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. Stitching a protein together: A synthesis approach is reported using selenazolidine and deselenization to access a protein with non-strategically placed cysteine residues. The challenging human phosphohistidine phosphatase 1 (PHPT1) protein, a 125-residue enzyme with three cysteine residues near the C-terminus, was used as a model system.
Bibliographical notePublisher Copyright:
© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
- native chemical ligation
- phosphohistidine phosphatase 1
- post-translational modifications