In a lot of cases active biomolecules are complexes of higher order, thus methods capable of counting the number of building blocks and elucidating their geometric arrangement are needed. Therefore, we experimentally validate here spin-counting via 4-pulse electron-electron double resonance (PELDOR) on well-defined test samples. Two biradicals, a symmetric and an asymmetric triradical, and a tetraradical were synthesized in a convergent reaction scheme via palladium-catalyzed cross-coupling reactions. PELDOR was then used to obtain geometric information and the number of spin centers per molecule in a single experiment. The measurement yielded the expected distances (2.2-3.8 nm) and showed that different spin-spin distances in one molecule can be resolved even if the difference amounts to only 5 A. The number of spins n has been determined to be 2.1 in both biradicals, to 3.1 and 3.0 in the symmetric and asymmetric triradicals, respectively, and to 3.9 in the tetraradical. The overall error of PELDOR spin-counting was found to be 5% for up to four spins. Thus, this method is a valuable tool to determine the number of constituting spin-bearing monomers in biologically relevant homo- and heterooligomers and how their oligomerization state and geometric arrangement changes during function.

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.

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.

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The synthesis and isolation of 12 alpha-aryl, beta, beta'-disilyl-substituted vinyl cations 1b-l, 7, and 8 with the tetrakis(pentafluorophenyl)borate counteranion is reported. The vinyl cations are characterized by NMR spectroscopy and are identified by their specific NMR chemical shifts (delta13C(C(+)) = 178.1-194.5; delta13C (Cbeta) = 83.3-89.9; delta13C (Cipso)) = 113.6-115.2; delta (29)Si = 25.0-12.0), supported by density functional calculations at the B3LYP/6-311G(2d,p)//B3LYP/6-31G(d) level. All cations are found to be stable at room temperature in solution and in the solid state. The NMR chemical shifts as well as J-coupling data indicate for vinyl cations, 1b-l, 7, and 8, the occurrence of substantial stabilization through pi-resonance via the aryl substituents and through sigma-delocalization via the beta-silyl groups. For vinyl cation 8, the free enthalpy of stabilization via pi-resonance by the alpha-ferrocenyl substituent is determined by temperature-dependent (29)Si NMR spectroscopy to be DeltaG++ = (48.9 +/- 4.2) kJ mol(-1). A Hammett-type analysis, which relates the (1)J(SiC(beta)) coupling constant and the low-field shift of the (29)Si NMR signal upon ionization, Deltadelta (29)Si, with the electron-donating ability of the aryl group, indicates an inverse relation between the extent of Si-C hyperconjugation and pi-donation. The computed structures (at B3LYP/6-31G(d)) of the vinyl cations 1a-l, 7, and 8 reveal the consequences of Si-C hyperconjugation and of pi-resonance interactions with the aryl groups. The structures, however, fail to express the interplay between sigma-delocalization and pi-conjugation in that the calculated Si-C bond lengths and the C+-C(ipso) bond lengths do not vary as a function of the substituent.

We demonstrate the ability of pulsed electron double resonance (PELDOR) experiments to determine the orientation of spin labels in biological macromolecules. Thus, the distance information usually obtained from PELDOR data can be complemented by the mutual orientation of macromolecular domains. A method to determine the angle beta between the spin label normal and the interspin axis is proposed and analyzed mathematically. The obtained analytical expression allows extraction of angles beta without a fitting procedure if these angles are equal for both nitroxide of biradical. The method was applied to the experimental data gathered on ten spin-labeled DNA samples. The angles estimated from the PELDOR data are in excellent agreement with literature values.

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.

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.