title: Directed evolution of the suicide protein O⁶-alkylguanine-DNA alkyltransferase for increased reactivity results in an alkylated protein with exceptional stability.
abstract: Here we present a biophysical, structural, and computational analysis of the directed evolution of the human DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) into SNAP-tag, a self-labeling protein tag. Evolution of hAGT led not only to increased protein activity but also to higher stability, especially of the alkylated protein, suggesting that the reactivity of the suicide enzyme can be influenced by stabilizing the product of the irreversible reaction. Whereas wild-type hAGT is rapidly degraded in cells after alkyl transfer, the high stability of benzylated SNAP-tag prevents proteolytic degradation. Our data indicate that the intrinstic stability of a key α helix is an important factor in triggering the unfolding and degradation of wild-type hAGT upon alkyl transfer, providing new insights into the structure-function relationship of the DNA repair protein.
authors.affiliation: Laboratory of Protein Engineering, Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Birgit
authors.initials: B
authors.lastname: Mollwitz
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Brunk
authors.forename: Simone
authors.initials: S
authors.lastname: Schmitt
authors.forename: Florence
authors.initials: F
authors.lastname: Pojer
authors.forename: Michael
authors.initials: M
authors.lastname: Bannwarth
authors.forename: Marc
authors.initials: M
authors.lastname: Schiltz
authors.forename: Ursula
authors.initials: U
authors.lastname: Rothlisberger
authors.forename: Kai
authors.initials: K
authors.lastname: Johnsson
mesh_terms: D000478:Alkylation / Q000235:genetics; D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004260:DNA Repair / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D055550:Protein Stability; D017433:Protein Structure, Secondary / Q000235:genetics; D058767:Protein Unfolding; D013329:Structure-Activity Relationship; D015854:Up-Regulation / Q000235:genetics
chemical_list: D019853:O(6)-Methylguanine-DNA Methyltransferase