Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein-intein junction

Proc Natl Acad Sci U S A. 2004 Apr 27;101(17):6397-402. doi: 10.1073/pnas.0306616101. Epub 2004 Apr 15.


Protein splicing is a posttranslational autocatalytic process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. Although we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how these are catalyzed and regulated is less well developed. In the current study, a combination of NMR spectroscopy and segmental isotopic labeling has been used to study the structure of an active protein splicing precursor, corresponding to an N-extein fusion of the Mxe GyrA intein. The (1)J(NC') coupling constant for the (-1) scissile peptide bond at the N-extein-intein junction was found to be approximately 12 Hz, which indicates that this amide is highly polarized, perhaps because of nonplanarity. Additional mutagenesis and NMR studies indicate that conserved box B histidine residue is essential for catalysis of the first step of splicing and for maintaining the (-1) scissile bond in its unusual conformation. Overall, these studies support the "ground-state destabilization" model as part of the mechanism of catalysis.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Isotopes
  • Mutagenesis, Site-Directed
  • Nuclear Magnetic Resonance, Biomolecular
  • Peptides / chemical synthesis
  • Peptides / chemistry*
  • Protein Conformation
  • Protein Splicing*


  • Isotopes
  • Peptides