Relating Intein Flexibility to the Temperature Dependence of Activity

Protein splicing is the self‐catalyzed removal of an intein, or intervening protein, in coordination with the ligation of the exteins, the flanking polypeptides. We are examining the DNA PolII inteins from the extremophiles Pyrococcus abyssi and Pyrococcus horikoshii. The inteins from P. abyssi and P. horikoshii are largely homologous except for the presence of a large, disorganized loop in the P. abyssi intein, in contrast to a smaller loop in the P. horikoshii intein. We have observed temperature dependence of the activity of the two inteins, which may be due to the difference in their structures. The smaller loop may allow for a more rigid structure in the P. horikoshii intein resulting in a higher temperature needed for protein splicing. The P. abyssi intein has a longer loop resulting in a less rigid structure, so a lower temperature is needed for protein splicing. To test this hypothesis, we used recombinant DNA to make a chimera of the P. abyssi intein with the P. horikoshii loop and vice versa, to determine whether the change in loop alters the temperature dependence of the inteins' activity. For the P. horikoshii expression vector containing the P. abyssi loop, we successfully created the chimeric gene and discovered that the protein expressed well and was larger than the starting protein. We tested for activity and found that the chimeric intein spliced more slowly than the native intein. For the P. abyssi gene containing the P. horikoshii loop, we are in the midst of preparation of the expression vector. This material is based upon work supported by the National Science Foundation under grant MCB‐1244089 and by the Camille and Henry Dreyfus Foundation.