Journal
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Volume 55, Issue 3, Pages 992-995Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.201509378
Keywords
native chemical ligation; phosphohistidine; phosphohistidine phosphatase1; post-translational modifications; selenocysteine
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Funding
- Israel Science Foundation
- German-Israeli Foundation for Scientific Research and Development (GIF)
- PBC fellowship, Council for higher Education, Israel
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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 phosphatase1 (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 -thienyl-l-alanine. The activity of both proteins was studied and compared, providing insights into the enzyme active site.
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