the target molecules, ORL-1, is a receptor of an opioid peptide, nociceptin. The strategy employed for the synthesis of the C-terminal region of ORL-1 is the native chemical ligation. The coupling between ORL-1(288-328) and ORL-1(329-370) was carried out in SDS solution. Under the defined conditions, ORL-1(288-370) that contained one transmembrane region and the C-terminal cytosolic tail region was obtained in the yield of 47%. The route to accomplish the total synthesis of ORL-1 is under investigation based on the information obtained by the research project mentioned above. Using synthetic membrane proteins and their related peptides, structural and functional analyses in terms of peptide-peptide and peptide-membrane interactions are carred out.

Synthesis of Post-translationally Modified Histone H3

Histones can undergo post-translational modifications such as methylation, acetylation or/and phosphorylation. These modifications are believed to play an essentially important role in gene regulation in an epigenetic manner. Focusing on the N-terminal region of histone H3, we searched a strategy that would provide a synthetic procedure for post-translationally modified histones. In the near future, a variety of the modified histone H3 with a full sequence will be synthesized.

0The chemically synthesized transmembrane-juxtamembrane regions of fibroblast growth factor receptor 3 and thrombopoietin receptor are subjected to structural analyses using spectroscopic studies such as solid state NMR.

Bibliography

M. Ahmed, J. Davis, D. Aucoin, T. Sato, S. Ahuja, S. Aimoto, J. I. Elliott, W. E. Van Nostrand, S. O. Smith Structural conversion of neurotoxic amyloid-β(1-42) oligomers to fibrils Nat. Struct. Mol. Biol., Published online 11 April , (2010)

Toru Kawakami, Saburo Aimoto The Use of a Cysteinyl Prolyl Ester (CPE) Autoactivating Unit in Peptide Ligation Reactions Tetrahedron 65 , 3871 - 3877 (2009)

T. Sato, T.-C.Tang, G. Reubins, J.Z. Fei, T. Fujimoto, P. Kienlen-Campard, S. N. Constantinescu, J.-N. Octave, S. Aimoto and S. O. Smith A helix-to-coil transition at the ε-cut site in the transmembrane dimer of the amyloid precursor protein is required for proteolysis Proc. Natl Acad. Sci. USA 106 , 1421 - 1426 (2009)

P. Kienlen-Campard, B. Tasiaux, J. Van Hees, M. Li, S. Huysseune, T. Sato, J. Z. Fei, S. Aimoto, P. J. Courtoy, S. O. Smith, S. N. Constantinescu, J. N. Octave Amyloidogenic processing but not aicd production requires a precisely oriented APP dimer assembled by transmembrane GXXXG motifs J. Biol. Chem. 283 , 7733 - 7744 (2008)

Contact

Saburo Aimoto
Institute for Protein Research
Osaka Univeristy
3-2 Yamadaoka
Suita, Osaka 565
Japan

http://www.protein.osaka-u.ac.jp/organic/orgj.htm

http://www.protein.osaka-u.ac.jp/organic/orgj.htm