Directed Evolution of the Suicide Protein O6-Alkylguanine-DNA Alkyltransferase for Increased Reactivity Results in an Alkylated Protein with Exceptional Stability
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- 作者：Birgit Mollwitz ; Elizabeth Brunk ; Simone Schmitt ; Florence Pojer ; Michael Bannwarth ; Marc Schiltz ; Ursula Rothlisberger ; Kai Johnsson
- 刊名：Biochemistry
- 出版年：2012
- 出版时间：February 7, 2012
- 年：2012
- 卷：51
- 期：5
- 页码：986-994
- 全文大小：529K
- 年卷期：v.51,no.5(February 7, 2012)
- ISSN：1520-4995
文摘
Here we present a biophysical, structural, and computational analysis of the directed evolution of the human DNA repair protein O6-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 伪 helix is an important factor in triggering the unfolding and degradation of wild-type hAGT upon alkyl transfer, providing new insights into the structure鈥揻unction relationship of the DNA repair protein.