Branched intermediate formation stimulates peptide bond cleavage in protein splicing

Silvia Frutos, Michael Goger, Baldissera Giovani, David Cowburn, Tom W. Muir

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

Protein splicing is a post-translational modification in which an intein domain excises itself out of a host protein. Here, we investigate how the steps in the splicing process are coordinated so as to maximize the production of the final splice products and minimize the generation of undesired cleavage products. Our approach has been to prepare a branched intermediate (and analogs thereof) of the Mycobacterium xenopi DNA gyrase A (Mxe GyrA) intein using protein semisynthesis. Kinetic analysis of these molecules indicates that the high fidelity of this protein-splicing reaction results from the penultimate step in the process (intein-succinimide formation) being rate-limiting. NMR experiments indicate that formation of the branched intermediate affects the local structure around the amide bond that is cleaved during succinimide formation. We propose that this structural change reflects a reorganization of the catalytic apparatus to accelerate succinimide formation at the C-terminal splice junction.

Original languageEnglish (US)
Pages (from-to)527-533
Number of pages7
JournalNature Chemical Biology
Volume6
Issue number7
DOIs
StatePublished - Jul 2010

Fingerprint

Inteins
Protein Splicing
Peptides
Mycobacterium xenopi
DNA Gyrase
Post Translational Protein Processing
Amides
Proteins
succinimide

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

Cite this

Branched intermediate formation stimulates peptide bond cleavage in protein splicing. / Frutos, Silvia; Goger, Michael; Giovani, Baldissera; Cowburn, David; Muir, Tom W.

In: Nature Chemical Biology, Vol. 6, No. 7, 07.2010, p. 527-533.

Research output: Contribution to journalArticle

Frutos, Silvia ; Goger, Michael ; Giovani, Baldissera ; Cowburn, David ; Muir, Tom W. / Branched intermediate formation stimulates peptide bond cleavage in protein splicing. In: Nature Chemical Biology. 2010 ; Vol. 6, No. 7. pp. 527-533.
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