title: Intein lacking conserved C-terminal motif G retains controllable N-cleavage activity.
abstract: A split-intein consists of two complementary fragments (N-intein and C-intein) that can associate to carry out protein trans-splicing. The Ssp GyrB S11 split-intein is an engineered unconventional split-intein consisting of a 150-amino-acid N-intein and an extremely small six-amino-acid C-intein, which comprises the conserved intein motif G. Here, we show that fusion proteins containing the 150-amino-acid N-intein could be triggered to undergo controllable N-cleavage in vitro when the six-amino-acid C-intein or a derivative thereof was added as a synthetic peptide in trans. More importantly, we discovered, unexpectedly, that the 150-amino-acid N-intein could be induced by strong nucleophiles to undergo N-cleavage in vitro, and in Escherichia coli cells, in the absence of the motif G-containing six-amino-acid C-intein. This finding indicated that the first step of the protein splicing mechanism (acyl shift) could occur in the absence of the entire motif G. Extensive kinetic analyses revealed that both the motif G residues and the Ser+1 residue positively influenced N-cleavage rate constants and yields. The 150-amino-acid N-intein could also tolerate various unrelated sequences appended to its C-terminus without disruption of the N-cleavage function, suggesting that the catalytic pocket of the intein has considerable structural flexibility. Our findings reveal interesting insights into intein structure-function relationships, and demonstrate a new and potentially more useful method of controllable, intein-mediated N-cleavage for protein engineering applications.
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada. gerrit.volkmann@uni-muenster.de
authors.forename: Gerrit
authors.initials: G
authors.lastname: Volkmann
authors.forename: Xiang-Qin
authors.initials: XQ
authors.lastname: Liu
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002384:Catalysis; D027081:DNA Gyrase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D019811:Hydroxylamine / Q000494:pharmacology; D047668:Inteins; D007700:Kinetics; D015080:Mesna / Q000494:pharmacology; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D054730:Protein Interaction Domains and Motifs; D019154:Protein Splicing / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019163:Reducing Agents / Q000494:pharmacology; D046939:Synechocystis / Q000201:enzymology; D015032:Zinc / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D004792:Enzyme Precursors; D050505:Mutant Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019163:Reducing Agents; D019811:Hydroxylamine; D027081:DNA Gyrase; D015032:Zinc; D015080:Mesna; D004229:Dithiothreitol