High-frequency pulsed electron-electron double resonance spectroscopy on DNA duplexes using trityl tags and shaped microwave pulses.

Accurate distances between two trityl paramagnetic tags site-specifically attached to DNA duplexes were measured by pulsed electron-electron double resonance spectroscopy at 180 and 260 GHz microwave frequencies. Up to a threefold increase in the sensitivity of 260 GHz PELDOR measurements was achieved by using shaped broad-band microwave pulses.

Nitroxide-nitroxide and nitroxide-metal distance measurements in transition metal complexes with two or three paramagnetic centres give access to thermodynamic and kinetic stabilities.

Fundamentally, the stability of coordination complexes and of templated (bio)macromolecular assemblies depends on the thermodynamic and kinetic properties of the intermediates and final complexes formed. Here, we used pulse EPR (electron paramagnetic resonance) spectroscopy to determine the stabilities of nanoscopic assemblies formed between one or two nitroxide spin-labelled tridentate 2,2':6',2''-terpyridine (tpy) ligands and divalent metal ions (FeII, ZnII, CoII and CuII). In three distinct approaches we exploited (a) the modulation depth of pulsed electron-electron double resonance (PELDOR) experiments in samples with increasing metal-to-ligand ratios, (b) the frequencies of PELDOR under broadband excitation using shaped pulses and (c) the distances recovered from well-resolved PELDOR data in fully deuterated solvents measured at 34 GHz. The results demonstrate that PELDOR is highly sensitive to resolving the stability of templated dimers and allows to readily distinguish anti-cooperative binding (for CuII ions) from cooperative binding (for CoII or FeII ions). In the case of paramagnetic ions (CoII and CuII) the use of broadband PELDOR allowed to identify the cooperativity of binding from the time domain and distance data. By using a second labelled tpy ligand and by mixing two homoleptic complexes of the same metal centre we could probe the kinetic stability on a timescale of tens of seconds. Here, tpy complexes of CuII and ZnII were found to be substitutionally labile, CoII showed very slow exchange and FeII was inert under our conditions. Not only do our chemical models allow studying metal-ligand interactions via PELDOR spectroscopy, the design of our study is directly transferable to (bio)macromolecular systems for determining the kinetic and thermodynamic stabilities underpinning (templated) multimerisation. Considering the limited methods available to obtain direct information on the composition and stability of complex assemblies we believe our approach to be a valuable addition to the armoury of methods currently used to study these systems.

Determination of helix orientations in a flexible DNA by multi-frequency EPR spectroscopy.

Distance measurements are performed between a pair of spin labels attached to nucleic acids using Pulsed Electron-Electron Double Resonance (PELDOR, also called DEER) spectroscopy which is a complementary tool to other structure determination methods in structural biology. The rigid spin label Ç, when incorporated pairwise into two helical parts of a nucleic acid molecule, allows the determination of both the mutual orientation and the distance between those labels, since Ç moves rigidly with the helix to which it is attached. We have developed a two-step protocol to investigate the conformational flexibility of flexible nucleic acid molecules by multi-frequency PELDOR. In the first step, a library with a broad collection of conformers, which are in agreement with topological constraints, NMR restraints and distances derived from PELDOR, was created. In the second step, a weighted structural ensemble of these conformers was chosen, such that it fits the multi-frequency PELDOR time traces of all doubly Ç-labelled samples simultaneously. This ensemble reflects the global structure and the conformational flexibility of the two-way DNA junction. We demonstrate this approach on a flexible bent DNA molecule, consisting of two short helical parts with a five adenine bulge at the center. The kink and twist motions between both helical parts were quantitatively determined and showed high flexibility, in agreement with a Förster Resonance Energy Transfer (FRET) study on a similar bent DNA motif. The approach presented here should be useful to describe the relative orientation of helical motifs and the conformational flexibility of nucleic acid structures, both alone and in complexes with proteins and other molecules.

Flexibility and conformation of the cocaine aptamer studied by PELDOR.

The cocaine aptamer is a DNA three-way junction that binds cocaine at its helical junction. We studied the global conformation and overall flexibility of the aptamer in the absence and presence of cocaine by pulsed electron-electron double resonance (PELDOR) spectroscopy, also called double electron-electron resonance (DEER). The rigid nitroxide spin label Ç was incorporated pairwise into two helices of the aptamer. Multi-frequency 2D PELDOR experiments allow the determination of the mutual orientation and the distances between two Çs. Since Ç is rigidly attached to double-stranded DNA, it directly reports on the aptamer dynamics. The cocaine-bound and the non-bound states could be differentiated by their conformational flexibility, which decreases upon binding to cocaine. We observed a small change in the width and mean value of the distance distribution between the two spin labels upon cocaine binding. Further structural insights were obtained by investigating the relative orientation between the two spin-labeled stems of the aptamer. We determined the bend angle between this two stems. By combining the orientation information with a priori knowledge about the secondary structure of the aptamer, we obtained a molecular model describing the global folding and flexibility of the cocaine aptamer.

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.

RIDME spectroscopy on high-spin Mn2+ centers.

Pulsed EPR dipolar spectroscopy is a powerful tool for determining the structure and conformational dynamics of biological macromolecules, as it allows precise measurements of distances in the range of 1.5-10 nm. Utilization of high-spin Mn2+ species as spin probes for distance measurements is of significant interest, because they are biologically compatible and endogenous in numerous biological systems. However, to date dipolar spectroscopy experiments with this kind of species have been underexplored. Here we present pulsed electron electron double resonance (PELDOR also called DEER) and relaxation-induced dipolar modulation enhancement (RIDME) experiments, which have been performed at W-band (94 GHz) and J-band frequencies (263 GHz) on a bis-MnDOTA (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) model system. The distances obtained from these experiments are in good agreement with predictions. RIDME experiments reveal a significantly higher modulation depth compared to PELDOR, which is an important consideration for biological samples. These experiments also feature higher harmonics of the dipolar coupling frequency due to effective multiple-quantum relaxation of high-spin Mn2+ as well as the multiple-component background function. Harmonics of the dipolar coupling frequency were taken into account by including additional terms in the kernel function of Tikhonov regularization analysis.

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+).

Pulsed EPR dipolar spectroscopy at Q- and G-band on a trityl biradical.

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.

Single crystals of an ionic anthracene aggregate with a triplet ground state

Crystalline supramolecular aggregates consisting of charged organic molecules, held together through metal-cluster-mediated Coulomb interactions, have attracted interest owing to their unusual structural, chemical and electronic properties. Aggregates containing metal cation clusters 'wrapped' by lipophilic molecular anions have, for example, been shown to be kinetically stable and soluble in nonpolar liquids such as saturated hydrocarbons. The formation of supramolecular aggregates can even be exploited to generate aromatic hydrocarbons that carry four negative charges and crystallize in the form of organic poly(metal cation) clusters or helical polymers. Here we report the anaerobic crystallization of an ionic organic aggregate--a contact ion septuple consisting of a fourfold negatively charged 'tripledecker' of three anthracene molecules bridged by four solvated potassium cations. Its electronic ground state is shown experimentally, using temperature-dependent electron paramagnetic resonance spectroscopy, to be a triplet. Although the spins in this biradical ionic solid are separated by a considerable distance, density functional theory calculations indicate that the triplet ground state is 84 kJ mol(-1) more stable than the first excited singlet state. We expect that the successful crystallization of the ionic solid we report here, and that of a covalent organic compound with a triplet ground state at room temperature, will stimulate further attempts to develop new triplet-ground-state materials for practical use.

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.

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.

Continuous-flow DNP polarizer for MRI applications at 1.5 T.

Here we describe a new hyperpolarization approach for magnetic resonance imaging applications at 1.5 T. Proton signal enhancements of more than 20 were achieved with a newly designed multimode microwave resonator situated inside the bore of the imager and used for Overhauser dynamic nuclear polarization of the water proton signal. Different from other approaches in our setup the hyperpolarization is achieved continuously by liquid water flowing through the polarizer under continuous microwave excitation. With an available flow rate of up to 1.5 ml/min, which should be high enough for DNP MR angiography applications in small animals like mice and rats. The hyperpolarized liquid cooled to physiological temperature can be routed by a mechanical switch to a quartz capillary for injection into the blood vessels of the target object. This new approach allows hyperpolarization of protons without the need of an additional magnet and avoids the losses arising from the transfer of the hyperpolarized solution between magnets. The signal-to-noise improvement of this method is demonstrated on two- and three-dimensional phantoms of blood vessels.

Orientation selection in photosynthetic PS I multilayers: structural investigation of the charge separated state P(700)(+z.rad;)A(1)(-z.rad;) by high-field/high-frequency time-resolved EPR at 3.4 T/95 GHz.

The radical-pair state of the primary electron donor and the secondary electron acceptor (P(700)(+z.rad;)A(1)(-z.rad;)) of the photosynthetic reaction center (RC) photosystem I (PS I) of Synechocystis PCC 6803 was studied by time-resolved electron paramagnetic resonance (TREPR) at high field/high frequency (3.4 T/95 GHz) using orientation selection in multilayers. The goal of the present article is to work out the basis for future studies, in which the improved resolution of such multilayers may be used to detect mutation-induced structural changes of PS I in membrane preparations. This approach is particularly interesting for systems that cannot be prepared as single crystals. However, in order to use such multilayers for structural investigations of protein complexes, it is necessary to know their orientation distribution. PS I was chosen as a test example because the wild type was recently crystallized and its X-ray structure determined to 2.5 A resolution [Nature 411 (2001) 909]. On the basis of our experimental results we determined the orientation distribution. Furthermore, a simulation model for the general case in which the orientation distribution is not axially symmetric about the C(2) symmetry axis of the RC is developed and discussed. Spectra simulations show that changes in the TREPR spectra of PS I are much more significant for these oriented multilayers than for disordered samples. In this way the use of oriented multilayers, in conjunction with multifrequency TREPR measurements on oriented as well as on disordered samples, is a promising approach for studies of structural changes of PS I systems that are induced by point mutations.

Conformational dynamics of nucleic acid molecules studied by PELDOR spectroscopy with rigid spin labels.

Nucleic acid molecules can adopt a variety of structures and exhibit a large degree of conformational flexibility to fulfill their various functions in cells. Here we describe the use of Pulsed Electron-Electron Double Resonance (PELDOR or DEER) to investigate nucleic acid molecules where two cytosine analogs have been incorporated as spin probes. Because these new types of spin labels are rigid and incorporated into double stranded DNA and RNA molecules, there is no additional flexibility of the spin label itself present. Therefore the magnetic dipole-dipole interaction between both spin labels encodes for the distance as well as for the mutual orientation between the spin labels. All of this information can be extracted by multi-frequency/multi-field PELDOR experiments, which gives very precise and valuable information about the structure and conformational flexibility of the nucleic acid molecules. We describe in detail our procedure to obtain the conformational ensembles and show the accuracy and limitations with test examples and application to double-stranded DNA.

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.

Sterically shielded spin labels for in-cell EPR spectroscopy: analysis of stability in reducing environment.

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.

Fourier-transform EPR at high-field/high-frequency (3.4 T/95 GHz) using broadband stochastic microwave excitation.

Stochastic excitation with a full-width-half-maximum bandwidth of 250 MHz was used to perform Fourier-transform (FT) high-field/high-frequency electron paramagnetic resonance (EPR) at 3.4T/95 GHz (W-band). Thereby, the required microwave peak power is reduced by a factor of tau(p)/T1 as compared to equivalent pulsed FT EPR in which the spin system with spin-lattice relaxation time T1 is excited by a single microwave pulse of length tau(p). Stochastic EPR is particularly interesting under high-field/high-frequency conditions, because the limited output power of mm microwave sources, amplifiers, and mixers makes pulse FT EPR in that frequency domain impossible, at least for the near future. On the other hand, FT spectroscopy offers several advantages compared to field-swept magnetic resonance methods, as is demonstrated by its success in NMR and X-band EPR. In this paper we describe a novel stochastic W-band microwave bridge including a bimodal induction mode transmission resonator that serves for decoupling the microwave excitation and signal detection. We report first EPR measurements and discuss experimental difficulties as well as achieved sensitivity. Moreover, we discuss future improvements and the possibility for an application of stochastic W-band FT EPR to transient signals such as those of photoexcited radical pairs in photosynthetic reaction centers.

Relaxation filtered hyperfine spectroscopy (REFINE).

A simple and general applicable method to separate spectrally overlapping hyperfine spectra of two paramagnetic compounds is presented. Overlapping spectral contributions from different paramagnetic species are a common situation in electron paramagnetic resonance (EPR) spectroscopy, resulting in complicate EPR spectra of metal enzymes, organic radicals or in the field of material sciences. On the other hand, the longitudinal relaxation times T1 of these species contributing to the overall EPR signal can vary by several orders of magnitude, depending on the paramagnetic component under study. These differences can be used to selectively study individual species by using an inversion-recovery preparation sequence as a filter. Here, we demonstrate the possibility to separate hyperfine spectra of two spectrally overlapping paramagnetic species by combining an inversion-recovery based relaxation filter together with ESEEM or ENDOR hyperfine spectroscopy (REFINE). The feasibility of the presented method is demonstrated on model compounds and the necessary requirements are discussed.

A new facile method for spin-labeling of oligonucleotides.

A new facile method for spin-labeling suitable for DNA and RNA oligonucleotides is presented. The nitroxide 3-ethenyl-2,2,5,5-tetramethyl-pyrrolin-1-yloxy was directly introduced during automated solid-phase synthesis by a Pd(0) cross coupling reaction. The main advantages of this procedure are the small amount of spin-label needed for the derivatisation of the oligonucleotide and the high coupling efficiency on the solid phase.