Grytz C M, Kazemi S, Marko A, Cekan P, Güntert P, Sigurdsson S Th, Prisner T F
Institute of Physical and Theoretical Chemistry, Goethe University, Max-von-Laue-Str. 7, 60438 Frankfurt am Main, Germany.
Phys Chem Chem Phys. 2017 Nov 15;19(44):29801-29811. doi: 10.1039/c7cp04997h.
Distance measurements are performed between a pair of spin labels attached to nucleic acids using Pulsed Electron-Electron Double Resonance (PELDOR, also called DEER) spectroscopy which is a complementary tool to other structure determination methods in structural biology. The rigid spin label Ç, when incorporated pairwise into two helical parts of a nucleic acid molecule, allows the determination of both the mutual orientation and the distance between those labels, since Ç moves rigidly with the helix to which it is attached. We have developed a two-step protocol to investigate the conformational flexibility of flexible nucleic acid molecules by multi-frequency PELDOR. In the first step, a library with a broad collection of conformers, which are in agreement with topological constraints, NMR restraints and distances derived from PELDOR, was created. In the second step, a weighted structural ensemble of these conformers was chosen, such that it fits the multi-frequency PELDOR time traces of all doubly Ç-labelled samples simultaneously. This ensemble reflects the global structure and the conformational flexibility of the two-way DNA junction. We demonstrate this approach on a flexible bent DNA molecule, consisting of two short helical parts with a five adenine bulge at the center. The kink and twist motions between both helical parts were quantitatively determined and showed high flexibility, in agreement with a Förster Resonance Energy Transfer (FRET) study on a similar bent DNA motif. The approach presented here should be useful to describe the relative orientation of helical motifs and the conformational flexibility of nucleic acid structures, both alone and in complexes with proteins and other molecules.
使用脉冲电子-电子双共振（PELDOR，也称为DEER）光谱法对连接在核酸上的一对自旋标记物进行距离测量，这是结构生物学中其他结构测定方法的一种补充工具。刚性自旋标记物Ç成对掺入核酸分子的两个螺旋部分时，由于它与所连接的螺旋一起刚性移动，因此可以确定这些标记物之间的相互取向和距离。我们开发了一种两步法，通过多频PELDOR研究柔性核酸分子的构象灵活性。第一步，创建了一个包含大量构象体的文库，这些构象体符合拓扑约束、NMR限制以及从PELDOR得出的距离。第二步，选择这些构象体的加权结构集合，使其同时符合所有双Ç标记样品的多频PELDOR时间轨迹。这个集合反映了双向DNA连接的整体结构和构象灵活性。我们在一个柔性弯曲的DNA分子上展示了这种方法，该分子由两个短螺旋部分组成，中间有一个五个腺嘌呤的凸起。定量测定了两个螺旋部分之间的扭结和扭曲运动，结果显示出高灵活性，这与对类似弯曲DNA基序的Förster共振能量转移（FRET）研究结果一致。本文提出的方法对于描述螺旋基序的相对取向以及核酸结构的构象灵活性应该是有用的，无论是单独的核酸结构，还是与蛋白质和其他分子形成的复合物中的核酸结构。
