Chemical Synthesis of the 20 kDa Heme Protein Nitrophorin 4 by α-Ketoacid-Hydroxylamine (KAHA) Ligation
The chemical synthesis of the 184-residue ferric heme-binding protein nitrophorin 4 was accomplished by sequential couplings of five unprotected peptide segments using α-ketoacid-hydroxylamine (KAHA) ligation reactions. The fully assembled protein was folded to its native structure and coordinated to the ferric heme b cofactor. The synthetic holoprotein, despite four homoserine residues at the ligation sites, showed identical properties to the wild-type protein in nitric oxide binding and nitrite dismutase reactivity. This work establishes the KAHA ligation as a valuable and viable approach for the chemical synthesis of proteins up to 20 kDa and demonstrates that it is well-suited for the preparation of hydrophobic protein targets.
It′s all coming together! The 20 kDa heme protein nitrophorin 4 was synthesized by sequential KAHA ligations using a convergent strategy. The folded synthetic protein readily coordinates the heme cofactor, and the holoprotein shows identical biological properties to the wild-type protein for NO binding and nitrite dismutase reactivity.
a-ketoacid-hydroxylamine (KAHA) ligation という、アルファケト酸とヒドロキシアミンからペプチド結合を形成する反応を鍵として、5つのペプチド鎖をくっつけることで、184残基、20kDaのタンパク質を人工的に合成し、そこにHemeを取り込ませ、ナイトライトジムスターゼ活性を持った人工酵素としています。
亜硝酸イオンの不均化
3 NO2– + 2 H+ <=> 2 NO + NO3– + H2O
この、KAHA ligationは、2006年にDr. ボーディ(スイスETH)によって報告された手法で、アミンやカルボン酸残基を保護することなく、狙った残基同士を結合できる画期的な手法です。
反応機構とできた人工酵素の構造
参考
ChemStation:http://www.chem-station.com/odos/2009/07/bode-bode-peptide-synthesis.html
Angew 2006: http://onlinelibrary.wiley.com/doi/10.1002/ange.200503991/epdf
Angew 2012: http://onlinelibrary.wiley.com/doi/10.1002/ange.201107198/epdf
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