Flexibilities of isoindoline-derived spin labels for nucleic acids by orientation selective PELDOR.

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.

Advanced EPR Methods for Studying Conformational Dynamics of Nucleic Acids.

Pulsed electron paramagnetic resonance (EPR) spectroscopy has become an important tool for structural characterization of biomolecules allowing measurement of the distances between two paramagnetic spin labels attached to a biomolecule in the 2-8 nm range. In this chapter, we will focus on applications of this approach to investigate tertiary structure elements as well as conformational dynamics of nucleic acid molecules. Both aspects take advantage of using specific spin labels that are rigidly attached to the nucleobases, as they allow obtaining not only the distance but also the relative orientation between both nitroxide moieties with high accuracy. Thus, not only the distance but additionally the three Euler angles between both the nitroxide axis systems and the two polar angles of the interconnecting vector with respect to the nitroxide axis systems can be extracted from a single pair of spin labels. To extract all these parameters independently and unambiguously, a set of multifrequency/multifield pulsed EPR experiments have to be performed. We will describe the experimental procedure as well as newly developed spin labels, which are helpful to disentangle all these parameters, and tools which we have developed to analyze such data sets. The procedures and analyses will be illustrated by examples from our laboratory.

Pulsed electron-electron double resonance spectroscopy between a high-spin Mn(2+) ion and a nitroxide spin label.

Pulsed Electron-Electron Double Resonance (PELDOR) has attracted considerable attention for biomolecular applications, as it affords precise measurements of distances between pairs of spin labels in the range of 1.5-8 nm. Usually nitroxide moieties incorporated by site-directed spin labelling with cysteine residues are used as spin probes in protein systems. Recently, naturally occurring cofactors and metal ions have also been explored as paramagnetic spin species for such measurements. In this work we investigate the performance of PELDOR between a nitroxide spin label and a high-spin Mn(2+) ion in a synthetic model compound at Q-band (34 GHz) and G-band (180 GHz). We demonstrate that the distances obtained with high-frequency PELDOR are in good agreement with structural predictions. At Q-band frequencies experiments have been performed by probing either the high-spin Mn(2+) ion or the nitroxide spin label. At G-band frequencies we have been able to detect changes in the dipolar oscillation frequency, depending on the pump-probe positions across the g-tensor resolved nitroxide EPR spectrum. These changes result from the restricted mobility of the nitroxide spin label in the model compound. Our results demonstrate that the high-spin Mn(2+) ion can be used for precise distance measurements and open the doors for many biological applications, as naturally occurring Mg(2+) sites can be readily exchanged for Mn(2+).

High-field dynamic nuclear polarization in aqueous solutions.

Unexpected high DNP enhancements of more than 10 have been achieved in liquid water samples at room temperature and magnetic fields of 9.2 T (corresponding to 400 MHz (1)H NMR frequency and 260 GHz EPR frequency). The liquid samples were polarized in situ using a double-resonance structure, which allows simultaneous excitation of NMR and EPR transitions and achieves significant DNP enhancements at very low incident microwave power of only 45 mW. These results demonstrate the first important step toward the application of DNP to high-resolution NMR, increasing the sensitivity on biomolecules with small sample volumes and at physiologically low concentrations.

Nitroxide spin labeled RNA for long range distance measurements by EPR-PELDOR.

Long range distance measurement on RNA allow the determination of RNA folds. Here we report the site specific incorporation of nitroxide spin labels at U,C and A by "on column synthesis". PELDOR (Pulsed Electron Double Resonance) measurements of several RNAs in the range of 2-6 nm were successful.

Internal magnetic gradient fields in glass bead packs from numerical simulations and constant time diffusion spin echo measurements.

Internal magnetic field gradients in water saturated glass bead packs were studied by numerical simulations and a constant time spin echo (CTSE) experiment. The CTSE is comprised of two spin echo refocusing periods where each of the two evolution periods, tau1 and tau2, is varied so that the total evolution, 2(tau1 + tau2), is held constant. The experiment is similar to that introduced by Norwood and Quilter and allows the effects of dephasing due to diffusion in a magnetic field gradient to be separated from other relaxation mechanisms. In our experiments, the magnetic susceptibility difference between the pore fluid and glass beads creates the internal field gradient. CTSE measurements were performed at 7 T (300 MHz 1H) for water saturated in 50 microm diameter glass bead pack. We find that the internal gradients in the center of the pore bodies, where free diffusion applies, is in the range of 10 to 100 G/cm. This fluid volume accounts for < or =10% of the total pore volume. From direct numerical simulations of the internal magnetic field based on a first principles calculation, we find that the major fraction, >90%, of the pore volume has internal gradients of order 500 to 5,000 G/cm. Signals from water in these large gradients is not observed in our CTSE measurements.

ENDOR study of gamma-irradiated hydrated testosterone orthorhombic single crystals.

The molecular structure of free radicals formed in gamma-irradiated orthorhombic single crystals of hydrated testosterone was investigated by Electron Nuclear Double Resonance (ENDOR) spectroscopy. Only one kind of radical was observed, which is formed by addition of hydrogen atom to oxygen atom O(3). We observed interaction of the unpaired electron, which is delocalized on the carbons C(3), C(4) and C(5), with one alpha-proton in position 4 and with four unequivalent beta-protons connected with the carbon atoms C(2) and C(6). The matrices of the hyperfine couplings and the g-factor of the radical are given.