Premium
Protein splicing of a temperature‐dependent intein from an extreme thermophile
Author(s) -
Colelli Kathryn M.,
Reitter Julie N.,
Mills Kenneth V.
Publication year - 2012
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.26.1_supplement.756.3
Subject(s) - intein , protein splicing , rna splicing , mutant , chemistry , biochemistry , biology , gene , rna
Inteins are intervening polypeptides that are excised during the process of protein splicing. Protein splicing is a self‐catalyzed process in which the intein is removed and the flanking polypeptides are ligated (N‐ and C‐ exteins). The Pyrococcus abyssi ( Pab ) PolII intein only splices at elevated temperatures. An NMR structure of the intein recently was solved in a collaborator's lab (Du, Z., et al. (2011) Structural and Mutational Studies of a Hyperthermophilic Intein from DNA Polymerase II of Pyrococcus abyssi. J. Biol. Chem. 286(44) 38638–38648.). The NMR data suggest increased rigidity of the intein in comparison to a mesophilic intein from Mycobacterium tuberculosis ( Mtu ), as well as a β‐hairpin found only in the structures of thermophilic inteins. We have replaced the β‐hairpin from the Pab intein with a linker inspired by the Mtu intein sequence to determine if the β‐hairpin plays a role in the structural stability/rigidity of the intein. We also have designed a genetic screening system that links protein splicing to cell growth on kanamycin in order to select for mutants that allow for splicing at lower temperatures. This material is based upon work supported by the National Science Foundation under grant MCB‐0950245, the Camille and Henry Dreyfus Foundation (KVM), and the Arnold and Mae Beckman Foundation (KMC).