Pulsed Electron-electron Double Resonance Research Articles

Investigation of the Potra Domains from Cyanobacterial Omp85 by Peldor Spectroscopy

Omp85 proteins contain a C-terminal transmembrane beta-barrel and a soluble N terminus with a varying number of polypeptide-transport-associated (POTRA) domains.1 N-terminal POTRA domains (P1 and P2) of Omp85 from the cyanobacterium Anabaena sp. PCC 7120 might have functions in substrate recognition and heterooligomerization.2 P3 is implied in regulation of protein transport by its L1-loop. Molecular dynamics (MD) simulations predicted that P2 and P3 are fixed in orientation, consistent with a short connection and a large interface between both domains, and that there is a hinge between P1 and P2.2 In this study we used site-directed spin labeling (SDSL) to investigate the conformational flexibility between POTRA domains by PELDOR (pulsed electron-electron double resonance) spectroscopy.3-5 The experimental results will be compared with the MD calculations and X-ray structures.2 Further studies of the interaction of POTRA domains with chaperones and substrates are underway.

Manganese Cation Reports on the Number of Coordinated Phosphates from the Nucleotide at the Active Site in Myosin ATPase

ATP is an important cofactor for myosin ATPase. ATP binding initiates myosin conformation change (recovery stroke), priming it for force generation. In the presence of a divalent cation (usually Mg2+), myosin hydrolyzes ATP to ADP and phosphate, and their subsequent release presumably triggers the power stroke in actomyosin. What is the role of ATP hydrolysis in myosin? What is the relation between the kinetics of ATP hydrolysis and those of myosin conformation changes? To address these problems, we propose to use pulsed electron paramagnetic resonance (EPR) techniques and study the coordination environment of the divalent cation complexed with a nucleotide at the myosin active site. To enable the EPR investigations, Mn2+ can be used instead of Mg2+ without interruption of the myosin ATPase activity. In this preliminary work, we studied the myosin.Mn complexes with non-hydrolyzable nucleotide analogs, AMPPNP (triphosphate) and ADP.AlF4 (diphosphate), as well as Mn.nucleotide complexes without myosin. Mn2+ binding to myosin was monitored by pulsed electron-electron double resonance (ELDOR), which detected the magnetic dipole interaction between Mn2+ and a nitroxide spin probe attached to the A639C myosin mutant. The number of coordinated phosphates in myosin.Mn.AMPPNP complex determined by 31P pulsed electron-nuclear double resonance (ENDOR) was twice that detected for the myosin.Mn.ADP.AlF4 complex. These results demonstrate the potential of pulsed EPR for distinguishing between the myosin.ATP and myosin.ADP biochemical states and study the role of ATP hydrolysis in myosin structural kinetics.

Distance Determination in Multimeric Membrane Protein by Symmetry Constrained Analysis of Double Electron-Electron Resonance Spectroscopy

Distance determinations are often a core strategy to decipher the molecular mechanism by which membrane proteins execute their biological function. EPR spectroscopy and pulsed Double Electron-Electron Resonance (DEER) methods are unparalleled tools for the measurement of probe-based long-range distances in protein. However, distance determination from echo intensity modulation in pulsed DEER experiments is a moderately ill-posed problem. Here, we show that by using protein symmetry-based geometrical constraints in homo-oligomeric membrane proteins, we are able to greatly facilitate the fitting solution. We modeled the distance distribution with 2 Gaussians, with each mean distance identified by its Gaussian peak. During the fit process, the mean distance ratio was constrained to be within a specified tolerance of the theoretical distance ratio determined by the symmetry of the oligomeric assembly (1:1.4 for tetramers; 1:1.6 for pentamers). We compared this approach against the classical Tikhonov regularization, concluding that our method stabilizes a solution that is much easier to interpret in molecular modeling terms. Importantly, we also show that when the qualities of the dipolar evolution signal increases (more observed periods and higher SNR), there is little or no difference in the distance distribution obtained by our method vs. Tikhonov regularization. This suggests that the geometrically constrained fit does not artificially distort the distance distribution. Our approach was validated on 2 different ion channels of different oligomeric states: CorA, a homopentameric Mg2+ channel and KcsA, a homotetrameric K+ channel. In all cases, the distances obtained by DEER are in excellent agreement with respective crystal structures.

Spectroscopic selection of distance measurements in a protein dimer with mixed nitroxide and Gd3+ spin labels

The pulse DEER (Double Electron-Electron Resonance) technique is frequently applied for measuring nanometer distances between specific sites in biological macromolecules. In this work we extend the applicability of this method to high field distance measurements in a protein assembly with mixed spin labels, i.e. a nitroxide spin label and a Gd(3+) tag. We demonstrate the possibility of spectroscopic selection of distance distributions between two nitroxide spin labels, a nitroxide spin label and a Gd(3+) ion, and two Gd(3+) ions. Gd(3+)-nitroxide DEER measurements possess high potential for W-band long range distance measurements (6 nm) by combining high sensitivity with ease of data analysis, subject to some instrumental improvements.

Pulsed electron spin nutation spectroscopy of weakly exchange-coupled biradicals: a general theoretical approach and determination of the spin dipolar interaction

Weakly exchange-coupled biradicals have attracted much attention in terms of their DNP application in NMR spectroscopy for biological systems or the use of synthetic electron-spin qubits. Pulse-ESR based electron spin nutation (ESN) spectroscopy applied to biradicals is generally treated as transition moment spectroscopy from the theoretical side, illustrating that it is a powerful and facile tool to determine relatively short distances between weakly exchange-coupled electron spins. The nutation frequency as a function of the microwave irradiation strength ω(1) (angular frequency) for any cases of weakly exchange-coupled systems can be classified into three categories; D(12) (spin dipolar interaction)-driven, Δg-driven and ω(1)-driven nutation behaviour with the increasing strength of ω(1). For hetero-spin biradicals, Δg effects can be a dominating characteristic in the biradical nutation spectroscopy. Two-dimensional pulse-based electron spin nutation (2D-ESN) spectroscopy operating at the X-band can afford to determine small values of D(12) in weakly exchange-coupled biradicals in rigid glasses. The analytical expressions derived here for ω(1)-dependent nutation frequencies are based on only four electronic spin states relevant to the biradicals, while real biradical systems often have sizable hyperfine interactions. Thus, we have evaluated nuclear hyperfine effects on the nutation frequencies to check the validity of the present theoretical treatment. The experimental spin dipolar coupling of a typical TEMPO-based biradical 1, (2,2,6,6-tetra[((2)H(3))methyl]-[3,3-(2)H(2),4-(2)H(1),5,5-(2)H(2)]piperidin-N-oxyl-4-yl)(2,2,6,6-tetra[((2)H(3))methyl]-[3,3-(2)H(2),4-(2)H(1),5,5-(2)H(2),(15)N]piperidin-(15)N-oxyl-4-yl) terephthalate in a toluene glass, with a distance of 1.69 nm between the two spin sites is D(12) = -32 MHz (the effect of the exchange coupling J(12) is vanishing due to the homo-spin sites of 1, i.e.Δg = 0), while 0 < |J(12)|≦ 1.0 MHz as determined by simulating the random-orientation CW ESR spectra of 1. In addition, we have carried out Q-band pulsed ELDOR (ELectron-electron DOuble Resonance) experiments to confirm whether the obtained values for D(12) and J(12) are accurate. The distance is in a fuzzy region for the distance-measurements capability of the conventional, powerful ELDOR spectroscopy. The strong and weak points of the ESN spectroscopy with a single microwave frequency applicable to weakly exchange-coupled multi-electron systems are discussed in comparison with conventional ELDOR spectroscopy. The theoretical spin dipolar tensor and exchange interaction of the TEMPO biradical, as obtained by sophisticated quantum chemical calculations, agree with the experimental ones.

Photo-Induced Electron Spin Polarization in Chemical and Biological Reactions: Probing Structure and Dynamics of Transient Intermediates by Multifrequency EPR Spectroscopy

In this minireview, modern multifrequency electron paramagnetic resonance (EPR) spectroscopy, in particular, at high magnetic fields, is shown to provide detailed information about structure, motional dynamics and spin chemistry of transient radicals and radical pairs occurring in photochemical reactions. Examples discussed comprise spin-polarized radicals and radical pairs in disordered systems, such as ultraviolet-irradiated quinone and ketone compounds in fluid alcohol solutions, green-light initiated electron transfer in biomimetic porphyrin–quinone donor–acceptor model systems in frozen solution, aiming at artificial photosynthesis, and red-light initiated electron transfer in natural photosynthetic reaction-center protein complexes. The transient paramagnetic states exhibit characteristic electron polarization (CIDEP) effects originating from a triplet mechanism, a radical-pair mechanism or a correlated coupled radical-pair mechanism. They contain valuable information about structure and dynamics of the short-lived reaction intermediates. Moreover, the CIDEP effects can be exploited for signal enhancement. Continuous-wave and pulsed versions of time-resolved high-field EPR spectroscopy, such as transient EPR and electron spin-echo experiments, are compared with respect to their advantages and limitations for the specific photoreaction under study. Furthermore, orientation resolving W-band pulsed electron-electron double resonance (PELDOR) experiments on the spin-correlated coupled radical pair $$ {\text{P}}_{865}^{ \cdot + } $$ $$ {\text{Q}}_{\text{A}}^{ \cdot - } $$ in frozen solution reaction centers from the purple photosynthetic bacterium Rb. sphaeroides reveal details of distance and orientation of the pair partners in their charge-separated transient state. The results are compared with those of the ground-state P865QA. In conjunction with Q-band proton electron-nuclear double resonance (ENDOR) experiments the W-band PELDOR results provide decisive evidence that the local structure of the QA binding site does not change under photoreduction of the quinone—in agreement with earlier FTIR studies. The examples given demonstrate that multifrequency EPR experiments on disordered systems add heavily to the capabilities of “classical” spectroscopic and diffraction techniques for determining structure–dynamics–function relations of biochemical processes, since short-lived intermediates can be observed in real time while staying in their working states at biologically relevant time scales.

High-Field Dipolar Electron Paramagnetic Resonance (EPR) Spectroscopy of Nitroxide Biradicals for Determining Three-Dimensional Structures of Biomacromolecules in Disordered Solids

We consider the state-of-the-art capabilities and future perspectives of electron-spin triangulation by high-field/high-frequency dipolar electron paramagnetic resonance (EPR) techniques designed for determining the three-dimensional structure of large supra-molecular complexes dissolved in disordered solids. These techniques combine double site-directed spin labeling (SDSL) with orientation-resolving pulsed electron-electron double resonance (PELDOR) spectroscopy. In particular, we appraise the prospects of angular triangulation, which extends the more familiar distance triangulation. As a model case for spin-labeled proteins, the three-dimensional structures of two nitroxide biradicals with rather stiff bridging blocks and deuterated nitroxide headgroups have been derived. To this end we applied 95 GHz high-field electron dipolar EPR spectroscopy with the microwave pulse-sequence configurations for PELDOR and relaxation-induced dipolar modulation enhancement (RIDME). Various specific spectroscopic strategies are discussed to overcome the problems of overlapping spectra of the chemically identical nitroxide labels when attached to macromolecular systems. We conclude that due to the high detection sensitivity and spectral resolution the combination of SDSL with high-field RIDME/PELDOR stands out as an extremely powerful tool for 3D structure determination of large disordered systems. The approach compares favorably with other structure-determining magnetic-resonance methods. This holds true both for stable and transient radical-pair states. Angular constraints are provided in addition to distance constraints obtained for the same sample. Thereby, the number of necessary distance constraints is strongly reduced. Since each measurement of a distance constraint requires an additional doubly spin-labeled sample, the reduction of necessary distance constraints is another appealing aspect of orientation-resolving EPR spin triangulation for protein structure determination.

Nonselective excitation of pulsed ELDOR using multi-frequency microwaves

The use of a polychromatic microwave pulse to expand the pumping bandwidth in pulsed electron–electron double resonance (PELDOR) was investigated. The pumping pulse was applied in resonance with the broad (∼100 mT) electron paramagnetic resonance (EPR) signal of the manganese cluster of photosystem II in the S 2 state. The observation pulses were in resonance with the narrow EPR signal of the tyrosine radical, Y D . It was found that in the case of the polychromatic pumping pulse containing five harmonics with the microwave frequencies between 8.5 and 10.5 GHz the PELDOR effect corresponding to the dipole interaction between the Mn cluster and Y D was about 2.9 times larger than that achieved with a monochromatic pulse. In addition to the dipolar modulation, the nuclear modulation effects were observed. The effects could be suppressed by averaging the PELDOR trace over the time interval between the observation microwave pulses. The polychromatic excitation technique described will be useful for improving the PELDOR sensitivity in the measurements of long distances in biological samples, where the pair consists of a radical with a narrow EPR spectrum and slow phase relaxation, and a metal center that has a broad EPR spectrum and a short phase relaxation time.

Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy

A disulfide-linked nitroxide side chain (R1) is the most widely used spin label for determining protein topology, mapping structural changes, and characterizing nanosecond backbone motions by site-directed spin labeling. Although the internal motion of R1 and the number of preferred rotamers are limited, translating interspin distance measurements and spatial orientation information into structural constraints is challenging. Here, we introduce a highly constrained nitroxide side chain designated RX as an alternative to R1 for these applications. RX is formed by a facile cross-linking reaction of a bifunctional methanethiosulfonate reagent with pairs of cysteine residues at i and i + 3 or i and i + 4 in an α-helix, at i and i + 2 in a β-strand, or with cysteine residues in adjacent strands in a β-sheet. Analysis of EPR spectra, a crystal structure of RX in T4 lysozyme, and pulsed electron-electron double resonance (ELDOR) spectroscopy on an immobilized protein containing RX all reveal a highly constrained internal motion of the side chain. Consistent with the constrained geometry, interspin distance distributions between pairs of RX side chains are narrower than those from analogous R1 pairs. As an important consequence of the constrained internal motion of RX, spectral diffusion detected with ELDOR reveals microsecond internal motions of the protein. Collectively, the data suggest that the RX side chain will be useful for distance mapping by EPR spectroscopy, determining spatial orientation of helical segments in oriented specimens, and measuring structural fluctuations on the microsecond time scale.

Nitrogen nuclear spin flips in nitroxide spin probes of different sizes in glassy o-terphenyl: Possible relation with α- and β-relaxations

The pulsed electron-electron double resonance (ELDOR) technique was employed to study nitroxide spin probes of three different sizes dissolved in glassy o-terphenyl. A microwave pulse applied to the central hyperfine structure (hfs) component of the nitroxide electron paramagnetic resonance spectrum was followed by two echo-detecting pulses of different microwave frequency to probe the magnetization transfer (MT) to the low-field hfs component. The MT between hfs components is readily related to flips in the nitrogen nuclear spin, which in turn are induced by molecular motion. The MT on the time scale of tens of microseconds was observed over a wide temperature range, including temperatures near and well below the glass transition. For a bulky nitroxide, it was found that MT rates approach dielectric α (primary) relaxation frequencies reported for o-terphenyl in the literature. For small nitroxides, MT rates were found to match the frequencies of dielectric β (secondary) Johari-Goldstein relaxation. The most probable motional mechanism inducing the nitrogen nuclear spin flips is large-angle angular jumps, between some orientations of unequal occupation probabilities. The pulsed ELDOR of nitroxide spin probes may provide additional insight into the nature of Johari-Goldstein relaxation in glassy media and may serve as a tool for studying this relaxation in substances consisting of non-rigid molecules (such as branched polymers) and in heterogeneous and non-polar systems (such as a core of biological membranes).

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.

Pulsed EPR Analysis of the Photo-Induced Triplet Radical Pair in the BLUF Protein SyPixD: Determination of the Protein–Protein Distance and Orientation in the Oligomeric Protein

A photo-induced radical pair of FADH· and Y8· and in BLUF protein SyPixD was studied by pulsed electron paramagnetic resonance (EPR) spectroscopy. Blue light illumination at 150 K for 30 min followed by cooling to 50 K during illumination induced the stable radical pair. The EPR signal has been characterized by a Pake doublet signal with complete S = 1 spin state. The radical pair was utilized as a probe to analyze the oligomer of SyPixD. The relative arrangement of PixD proteins in the complex was investigated by pulsed electron–electron double resonance (PELDOR) with the orientation selection. Based on the decameric structure in the crystal, the possible structure for the PELDOR results was discussed.

Effect of Pumping Pulse Duration on Echo Signal Amplitude in Four-Pulse PELDOR

The impact of pumping pulse duration on four-pulse pulsed electron–electron double resonance (PELDOR) data was experimentally studied. For biradicals with known distances between two spin labels, it is shown that refocused echo amplitude decreases with increasing the pumping pulse duration and decreasing the distance between spin labels. The effect becomes substantial when the pumping pulse duration is comparable or exceeds the inverse value of the dipole–dipole interaction between spin labels. This effect is essential for determination of distance distribution between labels in double-labeled molecules and for determination of the number of labels in clusters of spin-labeled molecules. PELDOR signal distortion was observed when the pumping pulse position in the time scale coincided with those of the detecting pulses. An approach of signal correction to eliminate this distortion is proposed.

Pulsed electron–electron double resonance (PELDOR) distance measurements in detergent micelles

Pulsed electron−electron double resonance (PELDOR) spectroscopy is a powerful tool for measuring nanometer distances in spin-labeled systems. A common approach is doubly covalent spin-labeling of a macromolecule and measurement of the inter-spin distance, or to use singly-labeled components of a system that forms aggregates or oligomers. This situation has been described as a spin-cluster. The PELDOR signal, however, does not only contain the desired dipolar coupling between the spin-labels of the molecule or cluster under study. In samples of finite concentration the dipolar coupling between the spin-labels of the randomly distributed molecules or spin-clusters also contributes significantly. In homogeneous frozen solutions or lipid vesicle membranes this second contribution can be considered to be an exponential or stretched exponential decay, respectively. In this study, we show that this assumption is not valid in detergent micelles. Spin-labeled fatty acids that are randomly partitioned into different detergent micelles give rise to PELDOR time traces which clearly deviate from stretched exponential decays. The obtained signals can be modeled quantitatively based on the size of the micelles, their aggregation number, the spin-label concentration and the degree of spin-labeling. As a main conclusion a PELDOR signal deviating from a stretched exponential decay does not necessarily prove the observation of specific distance information on the molecule or cluster. These results are important for the interpretation of PELDOR experiments on membrane proteins or lipophilic peptides solubilized in detergent micelles or small vesicles, which often do not show pronounced dipolar oscillations in their time traces.

Pulsed electron–electron double resonance: beyond nanometre distance measurements on biomacromolecules

PELDOR (or DEER; pulsed electron-electron double resonance) is an EPR (electron paramagnetic resonance) method that measures via the dipolar electron-electron coupling distances in the nanometre range, currently 1.5-8 nm, with high precision and reliability. Depending on the quality of the data, the error can be as small as 0.1 nm. Beyond mere mean distances, PELDOR yields distance distributions, which provide access to conformational distributions and dynamics. It can also be used to count the number of monomers in a complex and allows determination of the orientations of spin centres with respect to each other. If, in addition to the dipolar through-space coupling, a through-bond exchange coupling mechanism contributes to the overall coupling both mechanisms can be separated and quantified. Over the last 10 years PELDOR has emerged as a powerful new biophysical method without size restriction to the biomolecule to be studied, and has been applied to a large variety of nucleic acids as well as proteins and protein complexes in solution or within membranes. Small nitroxide spin labels, paramagnetic metal ions, amino acid radicals or intrinsic clusters and cofactor radicals have been used as spin centres.

Studying Protein DNA Binding with PELDOR

Pulsed Electron-Electron Double Resonance (PELDOR or DEER) is an Electron Paramagnetic Resonance (EPR) method by which distance can be measured very precisely in the range from ∼1-8nm1. As Fluorescence methods, PELDOR usually requires labels but which are considerable smaller and rigid2 making a translation of distance into structure easier.To overcome challenging label synthesis, we introduce the next generation of labels for DNA/RNA which make use of non-covalent binding. We can show that the specificity and strength of binding is large enough to yield high-quality PELDOR data yielding not only distances but also information on orientations and dynamics. This non-covalent labelling concept proves also successful for the study of protein binding and concomitant oligonucleotide bending.Labeling the helicases Hel308 and PcrA, we have been able to resolve the ATP, ADP, and DNA induced conformational switching of these molecular motors.This clearly shows that PELDOR does not only provide mere distances but access to orientations, dynamics and changes between conformational states of large oligonucleotide/protein complexes difficult to access otherwise.1) O. Schiemann, T.F. Prisner Quart. Rev. Biophys. 2007, 40, 1.2) O. Schiemann, P. Cekan, D. Margraf, T.F. Prisner, S.T. Sigurdsson Angew. Chem. 2009, 121, 3342.

The Histone Chaperones Nap1 and Vps75 Bind Histones H3 and H4 in a Tetrameric Conformation

SummaryHistone chaperones physically interact with histones to direct proper assembly and disassembly of nucleosomes regulating diverse nuclear processes such as DNA replication, promoter remodeling, transcription elongation, DNA damage, and histone variant exchange. Currently, the best-characterized chaperone-histone interaction is that between the ubiquitous chaperone Asf1 and a dimer of H3 and H4. Nucleosome assembly proteins (Nap proteins) represent a distinct class of histone chaperone. Using pulsed electron double resonance (PELDOR) measurements and protein crosslinking, we show that two members of this class, Nap1 and Vps75, bind histones in the tetrameric conformation also observed when they are sequestered within the nucleosome. Furthermore, H3 and H4 trapped in their tetrameric state can be used as substrates in nucleosome assembly and chaperone-mediated lysine acetylation. This alternate mode of histone interaction provides a potential means of maintaining the integrity of the histone tetramer during cycles of nucleosome reassembly.

Enzyme Kinetics Studies of Spin-Labeled Active Human Immunodeficiency Virus Type 1 protease

The immunodeficiency virus type 1 protease (HIV-1PR) is a homodimer comprised of 99 amino acid subunits, it functions as an aspartic protease that plays a crucial as role in the maturation of HIV-1. The protease post-translationally cleaves viral polyproteins gag and gag-pol, which makes it a target of AIDS antiviral therapy. The protease has an active site comprised of a D25 residue from each subunit. Accessibility of large, polypeptide substrate to the active site is mediated by a conformational change of two B-hairpin flaps that exposes the active site. The pulsed EPR technique double electron-electron resonance (DEER) has been utilized to measure distances between selected sites on the protease. For spin label incorporation the mutation K55C selected to generate distance profiles. We have shown previously, via traditional SDSL-EPR approaches, that the FDA-approved HIV-1PR inhibitors induce changes in the conformational sampling of the flap regions of the protease. Work described here will determine if spin label incorporation perturbs the kinetics of HIV-1PR constructs analyzed via DEER analysis. Data will ultimately yield rate constants for spin labeled HIV-1PR that will be compared to unlabeled HIV-1PR rate constants to determine if spin label incorporation perturb subtype constructs.

Studying biomolecular complexes with pulsed electron–electron double resonance spectroscopy

The function of biomolecules is intrinsically linked to their structure and the complexes they form during function. Techniques for the determination of structures and dynamics of these nanometre assemblies are therefore important for an understanding on the molecular level. PELDOR (pulsed electron-electron double resonance) is a pulsed EPR method that can be used to reliably and precisely measure distances in the range 1.5-8nm, to unravel orientations and to determine the number of monomers in complexes. In conjunction with site-directed spin labelling, it can be applied to biomolecules of all sizes in aqueous solutions or membranes. PELDOR is therefore complementary to the methods of X-ray crystallography, NMR and FRET (fluorescence resonance energy transfer) and is becoming a powerful method for structural determination of biomolecules. In the present review, the methods of PELDOR are discussed and examples where PELDOR has been used to obtain structural information on biomolecules are summarized.

PELDOR analysis of enzyme-induced structural changes in damaged DNA duplexes

PELDOR (pulsed electron-electron double resonance) spectroscopy was applied to determine spin-spin distances in spin-labeled DNA duplexes (13-mer and 17-mer) containing the damaged sites 8-oxoguanine or uncleavable abasic site analogue tetrahydrofuran. The lesions were located in one strand of the DNA, and two nitroxyl spin labels were attached at the 5'- and 3'-ends of the complementary strand. PELDOR data allow us to obtain distances between the two spin labels in DNAs, which turned out to be around 5 nm for the 13-mer DNA and around 6 nm for 17-mer DNA. Results of PELDOR measurements were supported by molecular dynamics calculations. Study of the interaction of DNA fragments with DNA repair enzyme 8-oxoguanine-DNA glycosylase from E. coli (Fpg protein) showed that this interaction leads to a noticeable decrease of the distance between spin labels, which indicates the enzyme-induced bending of the DNA duplex. This bending may be important for the mechanisms of recognition of damaged sites by DNA repair enzymes.