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

Alessandra Romanelli, Alexander Shekhtman, David Cowburn, Tom W. Muir

Research output: Contribution to journalArticle

125 Scopus citations

Abstract

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 1JNC' coupling constant for the (-1) scissile peptide bond at the N-extein-intein junction was found to be ≈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.

Original languageEnglish (US)
Pages (from-to)6397-6402
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number17
DOIs
StatePublished - Apr 27 2004
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • General

Fingerprint Dive into the research topics of 'Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein-intein junction'. Together they form a unique fingerprint.

  • Cite this