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Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels
Published on: July 4, 2016
Conformationally restricted isoindoline-derived spin labels in duplex DNA: distances and rotational flexibility by
Dnyaneshwar B Gophane1, Burkhard Endeward, Thomas F Prisner
1Department of Chemistry, Science Institute, University of Iceland, Dunhaga 3, 107 Reykjavik (Iceland), Fax: (+354)5528911.
Abstract:
Three structurally related isoindoline-derived spin labels that have different mobilities were incorporated into duplex DNA to systematically study the effect of motion on orientation-dependent pulsed electron-electron double resonance (PELDOR) measurements. To that end, a new nitroxide spin label, (ExIm)U, was synthesized and incorporated into DNA oligonucleotides. (ExIm)U is the first example of a conformationally unambiguous spin label for nucleic acids, in which the nitroxide N-O bond lies on the same axis as the three single bonds used to attach the otherwise rigid isoindoline-based spin label to a uridine base. Continuous-wave (CW) EPR measurements of (ExIm)U confirm a very high rotational mobility of the spin label in duplex DNA relative to the structurally related spin label (Im)U, which has restricted mobility due to an intramolecular hydrogen bond. The X-band CW-EPR spectra of (ExIm)U can be used to identify mismatches in duplex DNA. PELDOR distance measurements between pairs of the spin labels (Im)U, (Ox)U, and (ExIm)U in duplex DNA showed a strong angular dependence for (Im)U, a medium dependence for (Ox)U, and no orientation effect for (ExIm)U. Thus, precise distances can be extracted from (ExIm)U without having to take orientational effects into account.
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