Influence of Intein Flexibility on Temperature Dependence of Protein Splicing

Abstract

Protein splicing is a post‐translational modification by which intervening polypeptides called inteins catalyze their own excision from two flanking polypeptides, the exteins, concomitant with the ligation of the exteins. We studied the activity of DNA Polymerase II (PolII) inteins from the deep‐sea thermophiles Thermococcus kodakarensis (Tko), Thermococcus barophilus (Tba), Thermococcus sibiricus (Tsib), and Methanococcus infernus (Minf). Tko and Tba PolII inteins contain a homing endonuclease domain, while a flexible loop replaces this region in the Tsib and Minf inteins. This flexible loop, also found in Pyrococcus abyssi (Pab) and Pyrococcus horikoshii (Pho) PolII inteins, was hypothesized to be linked to the temperature dependent activity of these inteins. However, unlike Pab and Pho PolII inteins, we have shown that protein splicing and cleavage side reactions in the Tsib and Minf inteins occur in vivo in E. coli without the need for high temperature incubation in vitro. On the other hand, the Tba and Tko inteins splice poorly in E. coli and can be induced to splice in vitro at higher temperatures. We also have demonstrated that the Tba intein splices while the Tko intein undergoes a mixture of splicing and N‐terminal cleavage.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under grants MCB‐0950245, MCB‐1244089 and MCB‐1517138 and by a Henry Dreyfus Teacher‐Scholar Award (KVM).