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dc.contributor.authorMinteer, Christopher J.
dc.contributor.authorSiegart, Nicolle M.
dc.contributor.authorColelli, Kathryn M.
dc.contributor.authorLiu, Xinyue
dc.contributor.authorLinhardt, Robert J.
dc.contributor.authorWang, Chunyu
dc.contributor.authorGomez, Alvin V.
dc.contributor.authorReitter, Julie N.
dc.contributor.authorMills, Kenneth V.
dc.date2017
dc.date.accessioned2022-06-27T16:01:44Z
dc.date.available2022-06-27T16:01:44Z
dc.date.issued2017-02-28
dc.identifier.citationIntein-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
dc.identifier.issn15204995
dc.identifier.issn62960
dc.identifier.urihttps://doi.org/10.1021/acs.biochem.6b00894
dc.identifier.urihttps://hdl.handle.net/20.500.13015/5635
dc.descriptionBiochemistry, 56, 1042−1050
dc.descriptionNote : 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.
dc.description.abstractProtein 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.
dc.description.sponsorshipNational Science Foundation
dc.description.urihttps://login.libproxy.rpi.edu/login?url=https://doi.org/10.1021/acs.biochem.6b00894
dc.languageen_US
dc.language.isoENG
dc.relation.ispartofThe Linhardt Research Labs Online Collection
dc.relation.ispartofRensselaer Polytechnic Institute, Troy, NY
dc.relation.ispartofBiochemistry
dc.relation.urihttps://harc.rpi.edu/
dc.subjectBiology
dc.subjectChemistry and chemical biology
dc.subjectChemical and biological engineering
dc.subjectBiomedical engineering
dc.titleIntein-promoted cyclization of aspartic acid flanking the intein leads to atypical N-terminal cleavage
dc.typeArticle
dcterms.accessRightshttps://login.libproxy.rpi.edu/login?url=https://doi.org/10.1021/acs.biochem.6b00894
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dcterms.isVersionOfhttps://doi.org/10.1021/acs.biochem.6b00894
dc.rights.holderIn Copyright : this Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). https://rightsstatements.org/page/InC/1.0/
dc.creator.identifierhttps://orcid.org/0000-0003-2219-5833
dc.relation.departmentThe Linhardt Research Labs.
dc.relation.departmentThe Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
rpi.description.pages1042-1050
rpi.description.volume56


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