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Relating Intein Flexibility to the Temperature Dependence of Activity
Author(s) -
Yakely Anthony E.,
Bonano Alexander A.,
Jaramillo Mario V.,
Williams Jennie E.,
Reitter Julie N.,
Mills Kenneth V.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1083.2
Subject(s) - intein , pyrococcus horikoshii , protein splicing , chemistry , rna splicing , thermostability , genetics , biochemistry , biology , gene , rna , enzyme
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 Pol II 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, disordered 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. We successfully created the chimeric genes for both chimeric inteins and discovered that the proteins expressed well and were capable of splicing. For the P. abyssi intein with the P. horokoshii loop, we found that the chimeric intein spliced more than the native intein at a lower temperature but less at a higher temperature. For the P. horokoshii intein with the P. abyssi loop, we found that it displayed less splicing than the original protein at various temperatures. Comparative activity assays will be run to examine the temperature dependence of activity among the native and chimeric inteins. Support or Funding Information This material is based upon work supported by the National Science Foundation under grants MCB‐1244089 and MCB‐1517138 and by a Henry Dreyfus Teacher‐Scholar Award (KVM).