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Protein splicing is catalyzed by an intervening polypeptide, called an intein. Inteins facilitate their own excision from the flanking polypeptides, called exteins, as well as the ligation of the exteins. Three inteins interrupt the ribonucleotide reductase of the extreme thermophile Pyrococcus abyssi. We have separately over‐expressed each intein in E. coli as a fusion protein with flanking affinity domains. By site directed mutagenesis, we have altered the sequences flanking each intein to determine the flexibility of the intein insertion site. We have also made mutations to residues suspected of catalyzing the splicing steps in each intein. We plan to study the influence of these mutations on the rate and extent of splicing. This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.

Protein splicing is catalyzed by an intervening polypeptide, called an intein. Inteins facilitate their own excision from the flanking polypeptides, called exteins, as well as the ligation of the exteins. Three inteins interrupt the ribonucleotide reductase of the extreme thermophile Pyrococcus abyssi. We have separately over‐expressed each intein in E. coli as a fusion protein with flanking affinity domains. By site directed mutagenesis, we have altered the sequences flanking each intein to determine the flexibility of the intein insertion site. We have also made mutations to residues suspected of catalyzing the splicing steps in each intein, and plan to study the influence of these mutations on the rate and extent of splicing.This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.

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.

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