2 research outputs found
Internal Disulfide Bond Acts as a Switch for Intein Activity
Inteins
are intervening polypeptides that catalyze their own removal
from flanking exteins, concomitant to the ligation of the exteins.
The intein that interrupts the DP2 (large) subunit of DNA polymerase
II from Methanoculleus marisnigri (<i>Mma</i>) can promote protein splicing. However, protein splicing
can be prevented or reduced by overexpression under nonreducing conditions
because of the formation of a disulfide bond between two internal
intein Cys residues. This redox sensitivity leads to differential
activity in different strains of E. coli as well as in different cell compartments. The redox-dependent control
of in vivo protein splicing
in an intein derived from an anaerobe that can occupy multiple environments
hints at a possible physiological role for protein splicing
Intein-Promoted Cyclization of Aspartic Acid Flanking the Intein Leads to Atypical N‑Terminal Cleavage
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 <i>Pyrococcus abyssi</i> (<i>Pab</i> PolII
intein) can promote protein splicing <i>in vitro</i> 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