title: Chemical Synthesis of Proteins with Non-Strategically Placed Cysteines Using Selenazolidine and Selective Deselenization.
abstract: Although native chemical ligation has enabled the synthesis of hundreds of proteins, not all proteins are accessible through typical ligation conditions. The challenging protein, 125-residue human phosphohistidine phosphatase 1 (PHPT1), has three cysteines near the C-terminus, which are not strategically placed for ligation. Herein, we report the first sequential native chemical ligation/deselenization reaction. PHPT1 was prepared from three unprotected peptide segments using two ligation reactions at cysteine and alanine junctions. Selenazolidine was utilized as a masked precursor for N-terminal selenocysteine in the middle segment, and, following ligation, deselenization provided the native alanine residue. This approach was used to synthesize both the wild-type PHPT1 and an analogue in which the active-site histidine was substituted with the unnatural and isosteric amino acid β-thienyl-l-alanine. The activity of both proteins was studied and compared, providing insights into the enzyme active site.
authors.affiliation: The Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra, Givat Ram, Jerusalem, 91904, Israel.
authors.forename: Post Sai
authors.initials: PS
authors.lastname: Reddy
authors.affiliation: The Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra, Givat Ram, Jerusalem, 91904, Israel.
authors.forename: Shahar
authors.initials: S
authors.lastname: Dery
authors.affiliation: The Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra, Givat Ram, Jerusalem, 91904, Israel. Metanis@mail.huji.ac.il.
authors.forename: Norman
authors.initials: N
authors.lastname: Metanis
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D006860:Hydrogen Bonding; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D012643:Selenium / Q000737:chemistry*
chemical_list: D011506:Proteins; D012643:Selenium; D003545:Cysteine
keywords: native chemical ligation; phosphohistidine; phosphohistidine phosphatase 1; post-translational modifications; selenocysteine