Protein-induced changes in DNA structure and dynamics observed with noncovalent site-directed spin labeling and PELDOR
Protein-induced changes in DNA structure and dynamics observed with noncovalent site-directed spin labeling and PELDOR
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DOI：
10.1093/nar/gks817
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发表时间：
2013-01-01
影响因子：
14.9
通讯作者：
Schiemann, Olav
中科院分区：
文献类型：
--
作者：
Reginsson, Gunnar W.;Shelke, Sandip A.;Schiemann, Olav
定点自旋标记和脉冲电子-电子双共振（PELDOR或DEER）以前已成功地应用于研究核酸的结构和动力学。在特定位点的自旋标记核酸需要自旋标记物的共价连接，这涉及相当复杂和费力的化学合成。在这里，我们使用一种非共价标记策略，绕过共价标记化学，并表明结合特异性和效率足够大，使PELDOR或DEER测量的DNA双链体和DNA双链体结合的Lac阻遏蛋白。此外，标签的刚性不仅允许解析寡核苷酸的结构和动力学，而且还可以确定标签方向和蛋白质诱导的构象变化。结果证明，这种标记策略与PELDOR相结合，具有很大的潜力，研究寡核苷酸及其与各种配体的复合物的结构和动力学。
Site-directed spin labeling and pulsed electron-electron double resonance (PELDOR or DEER) have previously been applied successfully to study the structure and dynamics of nucleic acids. Spin labeling nucleic acids at specific sites requires the covalent attachment of spin labels, which involves rather complicated and laborious chemical synthesis. Here, we use a noncovalent label strategy that bypasses the covalent labeling chemistry and show that the binding specificity and efficiency are large enough to enable PELDOR or DEER measurements in DNA duplexes and a DNA duplex bound to the Lac repressor protein. In addition, the rigidity of the label not only allows resolution of the structure and dynamics of oligonucleotides but also the determination of label orientation and protein-induced conformational changes. The results prove that this labeling strategy in combination with PELDOR has a great potential for studying both structure and dynamics of oligonucleotides and their complexes with various ligands.