Continuous Wave Electron Paramagnetic Resonance Research Articles

Synthetic and Biophysical Studies on the Toxic Conformer in Amyloid β with the E22Δ Mutation in Alzheimer Pathology.

The toxic conformer of the 40- or 42-mer-amyloid β-proteins (Aβ) (Aβ40, Aβ42) with a turn at positions 22 and 23 plays a role in oligomer formation, leading to neurotoxicity as part of the pathogenesis of Alzheimer's disease (AD). A deletion mutant at Glu22 (E22Δ) of Aβ, known as an Osaka mutation, accelerates oligomerization. Although E22Δ-Aβ has not been found to be toxic to cultured neuronal cells and is instead synaptotoxic in long-term potentiation, there is no information on the toxic conformer of E22Δ-Aβ in AD. The site-directed spin labeling study of E22Δ-Aβ40 by continuous wave-electron spin resonance (CW-ESR) spectroscopy in part showed the spatial proximity between positions 10 and 35, which are characteristic of the toxic conformation of Aβ, indicating the existence of a toxic conformer of Aβ with the E22Δ mutation. To obtain structural insight, E22Δ-Aβ42 substitutes with proline (F20P, A21P, D23P, and V24P), in which proline is known as a turn inducer but is a β-sheet breaker, were synthesized. An enzyme immunoassay using the 24B3 antibody recognizing toxic conformer of Aβ was carried out. 24B3 reacted with these substitutes of E22Δ-Aβ42 as well as E22Δ-Aβ42 in a similar manner to WT-Aβ42. Notably, only A21P-E22Δ-Aβ42 exhibited strong neurotoxicity in rat primary neurons after 8 days of incubation, with potent high-order oligomerization compared with E22Δ-Aβ42. These results suggest that E22Δ-Aβ42 could enhance neurotoxicity by generating a toxic oligomer conformation with a turn near position 21.

N^N Pt(II) Bisacetylide Complexes with Oxoverdazyl Radical Ligands: Preparation, Photophysical Properties, and Magnetic Exchange Interaction between the Two Radical Ligands.

To study the effect of a stable radical on the photophysical properties of a phosphorescent Pt(II) coordination framework and the intramolecular magnetic interaction between radical ligands in the N^N Pt(II) bisacetylide complexes, we prepared a series of N^N Pt(II) bis(acetylide) complexes with oxoverdazyl radical acetylide ligands. The linker between the Pt(II) center and the spin carrier was systematically varied, to probe the effect on the sign and magnitude of the spin exchange interactions between the radical ligands and photophysical properties. The complexes were studied with steady-state and femtosecond/nanosecond transient absorption spectroscopy, continuous-wave electron paramagnetic resonance (EPR) spectroscopy, and density functional theory (DFT) computations. The transient absorption spectral studies show that the doublet excited state of the radicals are short-lived (τD ≈ 2 ps) and nonfluorescent. Moreover, the intrinsic long-lived triplet excited state (τT = 1.2 μs) of the Pt(II) coordination center was efficiently quenched by the radical (τT = 6.9 ps for one representative radical Pt(II) complex). The intramolecular magnetic interaction between the radical ligands through the diamagnetic Pt(II) atom was studied with temperature-dependent EPR spectroscopy; antiferromagnetic exchange interaction (-J S1S2, J = -5.4 ± 0.1 cm-1) for the complex with the shortest radical-radical distance through bridge fragments was observed. DFT computations give similar results for the sign and magnitude of the J values. For complexes with larger inter-radical distance, however, very weak coupling between the radical ligands was observed (|J| < 0.7 cm-1). Our results are useful for the study of the effect of a radical on the photophysical properties of the phosphorescent transition-metal complexes.

An EPR Study on the Interaction between the Cu(I) Metal Binding Domains of ATP7B and the Atox1 Metallochaperone

Copper’s essentiality and toxicity mean it requires a sophisticated regulation system for its acquisition, cellular distribution and excretion, which until now has remained elusive. Herein, we applied continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) spectroscopy in solution to resolve the copper trafficking mechanism in humans, by considering the route travelled by Cu(I) from the metallochaperone Atox1 to the metal binding domains of ATP7B. Our study revealed that Cu(I) is most likely mediated by the binding of the Atox1 monomer to metal binding domain 1 (MBD1) and MBD4 of ATP7B in the final part of its extraction pathway, while the other MBDs mediate this interaction and participate in copper transfer between the various MBDs to the ATP7B membrane domain. This research also proposes that MBD1-3 and MBD4-6 act as two independent units.

Iron Complexes of an Antiproliferative Aroyl Hydrazone: Characterization of Three Protonation States by Electron Paramagnetic Resonance Methods.

Tridentate aroyl hydrazones are effective metal chelators in biological settings, and their activity has been investigated extensively for medicinal applications in metal overload, cancer, and neurodegenerative diseases. The aroyl hydrazone motif is found in the recently reported prochelator (AH1-S)2, which has shown antiproliferative proapoptotic activity in mammalian cancer cell lines. Intracellular reduction of this disulfide prochelator leads to the formation of mercaptobenzaldehyde benzoylhydrazone chelator AH1 and to iron sequestration, which in turn impacts cell growth. Herein, we investigate the iron coordination chemistry of AH1 to determine the structural and spectroscopic properties of the iron complexes in the solid state and in solution. A neutral iron(III) complex of 2:1 ligand-to-metal stoichiometry was isolated and characterized fully to reveal two different binding modes for the tridentate AH1 ligand. Specifically, one ligand binds in the monoanionic keto form, whereas the other ligand coordinates as a dianionic enolate. Continuous-wave electron paramagnetic resonance experiments in frozen solutions indicated that this neutral complex is one of three low-spin iron(III) complexes observed depending on the pH of the solution. Electron spin echo envelope modulation (ESEEM) experiments allowed assignment of the three species to different protonation states of the coordinated ligands. Our ESEEM analysis provides a method to distinguish the coordination of aroyl hydrazones in the keto and enolate forms, which influences both the ligand field and overall charge of the complex. As such, this type of analysis could provide valuable information in a variety of studies of iron complexes of aroyl hydrazones, ranging from the investigation of spin-crossover behavior to tracking of their distribution in biological samples.

A Binuclear CuA Center Designed in an All α-Helical Protein Scaffold.

The primary and secondary coordination spheres of metal binding sites in metalloproteins have been investigated extensively, leading to the creation of high-performing functional metalloproteins; however, the impact of the overall structure of the protein scaffold on the unique properties of metalloproteins has rarely been studied. A primary example is the binuclear CuA center, an electron transfer cupredoxin domain of photosynthetic and respiratory complexes and, recently, a protein coregulated with particulate methane and ammonia monooxygenases. The redox potential, Cu-Cu spectroscopic features, and a valence delocalized state of CuA are difficult to reproduce in synthetic models, and every artificial protein CuA center to-date has used a modified cupredoxin. Here, we present a fully functional CuA center designed in a structurally nonhomologous protein, cytochrome c peroxidase (CcP), by only two mutations (CuACcP). We demonstrate with UV-visible absorption, resonance Raman, and magnetic circular dichroism spectroscopy that CuACcP is valence delocalized. Continuous wave and pulsed (HYSCORE) X-band EPR show it has a highly compact gz area and small Az hyperfine principal value with g and A tensors that resemble axially perturbed CuA. Stopped-flow kinetics found that CuA formation proceeds through a single T2Cu intermediate. The reduction potential of CuACcP is comparable to native CuA and can transfer electrons to a physiological redox partner. We built a structural model of the designed Cu binding site from extended X-ray absorption fine structure spectroscopy and validated it by mutation of coordinating Cys and His residues, revealing that a triad of residues (R48C, W51C, and His52) rigidly arranged on one α-helix is responsible for chelating the first Cu(II) and that His175 stabilizes the binuclear complex by rearrangement of the CcP heme-coordinating helix. This design is a demonstration that a highly conserved protein fold is not uniquely necessary to induce certain characteristic physical and chemical properties in a metal redox center.

Interaction of Alpha-Crystallin with Phospholipid Membranes

ABSTRACT Purpose/Aim: The amount of membrane-bound α-crystallin increases significantly with age and cataract formation, accompanied by a corresponding decline in the level of α-crystallin in the lens cytoplasm. The purpose of this research is to evaluate the binding affinity of α-crystallin to the phospholipid membranes as well as the physical properties of the membranes after α-crystallin binding. Materials and Methods: The continuous wave and saturation recovery electron paramagnetic resonance (EPR) methods were used to obtain the information about the binding affinity and the physical properties of the membrane. In this approach, the cholesterol analog spin label CSL was incorporated in the membrane and the binding of α-crystallin to the membrane was monitored by this spin label. Small uni-lamellar vesicles were prepared from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with 1% of CSL. The measured membrane properties included the mobility parameter, fluidity, and the oxygen transport parameter. Results: The binding affinity (Ka ) of α-crystallin with the POPC membrane was estimated to be 4.9 ± 2.4 µM−1. The profiles of mobility parameter showed that mobility parameter decreased with an increase in the binding of α-crystallin. The profiles of spin-lattice relaxation rate showed that the spin-lattice relaxation rate decreased with an increase in binding. These results show that the binding of α-crystallin makes the membrane more immobilized near the head group region of the phospholipids. Furthermore, the profiles of the oxygen transport parameter indicated that the oxygen transport parameter decreased with an increase of binding, indicating the binding of α-crystallin forms a barrier for the passage of non-polar molecules which supports the barrier hypothesis. Conclusions: The binding of α-crystallin to the membrane alters the physical properties of the membranes, and this plays a significant role in modulating the integrity of the membranes. EPR techniques are useful in studying α-crystallin membrane interactions.

Spin Crossover in Bipyridine Derivative Bridged One-Dimensional Iron(III) Coordination Polymer

Herein, the syntheses, solid-state molecular structures, and characterization of two types of one-dimensional FeIII coordination polymers showing thermally induced spin crossover are reported. The reaction of [Fe(acen)Cl] (acen2− = N,N′-ethylenebis(acetylacetonylideneaminate) with 3,3′-bpy or 4,4′-bpy (bpy = bipyridine) produced zigzag and linear one-dimensional chain complexes, [Fe(acen)(3,3′-bpy)][BPh4] (1) or [NEt3H][Fe(acen)(4,4′-bpy)][BPh4]2·0.5(4,4′-bpy) (2), respectively, as confirmed by single crystal X-ray diffraction analysis. Variable-temperature single crystal X-ray diffraction measurements, continuous-wave X-band electron paramagnetic resonance (EPR) spectra, 57Fe Mössßauer spectra, and DC magnetic susceptibility data revealed that complex 1 exhibited a gradual and complete spin crossover at a transition temperature of 212 K, while complex 2 undergoes an incomplete spin crossover even at 400 K.

Nanoscopic Characterization of Stearic Acid Release from Bovine Serum Albumin Hydrogels.

The release behavior of 16-doxyl stearic acid (16-DSA) from hydrogels made from bovine serum albumin (BSA) is characterized. 16-DSA serves as a model tracer molecule for amphiphilic drugs. Various hydrogel preparation procedures are tested and the fatty acid release from the different gels is compared in detail. These comparisons reach from the macroscopic level, the viscoelastic behavior via rheological characterization to changes on the nanoscopic level concerning the secondary structure of the protein during gelation through infrared (ATR-IR) spectroscopy. 16-DSA-BSA interaction via continuous wave electron paramagnetic resonance (CW EPR) spectroscopy in addition gives a nanoscopic view of small molecule-hydrogel interaction. The combined effects of fatty acid concentration, hydrogel incubation time, and gelation procedures on release behavior are studied via CW EPR spectroscopy and dynamic light scattering (DLS) measurements, which provide deep insight on the interaction of 16-DSA with BSA hydrogels and the nature and size of the released components, respectively. It is found that the release rate of the fatty acid from BSA hydrogels depends on and can thus be tuned through its loading percentage, duration of hydrogel formation and the type of gelation methods. All of the results confirm the potential of these gels as delivery hosts in pharmaceutical applications allowing the sustained release of drug.

Recent progress in EPR study of spin labeled polymers and spin probed polymer systems

AbstractThe EPR technique is commonly used for the detection and characterization of paramagnetic centers in chemical science. This method can provides a lot of information, such as identity, structure, dynamics, interaction, orientation, glass transition temperature, adsorption behavior, functionality, phase behavior, nano‐inhomogeneities, and conformation of the free‐radical portion of the polymer chain. Most polymers intrinsically possess diamagnetic properties, so in order to study polymers with EPR, paramagnetic centers need to be incorporated into the polymer systems. Spin labeling and spin probing are main methods of covalently attaching paramagnetic centers to polymer chains or embedding them in polymer matrices through non‐covalent interactions, respectively. Spin labeling and spin probing techniques for polymers and polymer systems (especially with nitroxide radicals) have also been studied, which have a profound impact on polymer science. This review focuses on the continuous wave EPR technique and introduces the recent advances in spin labeled polymers and spin probed polymer systems in EPR research.

193 nm excimer laser-induced color centers in Yb3+/Al3+/P5+-doped silica glasses

Yb3+, Yb3+/Al3+, and Yb3+/P5+ co-doped silica glasses, as well as pure silica, Al3+, and P5+ single-doped silica glasses, were prepared using a sol–gel method combined with high-temperature sintering. The glasses were irradiated with a 193 nm ArF excimer laser for different durations. The species of the 193 nm laser-induced color centers were identified by radiation-induced absorption (RIA), in-situ photoluminescence (PL), and continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopic techniques. To obtain the RIA and CW-EPR characteristics of each color center, the Gaussian decomposition of RIA and the line-shape simulation of the CW-EPR spectra were performed. The formation mechanism of the color centers was correlated with the glass microscopic structure, which was obtained by nuclear magnetic resonance (NMR) spectroscopy. The results show that the oxygen hole center and the Yb2+ ion pairs are primarily responsible for the radiation-induced darkening, and their formation is highly dependent on the charge balance between the Yb3+ ion and its ligand. This work provides insights into the underlying mechanism of pump light and/or external radiation-induced optical losses in Yb3+-doped silica fibers.

Structural Dynamics and Topology of the Inactive Form of S21 Holin in a Lipid Bilayer Using Continuous-Wave Electron Paramagnetic Resonance Spectroscopy.

The bacteriophage infection cycle plays a crucial role in recycling the world's biomass. Bacteriophages devise various cell lysis systems to strictly control the length of the infection cycle for an efficient phage life cycle. Phages evolved with lysis protein systems, which can control and fine-tune the length of this infection cycle depending on the host and growing environment. Among these lysis proteins, holin controls the first and rate-limiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time and concentration hence known as the simplest molecular clock. Pinholin S21 is the holin from phage Φ21, which defines the cell lysis time through a predefined ratio of active pinholin and antipinholin (inactive form of pinholin). Active pinholin and antipinholin fine-tune the lysis timing through structural dynamics and conformational changes. Previously we reported the structural dynamics and topology of active pinholin S2168. Currently, there is no detailed structural study of the antipinholin using biophysical techniques. In this study, the structural dynamics and topology of antipinholin S2168IRS in DMPC proteoliposomes is investigated using electron paramagnetic resonance (EPR) spectroscopic techniques. Continuous-wave (CW) EPR line shape analysis experiments of 35 different R1 side chains of S2168IRS indicated restricted mobility of the transmembrane domains (TMDs), which were predicted to be inside the lipid bilayer when compared to the N- and C-termini R1 side chains. In addition, the R1 accessibility test performed on 24 residues using the CW-EPR power saturation experiment indicated that TMD1 and TMD2 of S2168IRS were incorporated into the lipid bilayer where N- and C-termini were located outside of the lipid bilayer. Based on this study, a tentative model of S2168IRS is proposed where both TMDs remain incorporated into the lipid bilayer and N- and C-termini are located outside of the lipid bilayer. This work will pave the way for the further studies of other holins using biophysical techniques and will give structural insights into these biological clocks in molecular detail.

Development of planar microstrip resonators for electron spin resonance spectroscopy

This work focuses on the development of planar microwave resonators for use in electron spin resonance spectroscopic studies. Two half-wavelength microstrip resonators of different geometrical shapes, namely straight ribbon and omega, are fabricated on commercially available microwave laminates. Both resonators have a characteristic impedance of 50 Ω. We have performed electromagnetic field simulations for the resonators and have extracted design parameters that were used for fabrication. The effect of the geometry on the quasi-transverse electromagnetic (quasi-TEM) modes of the resonators is noted from simulation results. The fabrication is done using optical lithography in which laser printed photomasks are used. This prototyping technique allows us to fabricate resonators in a few hours with accuracy up to 6 mils. The resonators are characterized using a Vector Network Analyzer. The fabricated resonators are used in a home built low-temperature continuous wave electron spin resonance (CW-ESR) spectrometer which operates in S-band. It captures the absorption spectrum of the free radical DPPH, at both room temperature and 77 K. The measured g-factor using our resonators is consistent with the values reported in literature. Spin sensitivity of 1015 spins/gauss is achieved at 77 K. The designed resonators will be used in setting up a pulsed electron spin resonance spectrometer by suitably modifying the CW-ESR spectrometer.

Monitoring Protein-Protein Interactions in the Cyanobacterial Circadian Clock in Real Time via Electron Paramagnetic Resonance Spectroscopy.

The cyanobacterial circadian clock in Synechococcus elongatus consists of three proteins, KaiA, KaiB, and KaiC. KaiA and KaiB rhythmically interact with KaiC to generate stable oscillations of KaiC phosphorylation with a period of 24 h. The observation of stable circadian oscillations when the three clock proteins are reconstituted and combined in vitro makes it an ideal system for understanding its underlying molecular mechanisms and circadian clocks in general. These oscillations were historically monitored in vitro by gel electrophoresis of reaction mixtures based on the differing electrophoretic mobilities between various phosphostates of KaiC. As the KaiC phospho-distribution represents only one facet of the oscillations, orthogonal tools are necessary to explore other interactions to generate a full description of the system. However, previous biochemical assays are discontinuous or qualitative. To circumvent these limitations, we developed a spin-labeled KaiB mutant that can differentiate KaiC-bound KaiB from free KaiB using continuous-wave electron paramagnetic resonance spectroscopy that is minimally sensitive to KaiA. Similar to wild-type (WT-KaiB), this labeled mutant, in combination with KaiA, sustains robust circadian rhythms of KaiC phosphorylation. This labeled mutant is hence a functional surrogate of WT-KaiB and thus participates in and reports on autonomous macroscopic circadian rhythms generated by mixtures that include KaiA, KaiC, and ATP. Quantitative kinetics could be extracted with improved precision and time resolution. We describe design principles, data analysis, and limitations of this quantitative binding assay and discuss future research necessary to overcome these challenges.

A New Conducting Copolymer Bearing Electro-Active Nitroxide Groups as Organic Electrode Materials for Batteries.

To reduce the amount of conducting additives generally required for polynitroxide-based electrodes, a stable radical (TEMPO) is combined with a conductive copolymer backbone consisting of 2,7-bisthiophene carbazole (2,7-BTC), which is characterized by a high intrinsic electronic conductivity. This work deals with the synthesis of this new polymer functionalized by a redox nitroxide. Fine structural characterization using electron paramagnetic resonance (EPR) techniques established that: 1) the nitroxide radicals are properly attached to the radical chain (continuous wave EPR) and 2) the polymer chain has very rigid conformations leading to a set of well-defined distances between first neighboring pairs of nitroxides (pulsed EPR). The redox group combined with the electroactive polymer showed not only a very high electrochemical reversibility but also a perfect match of redox potentials between the de-/doping reaction of the bisthiophene carbazole backbone and the redox activity of the nitroxide radical. This new organic electrode shows a stable capacity (about 60 mAh g-1 ) and enables a strong reduction in the amount of carbon additive due to the conducting-polymer skeleton.

Structure and dynamics of spin label side chains in KvAP voltage-sensor domain: an all-atom MD simulation study

Site-directed spin labeling (SDSL) combined with continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy has become a useful approach to investigate the structural and dynamical properties of biomolecules, especially membrane protein. In this method, spin label side chains due to their motion highly depending on the local environment were employed as external molecules to probe the structure and dynamics of the membrane protein. The close relationships between the dynamics of the spin label side chain and protein structure have been studied for T4 lysozyme. Besides, molecular dynamics (MD) simulations were widely applied as a powerful tool to study the microscopic behavior of the proteins, completing experimental approaches. Here, we presented an all-atom MD simulation study of the nitroxide spin label (MTSSL) in the X-ray structure of a potassium channel voltage-sensor domain (VSD) from Aeropyrum Pernix (KvAP).

Using Electron Paramagnetic Resonance Spectroscopy to Determine the Role of Free‐Radical Protection in Tardigrade Survival Mechanisms

As aeronautic technology advances, so does the possibility of long‐term space travel and exploration. However, in order to make significant strides, it is first necessary to determine how to survive the antagonistic space environment, including temperature extremes, UVC radiation, and vacuum pressures, among others. Tardigrades are microscopic invertebrates that possess the ability to adapt to extreme environments through the formation of a tun, a dormant structure resulting from the downregulation of metabolism. This ability to survive under extreme conditions, including arctic temperatures, extreme heat, and a pure vacuum, makes tardigrades a useful model organism for space survival. By increasing our understanding of their survival mechanisms on a fundamental level, we may be able to generate technologies that protect humans in space from diseases caused by ROS and the damaging effects of oxidative stress.Previous research has examined the physiological response of tardigrades subjected to freezes and irradiation that model outer Earth conditions. In these studies, tardigrades were shown to enter tun formation through cryptobiosis, especially in response to physical stressors causing desiccation. Another study assessed tardigrades’ intergalactic survival capability by sending a culture into the vacuum of space, after which the cultures showed no loss of viability. We hypothesize that tardigrades have a specific, protective mechanism to respond to oxidative stress through the increased expression of superoxide dismutases (SODs) to break down potentially harmful ROS, which is supported by previous studies showing both the formation of superoxide radicals as well as an increase in SODs in desiccated animals.In this study, X‐band continuous wave electron paramagnetic resonance (EPR) spectroscopy was used to measure the presence of superoxide radicals in the tardigrade Hypsibius exemplaris before and after exposure to environmental stressors. Tardigrades were subjected to a combination of UVA and UVB irradiation before the addition of a spin probe, and the resulting EPR spectrum was compared to a nonirradiated H. exemplaris control. Results show that the tardigrades generated less ROS following environmental stress exposure, supporting the hypothesis of a protective mechanism to mediate radical production when subject to harsh conditions. Studies concerning other physical and chemical stressors, as well as examining radical production in tardigrades upon inducing tun formation, are ongoing. Further experiments also include determining specific activities of proteinaceous antioxidants like SOD before and after these exposures.Support or Funding InformationJ.R.C was supported the SURE Program funded through the West Virginia Research Challenge Fund, and administered by the West Virginia HEPC, DSR, Award Number DSR.17.09,the WV NASA Space Grant Consortium's NASA WV Space Grant Undergraduate Fellowship Program and from a Marshall University Creative Discover &amp; Undergraduate Research Award.D.R.J.K. and A.L.S. were supported by the National Science Foundation under Cooperative Agreement No. OIA‐1458952.

An improved adaptive signal averaging technique for noise reduction and tracking enhancements in continuous wave magnetic resonance.

We have significantly refined an adaptive signal averaging approach developed primarily for continuous wave electron paramagnetic resonance and electrically detected magnetic resonance measurements. This refinement overcomes several limitations and greatly simplifies the earlier approach. The new technique provides a large improvement in tracking and numerical stability and also features fewer adjustable parameters making this approach more user intuitive.

Paramagnetism and Relaxation Dynamics in Melanin Biomaterials.

Spectroscopical characterization of melanins is a prior requirement for the efficient tailoring of their radical scavenging, ultraviolet-visible radiation absorption, metal chelation, and natural pigment properties. Electron paramagnetic resonance (EPR), exploiting the common persistent paramagnetism of melanins, represents the elective standard for the structural and dynamical characterization of their constituting radical species. Although melanins are mainly investigated using X-band (9.5 GHz) continuous wave (CW)-EPR, an integration with the application of Q-band (34 GHz) in CW and pulse EPR for the discrimination of melanin pigments of different compositions is presented here. The longitudinal relaxation times measured highlight faster relaxation rates for cysteinyldopa melanin, compared to those of the most common dopa melanin pigment, suggesting pulse EPR spin-lattice relaxation time measurements as a complementary tool for characterization of pigments of interest for biomimetic materials engineering.

Cu2+-based distance measurements by pulsed EPR provide distance constraints for DNA backbone conformations in solution.

Electron paramagnetic resonance (EPR) has become an important tool to probe conformational changes in nucleic acids. An array of EPR labels for nucleic acids are available, but they often come at the cost of long tethers, are dependent on the presence of a particular nucleotide or can be placed only at the termini. Site directed incorporation of Cu2+-chelated to a ligand, 2,2′dipicolylamine (DPA) is potentially an attractive strategy for site-specific, nucleotide independent Cu2+-labelling in DNA. To fully understand the potential of this label, we undertook a systematic and detailed analysis of the Cu2+-DPA motif using EPR and molecular dynamics (MD) simulations. We used continuous wave EPR experiments to characterize Cu2+ binding to DPA as well as optimize Cu2+ loading conditions. We performed double electron-electron resonance (DEER) experiments at two frequencies to elucidate orientational selectivity effects. Furthermore, comparison of DEER and MD simulated distance distributions reveal a remarkable agreement in the most probable distances. The results illustrate the efficacy of the Cu2+-DPA in reporting on DNA backbone conformations for sufficiently long base pair separations. This labelling strategy can serve as an important tool for probing conformational changes in DNA upon interaction with other macromolecules.

Unusual 31P Hyperfine Strain Effects in a Conformationally Flexible Cu(II) Complex Revealed by Two-Dimensional Pulse EPR Spectroscopy.

Strain effects on g and metal hyperfine coupling tensors, A, are often manifested in Electron Paramagnetic Resonance (EPR) spectra of transition metal complexes, as a result of their intrinsic and/or solvent-mediated structural variations. Although distributions of these tensors are quite common and well understood in continuous-wave (cw) EPR spectroscopy, reported strain effects on ligand hyperfine coupling constants are rather scarce. Here we explore the case of a conformationally flexible Cu(II) complex, [Cu{Ph2P(O)NP(O)Ph2-κ2O,O'}2], bearing P atoms in its second coordination sphere and exhibiting two structurally distinct CuO4 coordination spheres, namely a square planar and a tetrahedrally distorted one, as revealed by X-ray crystallography. The Hyperfine Sublevel Correlation (HYSCORE) spectra of this complex exhibit 31P correlation ridges that have unusual inverse or so-called "boomerang" shapes and features that cannot be reproduced by standard simulation procedures assuming only one set of magnetic parameters. Our work shows that a distribution of isotropic hyperfine coupling constants (hfc) spanning a range between negative and positive values is necessary in order to describe in detail the unusual shapes of HYSCORE spectra. By employing DFT calculations we show that these hfc correspond to molecules showing variable distortions from square planar to tetrahedral geometry, and we demonstrate that line shape analysis of such HYSCORE spectra provides new insight into the conformation-dependent spectroscopic response of the spin system under investigation.