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• Will pulse EPR cross the diffusion between the experts and the users? • Hyperfine spectroscopy applications are likely to remain within the expert court. • Pulse-dipolar EPR can become a widely used technique beyond the experts circle. • A dedicated, low cost, bench top DEER spectrometer is envisioned. Application of EPR to biological systems includes many techniques and applications. In this short perspective, which dares to look into the future, I focus on pulse EPR, which is my field of expertise. Generally, pulse EPR techniques can be divided into two main groups: (1) hyperfine spectroscopy, which explores electron-nuclear interactions, and (2) pulse-dipolar (PD) EPR spectroscopy, which is based on electron-electron spin interactions. Here I focus on PD-EPR because it has a better chance of becoming a widely applied, easy-to-use table-top method to study the structural and dynamic aspects of bio-molecules. I will briefly introduce this technique, its current state of the art, the challenges it is facing, and finally I will describe futuristic scenarios of low-cost PD-EPR approaches that can cross the diffusion barrier from the core of experts to the bulk of the scientific community. [ABSTRACT FROM AUTHOR]

(100-x) mol % B2O3 x mol % Me2O (Me=Li,Na,K) glasses, exposed to γ-60Co irradiation to produce paramagnetic states, were characterized by W-band (95 GHz) pulse electron-nuclear double resonance (ENDOR) spectroscopy in order to characterize local structures occurring in the range of compositions between x=16 and x=25 at which the “boron oxide” anomaly occurs. The high resolution of nuclear frequencies allowed resolving the 7Li and 11B ENDOR lines. In the samples with x=16 and x=20 glasses, 11B hyperfine couplings of 16, 24, and 36 MHz were observed and attributed to the tetraborate, triborate, and boron oxygen hole center (BOHC) structures, respectively. The x=25 samples showed hyperfine couplings of 15 MHz for the tetraborate and 36 MHz for BOHC. Density functional theory (DFT) calculations predicted for these structures negative hyperfine couplings, which were confirmed by W-band ENDOR. This suggests that a spin polarization mechanism accounts for the negative hyperfine structure splitting. [ABSTRACT FROM AUTHOR]

Electron spin echo envelope modulation (ESEEM) induced by 27Al nuclei can be used to characterize the interactions of paramagnetic transition metal cations with the framework of various aluminosilicates. The quantitative analysis of such systems is complex due to the 27Al nuclear quadrupole interaction which is often large and unknown. In order to obtain a better understanding of the various spectral features of the 27Al modulation, the ESEEM of a S= (1)/(2) I= (5)/(2) spin system in orientationally disordered systems was investigated in the frequency domain. The relative contributions of the various electron-nuclear double resonance (ENDOR) frequencies to the Fourier transform (FT) ESEEM spectrum were studied as a function of the size of the nuclear quadrupole coupling constant and of the relative orientation of the nuclear quadrupole tensor with respect to the g-tensor. The parameters range investigated is that expected from Cu2+ interacting with framework Al in zeolites. Two approaches were employed in the calculations, exact diagonalization of the nuclear Hamiltonian and a second order perturbation treatment of the quadrupole interaction. It is found that the second order perturbation approach applies up to e2qQ/h<7 MHz. The conditions under which the FT-ESEEM spectrum is dominated by the | (1)/(2) >-|- (1)/(2) > ENDOR transitions were explored as well. This occurs when e2qQ/h is relatively large and it strongly depends on the orientation of the quadrupole tensor. When the FT-ESEEM can be described only by the | (1)/(2) >-|- (1)/(2) > ENDOR transitions, the simulations are significantly simplified since these can be calculated using the analytical expressions obtained by perturbation theory also for a quadrupole coupling constant as large as 10 MHz. [ABSTRACT FROM AUTHOR]

The effects of 27Al (I=5/2) nuclear quadrupole interaction on the electron spin echo modulation (ESEM) in the case of S=1/2 in disordered systems are analyzed using exact diagonalization of the spin Hamiltonian. The Hamiltonian parameters used in the calculations are those expected in Cu+2-exchanged zeolites. The g anisotropy is taken into account and the ESEM obtained using the spherical model approximation is compared with a trigonal correlated structure. It is found that in the 0.32–0.34 nm range of distances between the paramagnetic center and the Al nuclei all parameters related to the quadrupole tensor, namely the quadrupole interaction constant, the asymmetry parameter and the relative orientation with respect to the g tensor, have a substantial effect on the modulation depth. [ABSTRACT FROM AUTHOR]

Abstract: Anionic surfactant-templated mesoporous silicas (AMS) are synthesized with a co-structure directing agent (CSDA) that interacts with both the organic and inorganic components of the system. The AMS materials structure is controlled by pH. We investigated the formation of AMS cubic and hexagonal phases, prepared under the same conditions, except pH, by EPR spectroscopic measurements. We used silica-like and surfactant-like spin probes added to the reaction mixtures in minute amounts. Through the spin probes we resolved the specific interactions of the CSDA (N-trimethoxylsilylpropyl-N,N,N-trimethyl ammonium chloride (TMAPS)) with the surfactant (sodium myristate (C14AS)) and the polymerizing silica. We observed for both phases a fast formation of a mesophase upon addition of the silica precursor (TEOS, tetraethoxysilane) and the CSDA into the surfactant solution, attributed to the strong attraction between the CSDA and the anionic surfactant. This is then followed by a slow condensation of the silica. Electron spin echo envelope modulation (ESEEM) spectra of both spin probes in the as-synthesized materials indicated the presence of two types of CSDA molecules; one interacting with the surfactant and the other with the silica wall. Continuous wave EPR spectra showed different spin probe motilities in the two as-synthesized materials that indicated that the relative populations of the two CSDA types are different in the two phases. We attribute this difference to the pH differences in the reaction mixtures. A soft extraction of the surfactant from the pores did not alter the structure of the final materials, but it abolished the observed molecular level differences between them. The extraction allowed the pending ammonium groups to acquire a high degree of freedom and accessibility to water molecules. [Copyright &y& Elsevier]

A trinuclear manganese complex, [Mn[sub3]O(Me-salox)[sub3](2,4'-bpy)[sub3]- (dO[sub4])] ̇ 0.5MeCN (1̇0.5MeCN; Me-H[sub3]salox = 2-hydroxyphenyle- thanone oxime), has been synthesized and characterized structurally as a [Mn[supIII][sub3](μ[sub3]-O)][sup7+] core. Structural distortion by the twisting of the oxime ligand dominates ferromagnetic interactions within three Mn ions to give an S = 6 ground state as well as to enhance the anisotropy barrier U[subeff] to 37.5 K. [ABSTRACT FROM AUTHOR]

Abstract: The set-up of a new microwave bridge for a 95GHz pulse EPR spectrometer is described. The virtues of the bridge are its simple and flexible design and its relatively high output power (0.7W) that generates π pulses of 25ns and a microwave field, B 1 =0.71 mT. Such a high B 1 enhances considerably the sensitivity of high field double electron-electron resonance (DEER) measurements for distance determination, as we demonstrate on a nitroxide biradical with an interspin distance of 3.6nm. Moreover, it allowed us to carry out HYSCORE (hyperfine sublevel-correlation) experiments at 95GHz, observing nuclear modulation frequencies of 14N and 17O as high as 40MHz. This opens a new window for the observation of relatively large hyperfine couplings, yet not resolved in the EPR spectrum, that are difficult to observe with HYSCORE carried out at conventional X-band frequencies. The correlations provided by the HYSCORE spectra are most important for signal assignment, and the improved resolution due to the two dimensional character of the experiment provides 14N quadrupolar splittings. [Copyright &y& Elsevier]

Investigates the incorporation of Mn(II) into the mesoporous material MCM-41, synthesized under acidic conditions with [Si]/[H+]. Use of tetraethyl-orthosilicon as the silica source and cetyltrimethylammonium chloride or bromide as the structure-directing agent; Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy measurements.

Discusses the local environment of the manganese in MnAPSO-44. Application of magnetic resonance spectroscopic techniques; Probe of weak superhyperfine interactions between paramagnetic center and neighboring nuclear spins; Analysis of dipolar interaction; Spatial distribution of manganese.

Electron spectral diffusion (eSD) plays an important role in solid-state, static dynamic nuclear polarization (DNP) with polarizers that have inhomogeneously broadened EPR spectra, such as nitroxide radicals. It affects the electron spin polarization gradient within the EPR spectrum during microwave irradiation and thereby determines the effectiveness of the DNP process via the so-called indirect cross-effect (iCE) mechanism. The electron depolarization profile can be measured by electron-electron double resonance (ELDOR) experiments, and a theoretical framework for deriving eSD parameters from ELDOR spectra and employing them to calculate DNP profiles has been developed. The inclusion of electron depolarization arising from the 14N solid effect (SE) has not yet been taken into account in this theoretical framework and is the subject of the present work. The 14N SE depolarization was studied using W-band ELDOR of a 0.5mM TEMPOL solution, where eSD is negligible, taking into account the hyperfine interaction of both 14N and 1H nuclei, the long microwave irradiation applied under DNP conditions, and electron and nuclear relaxation. The results of this analysis were then used in simulations of ELDOR spectra of 10 and 20mM TEMPOL solutions, where eSD is significant using the eSD model and the SE contributions were added ad hoc employing the 1H and 14N frequencies and their combinations, as found from the analysis of the 0.5mM sample. This approach worked well for the 20mM solution, where a good fit for all ELDOR spectra recorded along the EPR spectrum was obtained and the inclusion of the 14N SE mechanism improved the agreement with the experimental spectra. For the 10mM solution, simulations of the ELDOR spectra recorded along the gz position gave a lower-quality fit than for spectra recorded in the center of the EPR spectrum. This indicates that the simple approach we used to describe the 14N SE is limited when its contribution is relatively high as the anisotropy of its magnetic interactions was not considered explicitly. [ABSTRACT FROM AUTHOR]

A new, triple resonance, pulse electron paramagnetic resonance (EPR) sequence is described. It provides spin links between forbidden electron spin transitions (ΔMS=±1, ΔMI≠0) and allowed nuclear spin transitions (ΔMI=±1), thus, facilitating the assignment of nuclear frequencies to their respective electron spin manifolds and paramagnetic centers. It also yields the relative signs of the hyperfine couplings of the different nuclei. The technique is based on the combination of electron-nuclear double resonance (ENDOR) and electron-electron double resonance (ELDOR)–detected NMR experiments in a way similar to the TRIPLE experiment. The feasibility and the information content of the method are demonstrated first on a single crystal of Cu-doped L-histidine and then on a frozen solution of a Cu-histidine complex. [ABSTRACT FROM AUTHOR]

Frequency-domain electron nuclear double resonance (ENDOR), two time-domain electron nuclear double resonance techniques, and electron spin echo envelope modulation spectroscopy are compared with respect to their merit in measurements of small hyperfine couplings to nuclei with intermediate gyromagnetic ratio such as 31P. The frequency-domain Mims ENDOR experiment is found to provide the most faithful line shapes. In the limit of long electron-nuclear distances of more than 0.5 nm, sensitivity of this experiment is optimized by matching the first interpulse delay to the transverse relaxation time of the electron spins. In the same limit, Mims ENDOR efficiency scales inversely with the sixth power of distance. Hyperfine splittings as small as 33 kHz can be detected, corresponding to an electron-31P distance of 1 nm. In systems, where a certain kind of nuclei is distributed in a plane, measurements of intermolecular hyperfine couplings can be analyzed in terms of a distance of closest approach of a paramagnetic center to that plane. By applying this technique to spin-labeled lipids in a fully hydrated lipid bilayer it is found that for a fraction of lipids, chain tilt angles can be 25° larger than the mean tilt angle of the lipid chains. This model of all-trans hydrocarbon chains with a broad distribution of tilt angles is also consistent with orientation selection effects in high-field ENDOR spectra. © 2005 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Nitroxide (NO) spin radicals are effective in characterizing structures, interactions and dynamics of biomolecules. The EPR applications in cell lysates or intracellular milieu require stable spin labels, but NO radicals are unstable in such conditions. We showed that the destabilization of NO radicals in cell lysates or even in cells is caused by NADPH/NADH related enzymes, but not by the commonly believed reducing reagents such as GSH. Maleimide stabilizes the NO radicals in the cell lysates by consumption of the NADPH/NADH that are essential for the enzymes involved in destabilizing NO radicals, instead of serving as the solo thiol scavenger. The maleimide treatment retains the crowding properties of the intracellular components and allows to perform long‐time EPR measurements of NO labeled biomolecules close to the intracellular conditions. The strategy of maleimide treatment on cell lysates for the EPR applications has been demonstrated on double electron‐electron resonance (DEER) measurements on a number of NO labeled protein samples. The method opens a broad application range for the NO labeled biomolecules by EPR in conditions that resemble the intracellular milieu. [ABSTRACT FROM AUTHOR]

Studies of protein structure and dynamics are usually carried out in dilute buffer solutions, conditions that differ significantly from the crowded environment in the cell. The double electron‐electron resonance (DEER) technique can track proteins' conformations in the cell by providing distance distributions between two attached spin labels. This technique, however, cannot access distances below 1.8 nm. Here, we show that GdIII‐19F Mims electron‐nuclear double resonance (ENDOR) measurements can cover part of this short range. Low temperature solution and in‐cell ENDOR measurements, complemented with room temperature solution and in‐cell GdIII‐19F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin‐labeled with rigid GdIII tags. The proteins were delivered into human cells via electroporation. The solution and in‐cell derived GdIII‐19F distances were essentially identical and lie in the 1–1.5 nm range revealing that both, GB1 and Ub, retained their overall structure in the GdIII and 19F regions in the cell. [ABSTRACT FROM AUTHOR]

Studies of protein structure and dynamics are usually carried out in dilute buffer solutions, conditions that differ significantly from the crowded environment in the cell. The double electron‐electron resonance (DEER) technique can track proteins' conformations in the cell by providing distance distributions between two attached spin labels. This technique, however, cannot access distances below 1.8 nm. Here, we show that GdIII‐19F Mims electron‐nuclear double resonance (ENDOR) measurements can cover part of this short range. Low temperature solution and in‐cell ENDOR measurements, complemented with room temperature solution and in‐cell GdIII‐19F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin‐labeled with rigid GdIII tags. The proteins were delivered into human cells via electroporation. The solution and in‐cell derived GdIII‐19F distances were essentially identical and lie in the 1–1.5 nm range revealing that both, GB1 and Ub, retained their overall structure in the GdIII and 19F regions in the cell. [ABSTRACT FROM AUTHOR]

[Display omitted] • Gd(III)–Gd(III) DEER background decay measurements show a shape dependence on temperature. • Analysis of the background decay shape provides the spin flip rate during evolution. • At the high end of the temperature range, the derived flip rate matches T 1 -1. • At the low end of the temperature range, the derived flip rate exceeds T 1 -1. • A second flip mechanism was found for Gd(III) The common approach to background removal in double electron–electron resonance (DEER) measurements on frozen solutions with a three-dimensional homogeneous distribution of doubly labeled biomolecules is to fit the background to an exponential decay function. Excluded volume effects or distribution in a dimension lower than three, such as proteins in a membrane, can lead to a stretched exponential decay. In this work, we show that in cases of spin labels with short spin–lattice relaxation time, up to an order of magnitude longer than the DEER trace length, relevant for metal-based spin labels, spin flips that take place during the DEER evolution time affect the background decay shape. This was demonstrated using a series of temperature-dependent DEER measurements on frozen solutions of a nitroxide radical, a Gd(III) complex, Cu(II) ions, and a bis-Gd(III) model complex. As expected, the background decay was exponential for the nitroxide, whereas deviations were noted for Gd(III) and Cu(II). Based on the theoretical approach of Keller et al. (Phys. Chem. Chem. Phys. 21 (2019) 8228–8245), which addresses the effect of spin–lattice relaxation-induced spin flips during the evolution time, we show that the background decay can be fitted to an exponent including a linear and quadratic term in t , which is the position of the pump pulse. Analysis of the data in terms of the probability of spontaneous spin flips induced by spin–lattice relaxation showed that this approach worked well for the high temperature range studied for Gd(III) and Cu(II). At the low temperature range, the spin flips that occured during the DEER evolution time for Gd(III) exceeded the measured spin–lattice relaxation rate and include contributions from spin flips due to another mechanisms, most likely nuclear spin diffusion. [ABSTRACT FROM AUTHOR]

The paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. Here we report a lanthanoid tag for selective reaction with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between the lanthanoid ion and the protein backbone. The tag is assembled on the protein in three steps, comprising (i) reaction with 4-fluoro-2,6-dicyanopyridine (FDCP); (ii) reaction of the cyano groups with α -cysteine, penicillamine or β -cysteine to complete the lanthanoid chelating moiety; and (iii) titration with a lanthanoid ion. FDCP reacts much faster with selenocysteine than cysteine, opening a route for selective tagging in the presence of solvent-exposed cysteine residues. Loaded with Tb3+ and Tm3+ ions, pseudocontact shifts were observed in protein NMR spectra, confirming that the tag delivers good immobilisation of the lanthanoid ion relative to the protein, which was also manifested in residual dipolar couplings. Completion of the tag with different 1,2-aminothiol compounds resulted in different magnetic susceptibility tensors. In addition, the tag proved suitable for measuring distance distributions in double electron–electron resonance experiments after titration with Gd3+ ions. [ABSTRACT FROM AUTHOR]

The paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. Here we report a lanthanoid tag that reacts selectively with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between lanthanoid ion and protein backbone. The tag is assembled on the protein in three steps, comprising (i) reaction with 4-fluoro-2,6-dicyanopyridine (FDCP), (ii) reaction of the cyano groups with α-cysteine, penicillamine or bcysteine to complete the lanthanide chelating moiety and (iii) titration with a lanthanoid ion. FDCP reacts much faster with selenocysteine than cysteine, opening a route for selective tagging in the presence of solvent-exposed cysteine residues. Loaded with Tb3+ and Tm3+ ions, pseudocontact shifts were observed in protein NMR spectra, confirming that the tag delivers good immobilisation of the lanthanoid ion relative to the protein, which was also manifested in residual dipolar couplings. Completion of the tag with different 1,2-amino thiol compounds resulted in different magnetic susceptibility tensors. addition, the tag proved suitable for measuring distance distributions in double electron-electron resonance experiments after titration with Gd3+ ions. [ABSTRACT FROM AUTHOR]

Gd3+-based spin labels are useful as an alternative to nitroxides for intramolecular distance measurements at high fields in biological systems. However, double electron-electron resonance (DEER) measurements using model Gd3+ complexes featured a low modulation depth and an unexpected broadening of the distance distribution for short Gd3+-Gd3+ distances, when analysed using the software designed for S = 1/2 pairs. It appears that these effects result from the different spectroscopic characteristics of Gd3+--the high spin, the zero field splitting (ZFS), and the flip-flop terms in the dipolar Hamiltonian that are often ignored for spin-1/2 systems. An understanding of the factors affecting the modulation frequency and amplitude is essential for the correct analysis of Gd3+-Gd3+ DEER data and for the educated choice of experimental settings, such as Gd3+ spin label type and the pulse parameters. This work uses time-domain simulations of Gd3+-Gd3+ DEER by explicit density matrix propagation to elucidate the factors shaping Gd3+ DEER traces. The simulations show that mixing between the |+½, -½> and |-½> +½i states of the two spins, caused by the flip-flop term in the dipolar Hamiltonian, leads to dampening of the dipolar modulation. This effect may be mitigated by a large ZFS or by pulse frequency settings allowing for a decreased contribution of the central transition and the one adjacent to it. The simulations reproduce both the experimental line shapes of the Fourier-transforms of the DEER time domain traces and the trends in the behaviour of the modulation depth, thus enabling a more systematic design and analysis of Gd3+ DEER experiments. [ABSTRACT FROM AUTHOR]

Abstract: Rapid freeze quench electron paramagnetic resonance (RFQ)-EPR is a method for trapping short lived intermediates in chemical reactions and subjecting them to EPR spectroscopy investigation for their characterization. Two (or more) reacting components are mixed at room temperature and after some delay the mixture is sprayed into a cold trap and transferred into the EPR tube. A major caveat in using commercial RFQ-EPR for high field EPR applications is the relatively large amount of sample needed for each time point, a major part of which is wasted as the dead volume of the instrument. The small sample volume (∼2μl) needed for high field EPR spectrometers, such as W-band (∼3.5T, 95GHz), that use cavities calls for the development of a microfluidic based RFQ-EPR apparatus. This is particularly important for biological applications because of the difficulties often encountered in producing large amounts of intrinsically paramagnetic proteins and spin labeled nucleic acid and proteins. Here we describe a dedicated microfluidic based RFQ-EPR apparatus suitable for small volume samples in the range of a few μl. The device is based on a previously published microfluidic mixer and features a new ejection mechanism and a novel cold trap that allows collection of a series of different time points in one continuous experiment. The reduction of a nitroxide radical with dithionite, employing the signal of Mn2+ as an internal standard was used to demonstrate the performance of the microfluidic RFQ apparatus. [Copyright &y& Elsevier]

We studied the structural evolution during the formation of large-pore cubic Ia3¯d silica-based mesoporous materials, synthesized with Pluroinc P123 and butanol as structure directing agents. We used cryogenic high resolution scanning electron microscopy (cryo-HRSEM) and freeze-fracture-replication (FFR) transmission electron microscopy (TEM). Typically a silica precursor is added to an acid-catalyzed solution of Pluronic P123 and butanol. The latter serves as a cosolute, which can be added either at the beginning of the reaction, or after precipitation and the formation of a hexagonal phase. In this study we focused on the structural evolution from the hexagonal phase to the final cubic phase in the two different reactions. The same structural evolution with different kinetics was detected for both reactions. Cryo-HRSEM and FFR-TEM images revealed that from the hexagonal phase a perforated layer (PL) phase is formed, which later evolves into a bicontinuous structure. The final cubic phase forms within the layers, maintaining their orientation. We suggest a formation mechanism involving cylinder merging for the hexagonal to PL transition. Upon additional polymerization of the silica, the PL phase relaxes into the stable Ia3¯d cubic phase. Another minor mechanism detected involves the direct transition between the hexagonal to the final cubic phase through cylinder branching. [ABSTRACT FROM AUTHOR]

The H5PV2Mo10O40 polyoxometalate and Pd/Al2O3 were used as co-catalysts under anaerobic conditions for the activation and oxidation of CO to CO2 by an electron transfer mechanism. Upon anaerobic reduction of H5PV2Mo10O40 with CO in the presence of Pd(0) two paramagnetic species were observed and characterized by continuous wave electron paramagnetic resonance (CW-EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopic measurements. Major species I (65—70%) is assigned to a species resembling a vanadyl cation that is supported on the polyoxotalate and showed a bonding interaction with 13CO. Minor species II (30—35%) is attributed to a reduced species where the vanadium(IV) atom is incorporated in the polyoxometalate framework but slightly distanced from the phosphate core. Under aerobic conditions, CO/O2, a nucleophilic oxidant was formed as elucidated by oxidation of thianthrene oxide as a probe substrate. Oxidation reactions performed on terminal alkenes such as 1-octene yielded a complicated mixture of products that was, however, clearly a result of alkene epoxidation followed by subsequent reactions of the intermediate epoxide. The significant competing reaction was a hydrocarbonylation reaction that yielded a ∼1:1 mixture of linear/branched carboxylic acids. [ABSTRACT FROM AUTHOR]

Concanavalin A is a member of the plant hemeagglutinin (or plant lectin) family that contains two metal binding sites; one, called S1, is occupied by Mn2+ and the other, S2, by Ca2+. 55Mn electron—nuclear double resonance (ENDOR) measurements were performed on a single crystal of concanavalin A at W-band (95 GHz, ~3.5 T) to determine the 55Mn nuclear quadrupole interaction in a protein binding site and its relation to structural parameters. Such measurements are easier at a high field because of the high sensitivity for size-limited samples and the reduction of second-order effects on the spectrum which simplifies spectral analysis. The analysis of the 55Mn ENDOR rotation patterns showed that two chemically inequivalent Mn2+ types are present at low temperatures, although the high-resolution X-ray structure reported only one site. Their quadrupole coupling constants, e2Qq/h, are significantly different; 10.7 ± 0.6 MHz for MnA2+ and only -2.7 ± 0.6 MHz for MnB2+. The ENDOR data also refined the hyperfine coupling determined earlier by single-crystal EPR measurements, yielding a small but significant difference between the two: -262.5 MHz for MnA2+ and -263.5 MHz for MnB2+. The principal z-axis for MnA2+ is not aligned with any of the Mn-ligand directions, but is 25° off the Mn-asp10 direction, and its orientation is different than that of the zero-field splitting (ZFS) interaction. Because of the small quadrupole interaction of MnB2+ the orientation dependence was very mild, leading to larger uncertainties in the asymmetry parameter. Nonetheless, there too z is not along the Mn-ligand bonds and is rotated 90° with respect to MnA. These results show, that similar to the ZFS, the quadrupolar interaction is highly sensitive to small differences in the coordination sphere of the Mn2+, and the resolution of the two types is in agreement with the earlier observation of a two-site conformational dynamic detected through the ZFS interaction, which is frozen out at low temperatures and averaged at room temperature. To account for the structural origin of the different e2Qq/h values, the electric field gradient tensor was calculated using the point-charge model. The calculations showed that a relatively small displacement of the oxygen ligand of asp10 can lead to differences on the order observed experimentally. [ABSTRACT FROM AUTHOR]

One- and two-dimensional electron-spin echo envelope modulation (ESEEM) spectra of Kramers’ multiplets in orientationally disordered systems are simulated using a simple mathematical model. A fairly general high-field spin Hamiltonian is considered with a general g-tensor and arbitrary relative orientations between all tensors involving the electron-spin S, the nuclear spin I, and their interaction. The zero field splitting (ZFS) and the nuclear quadrupole interactions are, however, approximated by their respective secular part in a way that retains all orientation dependencies and it is assumed that the nuclear quadrupole interaction is smaller than the hyperfine interaction. These approximations yield an effective sublevel nuclear Hamiltonian for each EPR transition and are sufficient to account for the most important characteristics of the ESEEM spectra of high electronic multiplets in orientationally disordered systems. Moreover, they allow to obtain some analytical expressions that for I=1/2 illuminate important aspects of 2D hyperfine sublevel correlation (HYSCORE) experiments in S=3/2, 5/2 systems. The pulses are considered as ideal and selective with respect to the different EPR transitions. The contributions of the latter to the echo intensity are weighed according to their different nutation angles and equilibrium Boltzmann populations. For simple axial cases with I=1/2, analytical expressions, analogous to the S=1/2 case, were derived for: (i) the modulation depth, (ii) the lineshapes of the HYSCORE cross-correlation ridges, and (iii) ENDOR powder pattern. Experimental results obtained from Mn(D2O)62+ and VO(D2O)52+ in frozen solutions are presented, compared, and analyzed in light of the theoretical part. [Copyright &y& Elsevier]

Examines the structure of copper(II)-histidine based complexes in frozen aqueous solutions. Components of the proton's hyperfine interaction and orientation of principal axis; Attachment of the amino and imino nitrogen to copper(II).

Double electron-electron resonance (DEER) is a pulse electron paramagnetic resonance (EPR) technique that measures distances between paramagnetic centres. It utilizes a four-pulse sequence based on the refocused Hahn spin echo. The echo decays with increasing pulse sequence length 2(τ1 Cτ2), where τ1 and τ2 are the two time delays. In DEER, the value of τ2 is determined by the longest inter-spin distance that needs to be resolved, and τ1 is adjusted to maximize the echo amplitude and, thus, sensitivity. We show experimentally that, for typical spin centres (nitroxyl, trityl, and Gd(III)) diluted in frozen protonated solvents, the largest refocused echo amplitude for a given τ2 is obtained neither at very short τ1 (which minimizes the pulse sequence length) nor at τ1 D τ2 (which maximizes dynamic decoupling for a given total sequence length) but rather at τ1 values smaller than τ2. Large-scale spin dynamics simulations based on the coupled cluster expansion (CCE), including the electron spin and several hundred neighbouring protons, reproduce the experimentally observed behaviour almost quantitatively. They show that electron spin dephasing is driven by solvent protons via the flip-flop coupling among themselves and their hyperfine couplings to the electron spin. [ABSTRACT FROM AUTHOR]

The complexity of the cellular medium can affect proteins’ properties, and, therefore, in-cell characterization of proteins is essential. We explored the stability and conformation of the first baculoviral IAP repeat (BIR) domain of X chromosome-linked inhibitor of apoptosis (XIAP), BIR1, as a model for a homodimer protein in human HeLa cells. We employed double electron–electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd3+ spin labels at three representative protein residues, C12 (flexible region), E22C, and N28C (part of helical residues 26 to 31) in the N-terminal region. In contrast to predictions by excluded-volume crowding theory, the dimer–monomer dissociation constant KD was markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was partially recapitulated under conditions of high salt concentrations, given that conserved salt bridges at the dimer interface are critically required for association. Unexpectedly, however, also the addition of the crowding agent Ficoll destabilized the dimer while the addition of bovine serum albumin (BSA) and lysozyme, often used to represent interaction with charged macromolecules, had no effect. Our results highlight the potential of DEER for in-cell study of proteins as well as the complexities of the effects of the cellular milieu on protein structures and stability. [ABSTRACT FROM AUTHOR]

Hsp90 plays a central role in cell homeostasis by assisting folding and maturation of a large variety of clients. It is a homo-dimer, which functions via hydrolysis of ATP-coupled to conformational changes. Hsp90's conformational cycle in the absence of cochaperones is currently postulated as apo-Hsp90 being an ensemble of "open"/"closed" conformations. Upon ATP binding, Hsp90 adopts an active ATP-bound closed conformation where the N-terminal domains, which comprise the ATP binding site, are in close contact. However, there is no consensus regarding the conformation of the ADP-bound Hsp90, which is considered important for client release. In this work, we tracked the conformational states of yeast Hsp90 at various stages of ATP hydrolysis in frozen solutions employing electron paramagnetic resonance (EPR) techniques, particularly double electron-electron resonance (DEER) distance measurements. Using rigid Gd(III) spin labels, we found the C domains to be dimerized with same distance distribution at all hydrolysis states. Then, we substituted the ATPase Mg(II) cofactor with paramagnetic Mn(II) and followed the hydrolysis state using hyperfine spectroscopy and measured the inter-N-domain distance distributions via Mn(II)-Mn(II) DEER. The point character of the Mn(II) spin label allowed us resolve 2 different closed states: The ATP-bound (prehydrolysis) characterized by a distance distribution having a maximum of 4.3 nm, which broadened and shortened, shifting the mean to 3.8 nm at the ADP-bound state (posthydrolysis). This provides experimental evidence to a second closed conformational state of Hsp90 in solution, referred to as "compact." Finally, the so-called high-energy state, trapped by addition of vanadate, was found structurally similar to the posthydrolysis state. [ABSTRACT FROM AUTHOR]

Electron-electron double resonance pulsed electron paramagnetic resonance (EPR) at 95 GHz (3.3 T) is used to follow the dynamics of the electron spin polarization during the first stages of dynamic nuclear polarization in solids. The experiments were performed on a frozen solution of Gd+3 (S=7/2) in water/glycerol. Focusing on the central |-1/2>→|+1/2> transition we measured the polarization transfer from the Gd3+ electron spin to the adjacent 1H protons. The dependence of the echo detected EPR signal on the length of the microwave irradiation at the EPR “forbidden” transition corresponding to an electron and a proton spin flip is measured for different powers, showing dynamics on the microsecond to millisecond time scales. A theoretical model based on the spin density matrix formalism is suggested to account for this dynamics. The central transition of the Gd3+ ion is considered as an effective S=1/2 system and is coupled to 1H (I=1/2) nuclei. Simulations based on a single electron-single nucleus four level system are shown to deviate from the experimental results and an alternative approach taking into account the more realistic multinuclei picture is shown to agree qualitatively with the experiments. [ABSTRACT FROM AUTHOR]

Mn 2+ often serves as a paramagnetic substitute to Mg 2+ , providing means for exploring the close environment of Mg 2+ in many biological systems where it serves as an essential co-factor. This applies to proteins with ATPase activity, where the ATP hydrolysis requires the binding of Mg 2+ -ATP to the ATPase active site. In this context, it is important to distinguish between the Mn 2+ coordination mode with free ATP in solution as compared to the protein bound case. In this work, we explore the Mn 2+ complexes with ATP, the non-hydrolysable ATP analog, AMPPNP, and ADP free in solution. Using W-band 31 P electron-nuclear double resonance (ENDOR) we obtained information about the coordination to the phosphates, whereas from electron-electron double resonance (ELDOR) – detected NMR (EDNMR) we determined the coordination to an adenosine nitrogen. The coordination to these ligands has been reported earlier, but whether the nitrogen and phosphate coordination is within the same nucleotide molecules or different ones is still under debate. By applying the correlation technique, THYCOS (triple hyperfine correlation spectroscopy), and measuring 15 N- 31 P correlations we establish that in Mn-ATP in solution both phosphates and a nitrogen are coordinated to the Mn 2+ ion. We also carried out DFT calculations to substantiate this finding. In addition, we expanded the understanding of the THYCOS experiment by comparing it to 2D-EDNMR for 55 Mn- 31 P correlation experiments and through simulations of THYCOS and 2D-EDNMR spectra with 15 N- 31 P correlations. [ABSTRACT FROM AUTHOR]

[Display omitted] • Enhanced echo sensitivity of higher spin transitions by polarisation transfer from the central transition of Gd(III). • Optimised arbitrary waveform generated shaped pulses allowing spin polarisation transfer. • Enhancement factor of over 2 obtained for polarised 1H Mims ENDOR experiments detecting on the |─3/2>→|─1/2 > transition of Gd(III). • Demonstrated polarisation transfer at Q- and W-band in Gd(III) complexes with different zero-field splitting parameters. • Spin dynamic simulations consistent with experimental enhancements seen. Half-Integer High Spin (HIHS) systems with zero-field splitting (ZFS) parameters below 1 GHz are generally dominated by the spin |─1/2>→|+1/2 > central transition (CT). Accordingly, most pulsed Electron Paramagnetic Resonance (EPR) experiments are performed at this position for maximum sensitivity. However, in certain cases it can be desirable to detect higher spin transitions away from the CT in such systems. Here, we describe the use of frequency swept Wideband, Uniform Rate, Smooth Truncation (WURST) pulses for transferring spin population from the CT, and other transitions, of Gd(III) to the neighbouring higher spin transition |─3/2>→|─1/2 > at Q- and W-band frequencies. Specifically, we demonstrate this approach to enhance the sensitivity of 1H Mims Electron-Nuclear Double Resonance (ENDOR) measurements on two model Gd(III) aryl substituted 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) complexes, focusing on transitions other than the CT. We show that an enhancement factor greater than 2 is obtained for both complexes at Q- and W-band frequencies by the application of two polarising pulses prior to the ENDOR sequence. This is in agreement with simulations of the spin dynamics of the system during WURST pulse excitation. The technique demonstrated here should allow more sensitive experiments to be measured away from the CT at higher operating temperatures, and be combined with any relevant pulse sequence. [ABSTRACT FROM AUTHOR]

Chirp and shaped pulses have been recently shown to be highly advantageous for improving sensitivity in DEER (double electron–electron resonance, also called PELDOR) measurements due to their large excitation bandwidth. The implementation of such pulses for pulse EPR has become feasible due to the availability of arbitrary waveform generators (AWG) with high sampling rates to support pulse shaping for pulses with tens of nanoseconds duration. Here we present a setup for obtaining chirp pulses on our home-built W-band (95 GHz) spectrometer and demonstrate its performance on Gd(III)-Gd(III) and nitroxide-nitroxide DEER measurements. We carried out an extensive optimization procedure on two model systems, Gd(III)-PyMTA–spacer–Gd(III)-PyMTA (Gd-PyMTA ruler; zero-field splitting parameter (ZFS) D ∼ 1150 MHz) as well as nitroxide–spacer–nitroxide (nitroxide ruler) to evaluate the applicability of shaped pulses to Gd(III) complexes and nitroxides, which are two important classes of spin labels used in modern DEER/EPR experiments. We applied our findings to ubiquitin, doubly labeled with Gd-DOTA-monoamide ( D ∼ 550 MHz) as a model for a system with a small ZFS. Our experiments were focused on the questions (i) what are the best conditions for positioning of the detection frequency, (ii) which pump pulse parameters (bandwidth, positioning in the spectrum, length) yield the best signal-to-noise ratio (SNR) improvements when compared to classical DEER, and (iii) how do the sample’s spectral parameters influence the experiment. For the nitroxide ruler, we report an improvement of up to 1.9 in total SNR, while for the Gd-PyMTA ruler the improvement was 3.1–3.4 and for Gd-DOTA-monoamide labeled ubiquitin it was a factor of 1.8. Whereas for the Gd-PyMTA ruler the two setups pump on maximum and observe on maximum gave about the same improvement, for Gd-DOTA-monoamide a significant difference was found. In general the choice of the best set of parameters depends on the D parameter of the Gd(III) complex. [ABSTRACT FROM AUTHOR]

Double-arm cyclen-based Gd3+ tags are shown to produce accurate nanometer scale Gd3+-Gd3+ distance measurements in double electron-electron resonance (DEER) experiments by confining the space accessible to the metal ion. The results show excellent agreement with predictions both for the maximum and width of the measured distance distributions. For distance measurements in proteins, the tags can be attached to two cysteine residues located in positions i and i+4, or i and i+8, of an α-helix. In the latter case, an additional mutation introducing an aspartic acid at position i+4 achieves particularly narrow distribution widths. The concept is demonstrated with cysteine mutants of T4 lysozyme and maltose binding protein. We report the narrowest Gd3+-Gd3+ distance distributions observed to date for a protein. By limiting the contribution of tag mobility to the distances measured, double-arm Gd3+ tags open new opportunities to study the conformational landscape of proteins in solution with high sensitivity. [ABSTRACT FROM AUTHOR]

The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in-cell distance measurements by the double electron-electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed GdIII spin label, a thiol-specific DOTA-derivative (DO3MA-3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in-cell distance measurements by the DEER method carried out at W-band frequencies. The high performance of DO3MA-3BrPy-GdIII is demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High-quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in-cell protein concentrations as low as 5-10 μ m. [ABSTRACT FROM AUTHOR]

The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in-cell distance measurements by the double electron-electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed GdIII spin label, a thiol-specific DOTA-derivative (DO3MA-3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in-cell distance measurements by the DEER method carried out at W-band frequencies. The high performance of DO3MA-3BrPy-GdIII is demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High-quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in-cell protein concentrations as low as 5-10 μ m. [ABSTRACT FROM AUTHOR]

The article reports on the investigation of the location and coordination shell of the copper (Cu) ions within the zeolite structure which can account for the catalytic activity. High-field electron-nuclear double resonance spectra of Cu ions in zeolite X are presented. Additional valuable constraints for density functional theory calculations can be provided by the determined tensors.

Mn2+ chelating tags for Mn2+–Mn2+ distance measurements by pulse EPR spectroscopy were developed. They feature a stable C–S conjugation to the protein, high reactivity towards cysteine thiols and short and rigid linkers that can be used in distance measurements with high resolution under reductive conditions. Double electron–electron resonance measurements at 95 GHz on ubiquitin labeled with these tags showed the expected narrow distance distribution. [ABSTRACT FROM AUTHOR]

Calmodulin (CaM) is a calcium-binding protein that regulates the function of many proteins by indirectly conferring Ca2+ sensitivity, and it undergoes a large conformational change on partners' binding. We compared the solution binding mode of the target peptides MARCKS and IQ by double electron-electron resonance (DEER) distance measurements and paramagnetic NMR. We combined nitroxide and Gd(III) spin labels, including specific substitution of one of the Ca2+ ions in the CaM mutant N60D by a Gd(III) ion. The binding of MARCKS to holo -CaM resulted neither in a closed conformation nor in a unique relative orientation between the two CaM domains, in contrast with the crystal structure. Binding of IQ to holo -CaM did generate a closed conformation. Using elastic network modeling and 12 distance restraints obtained from multiple holo -CaM/IQ DEER data, we derived a model of the solution structure, which is in reasonable agreement with the crystal structure. [Display omitted] • The target peptides MARCKS and IQ bind CaM both in its apo - and holo -states • In solution, the binding of MARCKS to holo -CAM does not generate a closed conformation • In solution, holo -CaM with bound IQ has a closed conformation, albeit heterogeneous • Gd(III)/nitroxide DEER distance restraints give a solution holo -CaM/IQ structural model Jash et al. track the conformational changes calmodulin undergoes on binding MARCKS and IQ peptides in solution and report significant differences in their binding modes. This is achieved by various Gd(III) and nitroxide spin labeling schemes for CaM and the peptides combined with EPR inter-spin distance and paramagnetic NMR measurements. [ABSTRACT FROM AUTHOR]

[Display omitted] • A methodological guide to deep neural networks in pulsed dipolar spectroscopy. • Neural networks are unexpectedly good at processing DEER and RIDME data. • Sparsely sampled datasets and uncertainty quantification are supported. • Unlike regularisation methods, DEERNet has no user-adjustable parameters. This is a methodological guide to the use of deep neural networks in the processing of pulsed dipolar spectroscopy (PDS) data encountered in structural biology, organic photovoltaics, photosynthesis research, and other domains featuring long-lived radical pairs and paramagnetic metal ions. PDS uses distance dependence of magnetic dipolar interactions; measuring a single well-defined distance is straightforward, but extracting distance distributions is a hard and mathematically ill-posed problem requiring careful regularisation and background fitting. Neural networks do this exceptionally well, but their "robust black box" reputation hides the complexity of their design and training – particularly when the training dataset is effectively infinite. The objective of this paper is to give insight into training against simulated databases, to discuss network architecture choices, to describe options for handling DEER (double electron-electron resonance) and RIDME (relaxation-induced dipolar modulation enhancement) experiments, and to provide a practical data processing flowchart. [ABSTRACT FROM AUTHOR]

Protein structure investigations are usually carried out in vitro under conditions far from their native environment in the cell. Differences between in-cell and in vitro structures of proteins can be generated by crowding effects, local pH changes, specific and nonspecific protein and ligand binding events, and chemical modifications. Double electronelectron resonance (DEER), in conjunction with site-directed spin-labeling, has emerged in the past decade as a powerful technique for exploring protein conformations in frozen solutions. The major challenges facing the application of this methodology to in-cell measurements are the instabilities of the standard nitroxide spin labels in the cell environment and the limited sensitivity at conventional X-band frequencies. We present a new approach for in-cell DEER distance measurement in human cells, based on the use of: (i) reduction resistant Gd3+ chelates as spin labels, (ii) high frequency (94.9 GHz) for sensitivity enhancement, and (iii) hypo-osmotic shock for efficient delivery of the labeled protein into the cell. The proof of concept is demonstrated on doubly labeled ubiquitin in HeLa cells. [ABSTRACT FROM AUTHOR]

ATP-dependent binding of the chaperonin GroEL to its cofactor GroES forms a cavity in which encapsulated substrate proteins can fold in isolation from bulk solution. It has been suggested that folding in the cavity may differ from that in bulk solution owing to steric confinement, interactions with the cavity walls, and differences between the properties of cavity-confined and bulk water. However, experimental data regarding the cavity-confined water are lacking. Here, we report measurements of water density and diffusion dynamics in the vicinity of a spin label attached to a cysteine in the Tyr71 → Cys GroES mutant obtained using two magnetic resonance techniques: electronspin echo envelope modulation and Overhauser dynamic nuclear polarization. Residue 71 in GroES is fully exposed to bulk water in free GroES and to confined water within the cavity of the GroEL—GroES complex. Our data show that water density and translational dynamics in the vicinity of the label do not change upon complex formation, thus indicating that bulk water-exposed and cavity-confined GroES surface water share similar properties. Interestingly, the diffusion dynamics of water near the GroES surface are found to be unusually fast relative to other protein surfaces studied. The implications of these findings for chaperonin-assisted folding mechanisms are discussed. [ABSTRACT FROM AUTHOR]

Highlights: [•] A triple resonance experiment that correlates ELDOR-detected NMR signals is described. [•] Theory and simulations to account for the cross peaks is presented. [•] Negative cross peaks appear between nuclear frequencies belonging to different M S manifolds. [•] Positive cross peaks are produced by of off resonance effects and nuclear relaxation. [•] Experiment is demonstrated on 14N of a nitroxide and 33S and 14N of Cu(II) type one center of Azurin. [ABSTRACT FROM AUTHOR]

High‐frequency double electron‐electron resonance (DEER) distance measurements using different Gd3+ tags (Gd‐DOTA and Gd‐C1) were carried out on transmembrane helical peptides (ca. 0.15 nmol; WALP peptides) in a model membrane. The ability to pick up small distance variations, the chemical flexibility of the tags, and the remarkable absolute sensitivity, make this approach attractive for studies of membrane proteins. [ABSTRACT FROM AUTHOR]

Abstandsmessungen an helicalen Transmembranpeptiden (ca. 0.15 nmol; WALP ‐ Peptide) in einer Modellmembran wurden mit der Doppel ‐ Elektron ‐ Elektron ‐ Resonanz (DEER) ‐ Technik bei hoher Frequenz unter Verwendung von Gd3+ ‐ Markern (Gd ‐ DOTA und Gd ‐ C1) durchgeführt. Der Ansatz ist attraktiv für Membranproteinstudien, da kleine Abstandsänderungen detektiert werden können und die Marker chemisch flexibel sind. [ABSTRACT FROM AUTHOR]

Nitroxide spin-labelled lipid analogues are often used to study model membrane properties using EPR spectroscopy. Whereas in liquid phase membranes the spin label assumes, on average, its putative location, in gel phases and frozen membrane, depending on its position along the acyl chain, it may exhibit a different average location. Here we used2H three-pulse Electron Spin Echo Envelope Modulation (ESEEM) of phospholipid spin probes, combined with various deuteration schemes to detect the effect of the model membrane curvature and cholesterol on vertical migrations of the spin label. We compared large and small unilamellar 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles with and without cholesterol (10%). The vertical displacement of the spin label was manifested as an apparently flat trans-membrane profile of water concentration and of label proximity to the head group choline. The spin-label propensity to migrate was found to increase with vesicle curvature and decrease in the presence of cholesterol. This in turn reflects the effect of packing and ordering of the membrane lipids. The results show that in curved vesicles lacking cholesterol, the label attached to carbon 16 may travel as far high along the membrane normal as the location of the label on carbon 5, due to the presence of U-shaped lipid conformations. This phenomenon must be taken into account when using spin-labelled lipids as membrane depth markers or to trace trans-membrane profiles. [ABSTRACT FROM PUBLISHER]

Abstract: The 13C solid state Dynamic Nuclear Polarization (DNP) mechanism using trityl radicals (OX63) as polarizers was investigated in the temperature range of 10–60K. The solutions used were 6M 13C urea in DMSO/H2O (50% v/v) with 15mM and 30mM OX63. The measurements were carried out at ∼3.5T, which corresponds to Larmor frequencies of 95GHz and 36MHz for the OX63 and the 13C nuclei, respectively. Measurements of the 13C signal intensity as a function of the microwave (MW) irradiation frequency yielded 13C DNP spectra with temperature dependent lineshapes for both samples. The maximum enhancement for the 30mM sample was reached at 40K, while that of the 15mM sample at 20–30K. Furthermore, the lineshapes observed showed that both the cross effect (CE) and the solid effect (SE) DNP mechanisms are active in this temperature range and that their relative contribution is temperature dependent. Simulations of the spectra with the relative contributions of the CE and SE mechanisms as a fit parameter revealed that for both samples the CE contribution decreases with decreasing temperature while the SE contribution increases. In addition, for the 15mM sample the contributions of the two mechanisms are comparable from 20K to 60K while for the 30mM the CE dominates in this range, as expected from the higher concentration. The steep decrease of the CE contribution towards low temperatures is however unexpected. The temperature dependence of the OX63 longitudinal relaxation, DNP buildup times and 13C spin lattice relaxation times did not reveal any obvious correlation with the DNP temperature dependence. A similar behavior of the CE and SE mechanism was observed for 1H DNP with the nitroxide radical TEMPOL as a polarizer. This suggests that this effect is a general phenomenon involving a temperature dependent competition between the CE and SE mechanisms, the source of which is, however, still unknown. [Copyright &y& Elsevier]

The organization and orientation of membrane-insertedhelices isimportant for better understanding the mode of action of membrane-activepeptides and of protein–membrane interactions. Here we reporton the application of ESEEM (electron spin–echo envelope modulation)and DEER (double electron–electron resonance) techniques toprobe the orientation and oligomeric state of an α-helical trans-membranemodel peptide, WALP23, under conditions of negative mismatch betweenthe hydrophobic cores of the model membrane and the peptide. UsingESEEM, we measured weak dipolar interactions between spin-labeledWALP23 and 2H nuclei of either the solvent (D2O) or of lipids specifically deuterated at the choline group. TheESEEM data obtained from the deuterated lipids were fitted using amodel that provided the spin label average distance from a layer of 2H nuclei in the hydrophilic region of the membrane and thedensity of the 2H nuclei in the layer. DEER was used toprobe oligomerization through the dipolar interaction between twospin-labels on different peptides. We observed that the center ofWALP23 does not coincide with the bilayer midplane and its N-terminusis more buried than the C-terminus. In addition, the ESEEM data fittingyielded a 2H layer density that was much lower than expected.The DEER experiments revealed the presence of oligomers, the presenceof which was attributable to the negative mismatch and the electrostaticdipole of the peptide. A discussion of a possible arrangement of theindividual helices in the oligomers that is consistent with the ESEEMand DEER data is presented. [ABSTRACT FROM AUTHOR]

Abstract: Double electron–electron resonance (DEER) at W-band (95GHz) was applied to measure the distance between a pair of nitroxide and Gd3+ chelate spin labels, about 6nm apart, in a homodimer of the protein ERp29. While high-field DEER measurements on systems with such mixed labels can be highly attractive in terms of sensitivity and the potential to access long distances, a major difficulty arises from the large frequency spacing (about 700MHz) between the narrow, intense signal of the Gd3+ central transition and the nitroxide signal. This is particularly problematic when using standard single-mode cavities. Here we show that a novel dual-mode cavity that matches this large frequency separation dramatically increases the sensitivity of DEER measurements, allowing evolution times as long as 12μs in a protein. This opens the possibility of accessing distances of 8nm and longer. In addition, orientation selection can be resolved and analyzed, thus providing additional structural information. In the case of W-band DEER on a Gd3+–nitroxide pair, only two angles and their distributions have to be determined, which is a much simpler problem to solve than the five angles and their distributions associated with two nitroxide spin labels. [Copyright &y& Elsevier]