Three segment ligation of a 104 kDa multi-domain protein by SrtA and OaAEP1.
Three segment ligation of a 104 kDa multi-domain protein by SrtA and OaAEP1.
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DOI：
10.1007/s10858-022-00409-w
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发表时间：
2023-04
影响因子：
2.7
通讯作者：
Latham, Michael P.
中科院分区：
文献类型：
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作者：
Azatian, Stephan B.;Canny, Marella D.;Latham, Michael P.
关键词：
核磁共振波谱是研究蛋白质结构和动力学的一种很好的工具，它可以加深对生物功能的理解。随着感兴趣的生物分子尺寸的增加，稀释核磁共振光谱中观察到的信号的数量以减少光谱重叠和提高分辨率可能变得有利。限制核磁共振数据中共振次数的一种方法是选择性地标记较大大分子中的较小结构域，这一过程称为分段同位素标记。已经描述了许多片段同位素标记的例子，其中蛋白质的两个片段通过化学或酶方法连接在一起，但对于三个或更多片段连接反应的描述要少得多。在这里，我们描述了一种酶分段标记方案，它结合了广泛使用的Sortase A和最近描述的OaAEP1，用于两个位点的连接策略。为了研究104 kDa DNA损伤修复蛋白Rad50中拟议的长程变构，我们将标记为Zn Hook结构域的侧链甲基连接在两个长片段之间，否则未标记。酶活性数据表明，连接反应造成的疤痕不影响Mre11-Rad50 DNA双链断裂修复复合体中的Rad50功能。最后，基于甲基的核磁共振波谱证实了全长连接蛋白的形成。我们的策略突出了OaAEP1在分段标记方面的优势，即更快的反应时间和更小的识别序列，并为在多位点分段标记反应中使用这两种酶提供了一个直接的模板。
NMR spectroscopy is an excellent tool for studying protein structure and dynamics which provides a deeper understanding of biological function. As the size of the biomolecule of interest increases, it can become advantageous to dilute the number of observed signals in the NMR spectrum to decrease spectral overlap and increase resolution. One way to limit the number of resonances in the NMR data is by selectively labeling a smaller domain within the larger macromolecule, a process called segmental isotopic labeling. Many examples of segmental isotopic labeling have been described where two segments of a protein are ligated together by chemical or enzymatic means, but there are far fewer descriptions of a three or more segment ligation reaction. Herein, we describe an enzymatic segmental labeling scheme that combines the widely used Sortase A and more recently described OaAEP1 for a two site ligation strategy. In preparation to study proposed long-range allostery in the 104 kDa DNA damage repair protein Rad50, we ligated side-chain methyl group labeled Zn Hook domain between two long segments of otherwise unlabeled P.furiosus Rad50. Enzymatic activity data demonstrated that the scars resulting from the ligation reactions did not affect Rad50 function within the Mre11-Rad50 DNA double strand break repair complex. Finally, methyl-based NMR spectroscopy confirmed the formation of the full-length ligated protein. Our strategy highlights the strengths of OaAEP1 for segmental labeling, namely faster reaction times and a smaller recognition sequence, and provides a straightforward template for using these two enzymes in multisite segmental labeling reactions.
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