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

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123 Citations (Scopus)

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

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Inteins
Protein Splicing
Protein Precursors
Catalysis
Peptides
Histidine
Amides
Mutagenesis
Introns
Magnetic Resonance Spectroscopy
Proteins

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

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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.",
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T2 - NMR evidence for an unusual peptide bond at the N-extein-intein junction

AU - Romanelli, Alessandra

AU - Shekhtman, Alexander

AU - Cowburn, David

AU - Muir, Tom W.

PY - 2004/4/27

Y1 - 2004/4/27

N2 - 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.

AB - 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.

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