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Protein Splicing of a Temperature‐Dependent Intein from an Extreme Thermophile
Author(s) -
Siegart Nicolle,
Colelli Kathryn,
Reitter Julie,
Mills Kenneth
Publication year - 2015
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.29.1_supplement.573.12
Subject(s) - intein , protein splicing , rna splicing , cleavage (geology) , chemistry , peptide bond , biochemistry , trans splicing , chemical biology , genetics , peptide , biology , gene , paleontology , rna , fracture (geology)
Inteins are intervening polypeptides that catalyze their own excision from the flanking polypeptides, or exteins, and the ligation of the exteins to form a functional protein, in a process called protein splicing. We are studying an intein that interrupts the DNA PolII from the archaebacteria Pyrococcus abyssi (Pab). The Pab PolII intein only splices at elevated temperatures. The intein contains an N‐terminal Cys and is preceded by Asn. The N‐terminal Cys typically acts as a nucleophile in the N‐S acyl shift of the first step in the canonical splicing mechanism. We have shown that protein cleavage occurs independently of the canonical splicing mechanism when the active site Cys is replaced by Ala, while the preceding Asn is replaced by Asp. In addition, this cleavage at the N‐terminus appears to be pH‐dependent, occurring under acidic conditions. We have investigated whether residues within the intein play a role in destabilizing the peptide bond and promoting cleavage. This material is based upon work supported by the National Science Foundation under grant MCB‐1244089 and by the Camille and Henry Dreyfus Foundation.