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The Influence of Conserved Residues and Structural Elements on the Stability and Activity of a Thermophilic Intein
Author(s) -
Minteer Christopher J.,
Colelli Katie M.,
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.1083.6
Subject(s) - intein , protein splicing , peptide bond , thermophile , rna splicing , chemistry , biochemistry , mutant , genetics , peptide , biology , gene , enzyme , rna
Protein splicing is a self‐catalyzed reaction where an intein is excised from precursor flanking polypeptides (exteins) and the exteins are ligated together to produce a mature protein. We are interested in how specific conserved residues catalyze this process and how structural domains affect intein structure. The structure of an intein from Pyrococcus abyssi (the Pab PolII intein) has a Hyperthermophilic/Thermophilic Hairpin (HTH), only found in the structures of thermophilic inteins. It may add thermal stability by extending the central beta sheet of the typical intein fold. Mutations to destabilize inter‐and intra‐strand salt bridges in the hairpin region may result in global destabilization, and were studied through assays to assess activity and susceptibility to proteolysis. We also are interested in the influence of conserved intein residues on activity. Residues in intein block B may serve to destabilize the scissile peptide bond at the N‐terminal splice junction. We have observed cleavage of that bond at low pH, independent of splicing steps, and are exploring whether block B residues are responsible. 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).