Laboratory of Protein Engineering, Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
Biochemistry. 2012 Feb 7;51(5):986-94. doi: 10.1021/bi2016537. Epub 2012 Jan 25.
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.
在这里，我们呈现了对人类 DNA 修复蛋白 O(6)-烷基鸟嘌呤-DNA 烷基转移酶（hAGT）定向进化为 SNAP 标签的生物物理、结构和计算分析，SNAP 标签是一种自我标记蛋白标签。hAGT 的进化不仅导致了蛋白活性的增加，而且还提高了稳定性，特别是烷基化蛋白的稳定性，这表明自杀酶的反应性可以通过稳定不可逆反应的产物来影响。野生型 hAGT 在烷基转移后在细胞内迅速降解，而苯甲基化 SNAP 标签的高稳定性可防止蛋白水解降解。我们的数据表明，关键 α 螺旋的固有稳定性是触发烷基转移后野生型 hAGT 解折叠和降解的重要因素，为 DNA 修复蛋白的结构-功能关系提供了新的见解。
