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Determination of helix orientations in a flexible DNA by multi-frequency EPR spectroscopy

Abstract

Distance measurements between a pair of spin labels attached to nucleic acids using Pulsed Electron-Electron Double Resonance (PELDOR, also called DEER) spectroscopy 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. To investigate the conformational flexibility of nucleic acids by multi-frequency PELDOR, we have developed a two-step protocol and applied it to a flexible two-way DNA junction. In the first step, a library was created with a broad collection of conformers that are in agreement with topological constraints, NMR restraints and distances derived from PELDOR. In the second step, a weighted structural ensemble of these conformers was chosen such that it fits simultaneously the multi-frequency PELDOR time traces of all doubly Ç-labeled samples. This ensemble reflects the global structure and the conformational flexibility of the two-way junction. We demonstrate this approach on a flexible bent DNA molecule, consisting of two short helical parts with a five adenine bulge in 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 com-plexes with proteins and other molecules.

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Publication details

The article was received on 24 Jul 2017, accepted on 09 Oct 2017 and first published on 09 Oct 2017


Article type: Paper
DOI: 10.1039/C7CP04997H
Citation: Phys. Chem. Chem. Phys., 2017, Accepted Manuscript
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    Determination of helix orientations in a flexible DNA by multi-frequency EPR spectroscopy

    C. M. Grytz, S. Kazemi, A. Marko, P. Cekan, P. Güntert, S. T. Sigurdsson and T. F. F. Prisner, Phys. Chem. Chem. Phys., 2017, Accepted Manuscript , DOI: 10.1039/C7CP04997H

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