Extein residues play an intimate role in the rate-limiting step of protein trans -splicing

Neel H. Shah, Ertan Eryilmaz, David Cowburn, Tom W. Muir

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Split inteins play an important role in modern protein semisynthesis techniques. These naturally occurring protein splicing domains can be used for in vitro and in vivo protein modification, peptide and protein cyclization, segmental isotopic labeling, and the construction of biosensors. The most well-characterized family of split inteins, the cyanobacterial DnaE inteins, show particular promise, as many of these can splice proteins in less than 1 min. Despite this fact, the activity of these inteins is context-dependent: certain peptide sequences surrounding their ligation junction (called local N-and C-exteins) are strongly preferred, while other sequences cause a dramatic reduction in the splicing kinetics and yield. These sequence constraints limit the utility of inteins, and thus, a more detailed understanding of their participation in protein splicing is needed. Here we present a thorough kinetic analysis of the relationship between C-extein composition and split intein activity. The results of these experiments were used to guide structural and molecular dynamics studies, which revealed that the motions of catalytic residues are constrained by the second C-extein residue, likely forcing them into an active conformation that promotes rapid protein splicing. Together, our structural and functional studies also highlight a key region of the intein structure that can be re-engineered to increase intein promiscuity.

Original languageEnglish (US)
Pages (from-to)5839-5847
Number of pages9
JournalJournal of the American Chemical Society
Volume135
Issue number15
DOIs
StatePublished - Apr 17 2013

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Trans-Splicing
Inteins
Protein Splicing
Proteins
Peptides
Kinetics
Cyclization
Structural dynamics
Biosensors
Biosensing Techniques
Labeling
Molecular Dynamics Simulation
Conformations
Molecular dynamics
Ligation

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Extein residues play an intimate role in the rate-limiting step of protein trans -splicing. / Shah, Neel H.; Eryilmaz, Ertan; Cowburn, David; Muir, Tom W.

In: Journal of the American Chemical Society, Vol. 135, No. 15, 17.04.2013, p. 5839-5847.

Research output: Contribution to journalArticle

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