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.
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, Nagoya 464-8602 (Japan).
Chunmao
C
He
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, Nagoya 464-8602 (Japan).
Sameer S
SS
Kulkarni
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, Nagoya 464-8602 (Japan).|Laboratorium für Organische Chemie, ETH Zurich, 8093 Zurich (Switzerland) http://www.bode.ethz.ch/
Frédéric
F
Thuaud
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, Nagoya 464-8602 (Japan). bode@org.chem.ethz.ch.|Laboratorium für Organische Chemie, ETH Zurich, 8093 Zurich (Switzerland) http://www.bode.ethz.ch/. bode@org.chem.ethz.ch.
Jeffrey W
JW
Bode
D018360:Crystallography, X-Ray; D006420:Hemeproteins / Q000138:chemical synthesis* / Q000737:chemistry; D019811:Hydroxylamine / Q000737:chemistry*; D007651:Keto Acids / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D012471:Salivary Proteins and Peptides / Q000138:chemical synthesis* / Q000737:chemistry
D006420:Hemeproteins; D007651:Keto Acids; D012471:Salivary Proteins and Peptides; C090560:nitrophorin; D019811:Hydroxylamine
heme proteins; ligation reactions; peptides; protein synthesis; solid-phase peptide synthesis