Protein splicing facilitated by highly similar inteins from two extreme thermophiles

Protein splicing is a post‐translational event by which an intervening polypeptide, the intein, facilitates its own excision from the flanking polypeptides, the exteins, and the ligation of the exteins. Splicing of the Pyrococcus abyssi ( Pab ) PolII intein is initiated by an increase in temperature, which allows us to measure the rate of individual steps of the splicing reaction in vitro. We explored the rate of the first step of splicing for versions of the Pab PolII intein modified by mutation at conserved residues Asp74 and Ser166. We found no influence on the rate of the first step of splicing, but mutations at Ser166 influence the efficiency of the overall splicing reaction. The Pyrococcus horikoshii PolII intein has 71% sequence identity with the Pab PolII intein, but lacks an unstructured loop found in the Pab PolII intein. We hypothesize that this loop plays a role either in protein stability or in allowing for a mechanism‐linked conformational change to coordinate the steps of splicing. We will compare the temperature dependence of the rate and extent of splicing reactions and of the overall fold for both inteins. This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and by the Camille and Henry Dreyfus Foundation.