Electron paramagnetic resonance (EPR) spectroscopy provides a variety of tools to study structures and structural changes of large biomolecules or complexes thereof. In order to unravel secondary structure elements, domain arrangements or complex formation, continuous wave and pulsed EPR methods capable of measuring the magnetic dipole coupling between two unpaired electrons can be used to obtain long-range distance constraints on the nanometer scale. Such methods yield reliably and precisely distances of up to 80 A, can be applied to biomolecules in aqueous buffer solutions or membranes, and are not size limited. They can be applied either at cryogenic or physiological temperatures and down to amounts of a few nanomoles. Spin centers may be metal ions, metal clusters, cofactor radicals, amino acid radicals, or spin labels. In this review, we discuss the advantages and limitations of the different EPR spectroscopic methods, briefly describe their theoretical background, and summarize important biological applications. The main focus of this article will be on pulsed EPR methods like pulsed electron-electron double resonance (PELDOR) and their applications to spin-labeled biosystems.

31P NMR revealed that the complex of p21ras with the GTP analog GppNHp.Mg2+ exists in two conformational states, states 1 and 2. In wild-type p21ras the equilibrium constant K1(12) between the two states is 1.09. The population of these states is different for various mutants but independent of temperature. The activation enthalpy delta H ++ and activation entropy delta S ++ for the conformational transitions were determined by full-exchange matrix analysis for wild-type p21ras and p21ras(S65P). For the wild-type protein one obtains delta H ++ = 89 +/- 2 kJ mol-1 and delta S ++ = 102 +/- 20 J mol-1 K-1 and for the mutant protein delta H ++ = 93 +/- 7 kJ mol-1 and delta S ++ = 138 +/- 30 J mol-1 K-1. The study of various p21ras mutants suggests that the two states correspond to different conformations of loop L2, with Tyr-32 in two different positions relative to the bound nucleotide. High-field EPR at 95 GHz suggest that the observed conformational transition does not directly influence the coordination sphere of the protein-bound metal ion. The influence of this transition on loop L4 was studied by 1H NMR with mutants E62H and E63H. There was no indication that L4 takes part in the transition described in L2, although a reversible conformational change could be induced by decreasing the pH value. The exchange between the two states is slow on the NMR time scale (< 10 s-1): at approximately pH 5 the population of the two states is equal. The interaction of p21ras-triphosphate complexes with the Ras-binding domain (RBD) of the effector protein c-Raf-1, Raf-RBD, and with the GTPase activating protein GAP was studied by 31P NMR spectroscopy. In complex with Raf-RBD the second conformation of p21ras (state 2) is stabilized. In this conformation Tyr-32 is located in close proximity to the phosphate groups of the nucleotide, and the beta-phosphate resonance is shifted upfield by 0.7 ppm. Spectra obtained in the presence of GAP suggest that in the ground state GAP does not interact directly with the nucleotide bound to p21ras and does not induce larger conformational changes in the neighborhood of the nucleotide. The experimental data are consistent with a picture where GAP accelerates the exchange process between the two states and simultaneously increases the population of state 1 at higher temperature.

A new polarizing agent with superior performance in dynamic nuclear polarization experiments is introduced, and utilizes two TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) moieties connected through a rigid spiro tether (see structure). The observed NMR signal intensities were enhanced by a factor of 1.4 compared to those of TOTAPOL, a previously described TEMPO-based biradical with a flexible tether.

Radical ions of organic dyes are highly reactive species and have been studied for decades by transient absorption spectroscopy and pulse radiolysis experiments in oxygen-depleted solution. Here we show by continuous wave EPR, absorption, and fluorescence experiments that the triplet state of rhodamine dyes can be photoreduced by thiols to form stable radical anions in aqueous solution with a lifetime of up to several hours. Our data demonstrate that reduction of the triplet state and photoinduced oxidation of reactive intermediates by oxygen represents a general mechanism for reversible photoswitching of dyes in aqueous thiol-containing solutions highlighting the key role of molecular oxygen for super-resolution fluorescence imaging. Since cells contain the thiol glutathione at millimolar concentrations and reactive oxygen species are formed as side products our findings are of consequence for live cell fluorescence microscopy.

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.

In the Spring of 2013, NMR spectroscopists convened at the Weizmann Institute in Israel to brainstorm on approaches to improve the sensitivity of NMR experiments, particularly when applied in biomolecular settings. This multi-author interdisciplinary Review presents a state-of-the-art description of the primary approaches that were considered. Topics discussed included the future of ultrahigh-field NMR systems, emerging NMR detection technologies, new approaches to nuclear hyperpolarization, and progress in sample preparation. All of these are orthogonal efforts, whose gains could multiply and thereby enhance the sensitivity of solid- and liquid-state experiments. While substantial advances have been made in all these areas, numerous challenges remain in the quest of endowing NMR spectroscopy with the sensitivity that has characterized forms of spectroscopies based on electrical or optical measurements. These challenges, and the ways by which scientists and engineers are striving to solve them, are also addressed.

A pulsed electron paramagnetic resonance (EPR) spectroscopic ruler for oligonucleotides was developed using a series of duplex DNAs. The spin-labeling is accomplished during solid-phase synthesis of the oligonucleotides utilizing a palladium-catalyzed cross-coupling reaction between 5-iodo-2'-deoxyuridine and the rigid spin-label 2,2,5,5-tetramethyl-pyrrolin-1-yloxyl-3-acetylene (TPA). 4-Pulse electron double resonance (PELDOR) was then used to measure the intramolecular spin-spin distances via the dipolar coupling, yielding spin-spin distances of 19.2, 23.3, 34.7, 44.8, and 52.5 A. Employing a full-atom force field with explicit water, molecular dynamic (MD) simulations on the same spin-labeled oligonucleotides in their duplex B-form gave spin-spin distances of 19.6, 21.4, 33.0, 43.3, and 52.5 A, respectively, in very good agreement with the measured distances. This shows that the oligonucleotides adopt a B-form duplex structure also in frozen aqueous buffer solution. It also demonstrates that the combined use of site-directed spin-labeling, PELDOR experiments, and MD simulations can yield a microscopic picture about the overall structure of oligonucleotides. The technique is also applicable to more complex systems, like ribozymes or DNA/RNA-protein complexes, which are difficult to access by NMR or X-ray crystallography.

Electron paramagnetic resonance (EPR) spectroscopy is a powerful and widely used technique for studying structure and dynamics of biomolecules under bio-orthogonal conditions. In-cell EPR is an emerging area in this field; however, it is hampered by the reducing environment present in cells, which reduces most nitroxide spin labels to their corresponding diamagnetic N-hydroxyl derivatives. To determine which radicals are best suited for in-cell EPR studies, we systematically studied the effects of substitution on radical stability using five different classes of radicals, specifically piperidine-, imidazolidine-, pyrrolidine-, and isoindoline-based nitroxides as well as the Finland trityl radical. Thermodynamic parameters of nitroxide reduction were determined by cyclic voltammetry; the rate of reduction in the presence of ascorbate, cellular extracts, and after injection into oocytes was measured by continuous-wave EPR spectroscopy. Our study revealed that tetraethyl-substituted nitroxides are good candidates for in-cell EPR studies, in particular pyrrolidine derivatives, which are slightly more stable than the trityl radical.