Dominik Margraf

Relative Orientation of Rigid Nitroxides by PELDOR: Beyond Distance Measurements in Nucleic Acids

Show me your angle: Incorporation of the rigid spin label C allows determination of both distance and orientation of two nitroxide spin labels in DNA by PELDOR experiments at common X-band frequencies. The orientational information is obtained by varying the position of the detection pulses over the nitroxide spectrum. Simulation of the set of time traces yields very precise distances and angles.

Conformational Flexibility of DNA

Pulsed Electron-Electron Double Resonance (PELDOR) on double-stranded DNA (ds-DNA) was used to investigate the conformational flexibility of helical DNA. Stretching, twisting, and bending flexibility of ds-DNA was determined by incorporation of two rigid nitroxide spin labels into a series of 20 base pair (bp) DNA duplexes. Orientation-selective PELDOR experiments performed at both X-band (9 GHz/0.3 T) and G-band (180 GHz/6.4 T) with spin label distances in the range of 2-4 nm allowed us to differentiate between different simple models of DNA dynamics existing in the literature. All of our experimental results are in full agreement with a dynamic model for ds-DNA molecules, where stretching of the molecule leads to a slightly reduced radius of the helix induced by a cooperative twist-stretch coupling.

Conformational flexibility of nitroxide biradicals determined by X-band PELDOR experiments

PELDOR (pulsed electron–electron double resonance) experiments have been performed at X-band (9 GHz) frequencies on a linear and a bent nitroxide biradical. All PELDOR time traces were recorded with the pump frequency νB set at the center of the nitroxide spectra to achieve maximum pumping efficiency, while the probe frequency νA was stepped between a frequency offset ΔνAB = νA − νB of +40 to +80 MHz. The modulation frequencies and the damping of the oscillations change as a function ΔνAB, whereas the modulation depth λ for our investigated systems was only very slightly altered. This can be explained by the selection of different orientations of nitroxide radicals with respect to the external magnetic field as a function of frequency offset. Quantitative simulations of the PELDOR time traces could be achieved for both molecules and for all offset frequencies using a simple geometric model, described by a free rotation of the nitroxide radical around its acetylene bond and a single bending mode of the interconnecting molecular bridge. The results show that the distribution function for the relative orientations of the nitroxides with respect to each other and with respect to the dipolar vector R deviates from a random distribution and thus has to be taken into account to quantitatively simulate the PELDOR traces. Vice versa, a quantitative simulation of PELDOR time traces with variable offset frequencies allows the determination of the conformational freedom of such molecules.

Molecular orientation studies by pulsed electron-electron double resonance experiments

Pulsed electron-electron double resonance (PELDOR) has proven to be a valuable tool to measure the distribution of long range distances in noncrystalline macromolecules. These experiments commonly use nitroxide spin labels as paramagnetic markers that are covalently attached to the macromolecule at specific positions. Unless these spin labels are flexible in such a manner that they exhibit an almost random orientation, the PELDOR signals will-apart from the interspin distance-also depend on the orientation of the spin labels. This effect needs to be considered in the analysis of PELDOR signals and can, moreover, be used to obtain additional information on the structure of the molecule under investigation. In this work, we demonstrate that the PELDOR signal can be represented as a convolution of a kernel function containing the distance distribution function and an orientation intensity function. The following strategy is proposed to obtain both functions from the experimental data. In a first step, the distance distribution function is estimated by the Tikhonov regularization, using the average over all PELDOR time traces with different frequency offsets and neglecting angular correlations of the spin labels. Second, the convolution relation is employed to determine the orientation intensity function, using again the Tikhonov regularization. Adopting small nitroxide biradical molecules as simple examples, it is shown that the approach works well and is internally consistent. Furthermore, independent molecular dynamics simulations are performed and used to calculate PELDOR signals, distance distributions, and orientational intensity functions. The calculated and experimental results are found to be in excellent overall agreement.

Structure and dynamics of nucleic acids.

In this chapter we describe the application of CW and pulsed EPR methods for the investigation of structural and dynamical properties of RNA and DNA molecules and their interaction with small molecules and proteins. Special emphasis will be given to recent applications of dipolar spectroscopy on nucleic acids.

Biphenyl-4,4′-diyl bis­(2,2,5,5-tetra­methyl-1-oxyl-3-pyrroline-3-carboxyl­ate)

In the title compound, C30H34N2O6, the complete molecule is generated by a crystallographic 2/m symmetry operation. The 1-oxyl-3-pyrroline-3-carboxylate group lies on a mirror plane. The dihedral angle between the ring planes of the biphenyl fragment is constrained by symmetry to be zero, resulting in rather short intramolecular H⋯H contact distances of 2.02 Å. In the crystal, molecules are connected along the a-axis direction by very weak intermolecular methyl–phenyl C—H⋯π interactions. The C—H bond is not directed to the center of the benzene ring, but mainly to one C atom [C—H⋯C(x − 1, y, z): H⋯C = 2.91 Å and C—H⋯C = 143°].

4,4′,4′′-(Methane­triyl)triphenyl tris­(2,2,5,5-tetra­methyl-1-oxyl-3-pyrroline-3-carboxyl­ate) benzene tris­olvate

In the asymmetric unit of the title compound, C46H52N3O9·3C6H6, two of the benzene solvent molecules are located in general positions and two are disposed about inversion centers. One of the benzene molecules on an inversion center was grossly disordered and was excluded using the SQUEEZE subroutine in PLATON [Spek (2009 ▶). Acta Cryst. D65, 148–155]. In addition, one of the 2,2,5,5-tetramethyl-1-oxyl-3-pyrrolin-3-ylcarbonyl groups is disordered over two orientations with refined occupancies of 0.506 (2) and 0.494 (2). The 1-oxyl-3-pyrroline-3-carboxylate groups are essentially planar, with mean deviations from the planes of 0.026 (2), 0.012 (2), 0.034 (4) and 0.011 (4) Å. In the crystal structure, molecules are connected by five weak intermolecular C—H⋯O and four weak intermolecular C—H⋯π(benzene) interactions.