N-methylcysteine-mediated total chemical synthesis of ubiquitin thioester

Lesly A. Erlich, K. S.Ajish Kumar, Mahmood Haj-Yahya, Philip E. Dawson, Ashraf Brik

Research output: Contribution to journalArticlepeer-review

91 Scopus citations

Abstract

Ubiquitin thioester is a key intermediate in the ubiquitylation of proteins and is formed enzymatically through the activation of α-COOH of ubiquitin in an ATP dependent manner using the E1 enzyme. The current methods used for the preparation of ubiquitin thioester rely on either the enzymatic machinery or on expressed protein ligation technology. In this article, we report a new chemical strategy, combining native chemical ligation and N-methylcysteine containing peptides, to chemically prepare ubiquitin thioester for the first time. The N-methylcysteine is utilized as an N→S acyl transfer device, and in its protected form serves as a latent thioester functionality. This enabled us to trigger the formation of ubiquitin thioester subsequent to the assembly of the ubiquitin polypeptide via native chemical ligation. The synthetic ubiquitin thioester showed a similar behavior in peptide ubiquitylation to the one obtained via expression. This approach should allow for higher flexibility in the chemical manipulation of ubiquitin thioester in a wide variety of ubiquitylated peptides and proteins for structural and biochemical analysis and for the synthesis of ubiquitin chains.

Original languageEnglish
Pages (from-to)2392-2396
Number of pages5
JournalOrganic and Biomolecular Chemistry
Volume8
Issue number10
DOIs
StatePublished - 21 May 2010

ASJC Scopus subject areas

  • Biochemistry
  • Physical and Theoretical Chemistry
  • Organic Chemistry

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