Structure of an engineered intein reveals thiazoline ring and provides mechanistic insight

C. Seth Pearson, Reza Nemati, Binbin Liu, Jing Zhang, Matteo Scalabrin, Zhong Li, Hongmin Li, Dan Fabris, Marlene Belfort, Georges Belfort

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N-terminal Lys-Cys splice junction. We identified conditions to stablize the thiazoline ring and provided the first crystallographic evidence, at 1.54 Å resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. In addition, the pivotal mutation maps to a highly conserved B-block threonine, which is now seen to play a causative role not only in ground-state destabilization of the scissile N-terminal peptide bond, but also in steering the tetrahedral intermediate toward thioester formation, giving new insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic ring opening under acidic conditions. A pH cycling strategy to control N-terminal cleavage is proposed, which may be of interest for biotechnological applications requiring a splicing activity switch, such as for protein recovery in bioprocessing.

Original languageEnglish (US)
Pages (from-to)709-721
Number of pages13
JournalBiotechnology and Bioengineering
Issue number4
StatePublished - Apr 2019
Externally publishedYes


  • cleavage control
  • molecular switch
  • protein splicing
  • thiazoline crystal structure

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology


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