TY - JOUR
T1 - Semisynthesis of a segmental isotopically labeled protein splicing precursor
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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U2 - 10.1073/pnas.0306616101
DO - 10.1073/pnas.0306616101
M3 - Article
C2 - 15087498
AN - SCOPUS:2342507637
SN - 0027-8424
VL - 101
SP - 6397
EP - 6402
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 17
ER -