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.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Mollwitz B,Brunk E,Schmitt S,Pojer F,Bannwarth M,Schiltz M,Rothlisberger U,Johnsson K

doi

10.1021/bi2016537

subject

Has Abstract

pub_date

2012-02-07 00:00:00

pages

986-94

issue

5

eissn

0006-2960

issn

1520-4995

journal_volume

51

pub_type

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