Protein splicing is the post‐translational excision of an intervening polypeptide (intein) from its flanking domains (the exteins), concomitant with extein ligation. The first step of splicing is an amide‐to‐thioester rearrangement of the peptide bond linking the N‐extein and intein. Class three inteins bypass this step. The Clostridium thermocellum TerA intein is class three, whereas the Thermobifida fusca Tfu2914 intein is a traditional intein despite having class three sequence motifs. The third step of splicing is Asn cyclization coupled to cleavage of the peptide bond linking the intein and C‐extein. Both the Methanoculleus marisnigri (Mma) and the Pyrococcus abyssi (Pab) PolII inteins promote efficient splicing with C‐terminal Gln in place of Asn. The activity of the Mma PolII intein may be regulated by disulfide bond formation. The Pab PolII intein splices only at elevated temperatures, allowing for isolation of a stable precursor and study each step of splicing in vitro. An NMR solution structure of the Pab PolII intein reveals a particularly rigid structure, a disordered loop absent in a highly similar P. horikoshii intein, and a β‐hairpin specific to thermophilic inteins.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and the Camille and Henry Dreyfus Foundation.
Protein splicing is a post‐translational event by which an intervening polypeptide, called an intein, facilitates its own excision from the flanking polypeptides, called the exteins, and the ligation of the exteins. The first step of protein splicing is an amide to ester or thioester rearrangement of the peptide bond linking the N‐extein and intein, facilitated by the N‐terminal Ser or Cys of the intein. The Clostridium thermocellum TerA protein is interrupted by an intein that lacks the N‐terminal nucleophile. The TerA intein splices as a class three intein by bypassing the first step in the protein splicing mechanism and using an internal Cys to facilitate attack at the N‐terminal splice junction. We have examined the influence of conserved residues on the catalysis of this alternative mechanism.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and the Camille and Henry Dreyfus Foundation.
Protein splicing is the post‐translational excision of an intervening polypeptide (intein) from its flanking domains (the exteins), concomitant with extein ligation. The third step of splicing is Asn cyclization coupled to cleavage of the peptide bond linking the intein and C‐extein. Both the Methanoculleus marisnigri (Mma) and the Pyrococcus abyssi (Pab) PolII inteins promote efficient splicing with C‐terminal Gln in place of Asn. The activity of both inteins is regulated by disulfide bond formation, which might suggest a regulatory role for splicing. The Pab PolII intein splices only at elevated temperatures, allowing for isolation of a stable precursor and for study of each step of splicing in vitro. An NMR solution structure of the Pab PolII intein reveals a particularly rigid structure, a disordered loop absent in a highly similar P. horikoshii intein, and a β‐hairpin specific to inteins from archaebacteria.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and the Camille and Henry Dreyfus Foundation.
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