title: Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein-intein junction.
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 (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.
authors.affiliation: Laboratory of Synthetic Protein Chemistry, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Romanelli
authors.forename: Alexander
authors.initials: A
authors.lastname: Shekhtman
authors.forename: David
authors.initials: D
authors.lastname: Cowburn
authors.forename: Tom W
authors.initials: TW
authors.lastname: Muir
mesh_terms: D007554:Isotopes; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D011487:Protein Conformation; D019154:Protein Splicing
chemical_list: D007554:Isotopes; D010455:Peptides