Pulsed electron paramagnetic resonance (EPR) spectroscopy is a valuable technique for the precise determination of distances between paramagnetic spin labels that are covalently attached to macromolecules. Nitroxides have commonly been utilised as paramagnetic tags for biomolecules, but trityl radicals have recently been developed as alternative spin labels. Trityls exhibit longer electron spin relaxation times and higher stability than nitroxides under in vivo conditions. So far, trityl radicals have only been used in pulsed EPR dipolar spectroscopy (PDS) at X-band (9.5 GHz), Ku-band (17.2 GHz) and Q-band (34 GHz) frequencies. In this study we investigated a trityl biradical by PDS at Q-band (34 GHz) and G-band (180 GHz) frequencies. Due to the small spectral width of the trityl (30 MHz) at Q-band frequencies, single frequency PDS techniques, like double-quantum coherence (DQC) and single frequency technique for refocusing dipolar couplings (SIFTER), work very efficiently. Hence, Q-band DQC and SIFTER experiments were performed and the results were compared; yielding a signal to noise ratio for SIFTER four times higher than that for DQC. At G-band frequencies the resolved axially symmetric g-tensor anisotropy of the trityl exhibited a spectral width of 130 MHz. Thus, pulsed electron electron double resonance (PELDOR/DEER) obtained at different pump-probe positions across the spectrum was used to reveal distances. Such a multi-frequency approach should also be applicable to determine structural information on biological macromolecules tagged with trityl spin labels.
Pulsed electron electron double resonance experiments with rigid spin labels can reveal very detailed information about the structure and conformational flexibility of nucleic acid molecules. On the other hand, the analysis of such data is more involved the distance and orientation information encoded in the time domain data need to be extracted and separated. In this respect studies with different spin labels with variable internal mobility are interesting and can help to unambiguously interpret the EPR data. Here orientation selective multi-frequency/multi-field 4-pulse PELDOR/DEER experiments with three recently presented semi-rigid or conformationally unambiguous isoindoline-derived spin labels were performed and simulated quantitatively by taking the spin label dynamics into account. PELDOR measurements were performed for a 20-mer dsDNA with two spin labels attached to two defined uridine derivatives. Measurements were recorded for different spin label positions within the double helical strand and for different magnetic field strengths. The experimental data sets were compared with simulations, taking into account the previously described dsDNA dynamics and the internal motions of the spin label itself, which had shown distinct differences between the three spin labels used. The (ExIm)U spin label shows a free rotation around a single bond, which averages out orientation effects, without influencing the distance distribution as it can occur in other spin labels. The (Im)U and (Ox)U spin label, on the other hand, show distinct orientation behaviour with minimal intrinsic motion. We could quantitatively determine this internal motion and demonstrate that the conformational dynamics of the nucleic acid and the spin label can be well separated by this approach.
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