Intein-promoted cyclization of aspartic acid flanking the intein leads to atypical N-terminal cleavage
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Authors
Minteer, Christopher J.
Siegart, Nicolle M.
Colelli, Kathryn M.
Liu, Xinyue
Linhardt, Robert J.
Wang, Chunyu
Gomez, Alvin V.
Reitter, Julie N.
Mills, Kenneth V.
Issue Date
2017-02-28
Type
Article
Language
ENG
Keywords
Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
Alternative Title
Abstract
Protein splicing is a post-translational reaction facilitated by an intein, or intervening protein, which involves the removal of the intein and the ligation of the flanking polypeptides, or exteins. A DNA polymerase II intein from Pyrococcus abyssi (Pab PolII intein) can promote protein splicing in vitro on incubation at high temperature. Mutation of active site residues Cys1, Gln185, and Cys+1 to Ala results in an inactive intein precursor, which cannot promote the steps of splicing, including cleavage of the peptide bond linking the N-extein and intein (N-terminal cleavage). Surprisingly, coupling the inactivating mutations to a change of the residue at the C-terminus of the N-extein (N-1 residue) from the native Asn to Asp reactivates N-terminal cleavage at pH 5. Similar “aspartic acid effects” have been observed in other proteins and peptides but usually only occur at lower pH values. In this case, however, the unusual N-terminal cleavage is abolished by mutations to catalytic active site residues and unfolding of the intein, indicating that this cleavage effect is mediated by the intein active site and the intein fold. We show via mass spectrometry that the reaction proceeds through cyclization of Asp resulting in anhydride formation coupled to peptide bond cleavage. Our results add to the richness of the understanding of the mechanism of protein splicing and provide insight into the stability of proteins at moderately low pH. The results also explain, and may help practitioners avoid, a side reaction that may complicate intein applications in biotechnology.
Description
Biochemistry, 56, 1042−1050
Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
Full Citation
Intein-promoted cyclization of aspartic acid flanking the intein leads to atypical N-terminal cleavage, C. J. Minteer, N. M. Siegart, K. M. Colelli, X. Liu, R. J. Linhardt, C. Wang, A. V. Gomez, J. N. Reitter, K. V. Mills, Biochemistry, 56, 1042−1050, 2017
Publisher
Terms of Use
Journal
Volume
Issue
PubMed ID
DOI
ISSN
15204995
62960
62960