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In the brains of Alzheimer's disease patients, fibrillar aggregates containing amyloid-beta (Aß) peptides are found, along with elevated concentrations of Cu(II) ions. The aggregation pathways of Aß peptides can be modulated by Cu(II) ions and is determined by the formation and nature of the Cu(II)-Aß complex. If spin-labeled, the Cu(II)-Aß complex contains two dipolar coupled paramagnetic centers, the spin label and the Cu(II) ion. Measurement of the dipolar coupling between these paramagnetic centers by relaxation-induced dipolar modulation enhancement (RIDME) allows to monitor the complex formation and thus opens a way to follow the Cu(II) transfer between peptides if a mixture of wild-type and spin-labeled ones is used. We evaluate this approach for a specific Cu(II)-Aß complex, the aggregation-inert Componentâ II. The kinetics of the Cu(II) transfer can be resolved by performing RIDME in a time-dependent manner. A temporal resolution of seconds has been achieved, with the potential to reach milliseconds, using a rapid-freeze quench device to stop the Cu(II) transfer in solution after defined incubation times.
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Péptidos beta-Amiloides , Cobre , Cobre/química , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Cinética , Humanos , Espectroscopía de Resonancia por Spin del ElectrónRESUMEN
Metal ion complexes frequently show substituted 1,4,7-triazacyclononane (tacn) as the ligand. Besides providing donor atoms for complex formation, tacn serves as a scaffold for equipping the complex with further functional units that are needed for the complementation and electronic tuning of the metal ion coordination sphere and/or add other features, e.g., light-absorbing antennas and groups for bioconjugation. To exploit the full potential of substituted tacn, strategies for directed syntheses of NO(R1,R1,R2) and NO(R1,R2,R3), i.e., tacn with two and even three different substituents R, are needed. Herein, we report a strategy that takes advantage of solid-phase synthesis in the assembly of the precursors NO(R1,R1,H) and NO(R1,R2,H). The assembly of NO(R1,R2,H) is based on a highly selective formation of NO(Cbz,tfAc,H), with Cbz being the link between tacn and solid phase. For this, tacn was loaded onto (4-nitrophenyl carbonate)-resin, thereby forming resin-bound (rb)-tacn, which corresponds to NO(Cbz,H,H) bound to the solid phase. Treatment of rb-tacn with ethyl trifluoroacetate gave rb-NO(tfAc,H), which corresponds to NO(Cbz,tfAc,H). With rb-tacn and rb-NO(tfAc,H) in hand, a variety of NO(R1,R1,H) and NO(R1,R2,H) were prepared, showing the broad applicability of the strategy with respect to the type of substituents and of reactions (nucleophilic substitution, reductive amination, aza-Michael addition, addition to epoxides, acylation). The study also identified limitations and points for improvement.
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Following the success of cryogenic EPR signal preamplification at X-band, we present a Q-band EPR cryoprobe compatible with a standard EPR resonator. The probehead is equipped with a cryogenic ultra low-noise microwave amplifier and its protection circuit that are placed close to the sample in the same cryostat. Our cryoprobe maintains the same sample access and tuning which is typical in Q-band EPR, as well as supports high-power pulsed experiments on typical samples. The performance of our setup is benchmarked against that of existing commercial and home-built Q-band spectrometers, using CW EPR and pulsed EPR/ENDOR experiments to reveal a significant sensitivity improvement which reduces the measurement time by a factor of about 40× at 6 K temperature at reduced power levels.
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The remarkably narrow central line in the electron paramagnetic resonance spectrum and the very weak zero-field splitting (ZFS) make [GdIII(NO3Pic)] ([GdIII(TPATCN)]) an attractive starting point for the development of spin labels. For retaining the narrow line of this parent complex when modifying it with a substituent enabling bioconjugation, alkyl with a somehow remote functional group as a substituent at the picolinate moiety was found to be highly suitable because ZFS stayed weak, even if the threefold axial symmetry was broken. The ZFS is so weak that hyperfine coupling and/or g-value variations noticeably determine the linewidth in Q band and higher fields when the biomolecule is protonated, which is the standard situation, and in W band and higher fields for the protonated complex in a fully deuterated surrounding. Clearly, [NDSE-{GdIII(NO3Pic)}], a spin label targeting the cysteines in a peptide, is at a limit of linewidth narrowing through ZFS minimization. The labeling reaction is highly chemoselective and, applied to a polyproline with two cysteine units, it took no more than a minute at 7 °C and pH 7.8. Subsequent disulfide scrambling is very slow and can therefore be prevented. Double electron-electron resonance and relaxation-induced dipolar modulation enhancement applied to the spin-labeled polyproline proved the spin label useful for distance determination in peptides.
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Cisteína , Gadolinio , Marcadores de Spin , Gadolinio/química , Espectroscopía de Resonancia por Spin del ElectrónRESUMEN
To characterize structure and molecular order in the nanometre range, distances between electron spins and their distributions can be measured via dipolar spin-spin interactions by different pulsed electron paramagnetic resonance experiments. Here, for the single-frequency technique for refocusing dipolar couplings (SIFTER), the buildup of dipolar modulation signal and intermolecular contributions is analysed for a uniform random distribution of monoradicals and biradicals in frozen glassy solvent by using the product operator formalism for electron spin S=1/2. A dipolar oscillation artefact appearing at both ends of the SIFTER time trace is predicted, which originates from the weak coherence transfer between biradicals. The relative intensity of this artefact is predicted to be temperature independent but to increase with the spin concentration in the sample. Different compositions of the intermolecular background are predicted in the case of biradicals and in the case of monoradicals. Our theoretical account suggests that the appropriate procedure of extracting the intramolecular dipolar contribution (form factor) requires fitting and subtracting the unmodulated part, followed by division by an intermolecular background function that is different in shape. This scheme differs from the previously used heuristic background division approach. We compare our theoretical derivations to experimental SIFTER traces for nitroxide and trityl monoradicals and biradicals. Our analysis demonstrates a good qualitative match with the proposed theoretical description. The resulting perspectives for a quantitative analysis of SIFTER data are discussed.
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Distance determination with pulsed EPR has become an important technique for the structural investigation of biomacromolecules, with double electron-electron resonance spectroscopy (DEER) as the most important method. GdIII-based spin labels are one of the most frequently used spin labels for DEER owing to their stability against reduction, high magnetic moment, and absence of orientation selection. A disadvantage of GdIII-GdIII DEER is the low modulation depth due to the broad EPR spectrum of GdIII. Here, we introduce laser-induced magnetic dipole spectroscopy (LaserIMD) with a spin pair consisting of GdIII(PymiMTA) and a photoexcited porphyrin as an alternative technique. We show that the excited state of the porphyrin is not disturbed by the presence of the GdIII complex and that herewith modulation depths of almost 40% are possible. This is significantly higher than the value of 7.2% that was achieved with GdIII-GdIII DEER.
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Porfirinas , Espectroscopía de Resonancia por Spin del Electrón , Marcadores de Spin , Fenómenos Magnéticos , Rayos LáserRESUMEN
The measurement of distances in proteins can be challenging in the 5-20 Å range, which is outside those accessible through conventional NMR and EPR methods. Recently it was demonstrated that distances in this range could be measured between a nitroxide as a paramagnetic spin label and a nearby fluorine atom (19F) as a nuclear spin label using high-field (W-band/3.4 T) ENDOR spectroscopy. Here we show that such measurements can also be performed using a gadolinium ion (Gd3+) as the paramagnetic tag. Gd3+ has two advantages. (i) A greater electronic spin (S = 7/2) and fast electronic spin-lattice (T1) relaxation, improving sensitivity by allowing data to be collected at lower temperatures. (ii) A narrow EPR signal for the -½ â ½ transition, and therefore no orientation selection artefacts. Signal intensities can be further enhanced by using a trifluoromethyl (C19F3) group instead of a single 19F atom. Using the protein calbindin D9k with a Ca2+ ion replaced by a Gd3+ ion and a trifluoromethylphenylalanine in position 50, we show that distances up to about 10 Å can be readily measured. Longer distances proved more difficult to measure due to variable electronic TM relaxation rates, which lead to broader Lorentzian ENDOR lineshapes. Gd3+ complexes (Gd3+ tags), which reliably display longer TM times, allow longer distances to be measured (8-16 Å). We also provide preliminary evidence that the intensity of ENDOR signals follows the predicted 1/r6 dependence, indicating that distances r > 20 Å can be measured by this method.
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Gadolinio , Proteínas , Espectroscopía de Resonancia por Spin del Electrón/métodos , Marcadores de Spin , Proteínas/química , Gadolinio/química , Espectroscopía de Resonancia MagnéticaRESUMEN
Relaxation-induced dipolar modulation enhancement (RIDME) is a pulse EPR technique that is particularly suitable to determine distances between paramagnetic centers with a broad EPR spectrum, e.g. metal-ion-based ones. As far as high-spin systems (S > ½) are concerned, the RIDME experiment provides not only the basic dipolar frequency but also its overtones, which complicates the determination of interspin distances. Here, we present and discuss in a step-by-step fashion an r.m.s.d.-based approach for the calibration of the overtone coefficients for a series of molecular rulers doubly labeled with Gd(III)-PyMTA tags. The constructed 2D total-penalty diagrams help revealing that there is no unique set of overtone coefficients but rather a certain pool, which can be used to extract distance distributions between high-spin paramagnetic centers, as determined from the RIDME experiment. This is of particular importance for comparing RIDME overtone calibration and distance distributions obtained in different labs.
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Espectroscopía de Resonancia por Spin del Electrón , Espectroscopía de Resonancia por Spin del Electrón/métodosRESUMEN
Our previous study on nitroxides in o-terphenyl (OTP) revealed two separable decoherence processes at low temperatures, best captured by the sum of two stretched exponentials (SSE) model. Dynamical decoupling (DD) extends both associated dephasing times linearly for 1 to 5 refocusing pulses [Soetbeer et al., Phys. Chem. Chem. Phys., 2018, 20, 1615]. Here we demonstrate an analogous DD behavior of water-soluble nitroxides in water-glycerol glass by using nitroxide and/or solvent deuteration for component assignment. Compared to the conventional Hahn experiment, we show that Carr-Purcell and Uhrig DD schemes are superior in resolving and identifying active dephasing mechanisms. Thereby, we observe a partial coherence loss to intramolecular nitroxide and trityl nuclei that can be alleviated, while the zero field splitting-induced losses for gadolinium labels cannot be refocused and contribute even at the central transition of this spin-7/2 system. Independent of the studied spin system, Uhrig DD leads to a characteristic convex dephasing envelope in both protonated water-glycerol and OTP glass, thus outperforming the Carr-Purcell scheme.
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Inspired by the considerable success of cryogenically cooled NMR cryoprobes, we present an upgraded X-band EPR probehead, equipped with a cryogenic low-noise preamplifier. Our setup suppresses source noise, can handle the high microwave powers typical in X-band pulsed EPR, and is compatible with the convenient resonator coupling and sample access found on commercially available spectrometers. Our approach allows standard pulsed and continuous-wave EPR experiments to be performed at X-band frequency with significantly increased sensitivity compared to the unmodified setup. The probehead demonstrates a voltage signal-to-noise ratio (SNR) enhancement by a factor close to 8× at a temperature of 6 K, and remains close to 2× at room temperature. By further suppressing room-temperature noise at the expense of reduced microwave power (and thus minimum π-pulse length), the factor of SNR improvement approaches 15 at 6 K, corresponding to an impressive 200-fold reduction in EPR measurement time. We reveal the full potential of this probehead by demonstrating such SNR improvements using a suite of typical hyperfine and dipolar spectroscopy experiments on exemplary samples.
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Gadolinium complexes are attracting increasing attention as spin labels for EPR dipolar distance measurements in biomolecules and particularly for in-cell measurements. It has been shown that flip-flop transitions within the central transition of the high-spin Gd3+ ion can introduce artefacts in dipolar distance measurements, particularly when measuring distances less than 3â¯nm. Previous work has shown some reduction of these artefacts through increasing the frequency separation between the two frequencies required for the double electron-electron resonance (DEER) experiment. Here we use a high-power (1â¯kW), wideband, non-resonant system operating at 94â¯GHz to evaluate DEER measurement protocols using two stiff Gd(III) rulers, consisting of two bis-Gd3+-PyMTA complexes, with separations of 2.1â¯nm and 6.0â¯nm, respectively. We show that by avoiding the -12â12 central transition completely, and placing both the pump and the observer pulses on either side of the central transition, we can now observe apparently artefact-free spectra and narrow distance distributions, even for a Gd-Gd distance of 2.1â¯nm. Importantly we still maintain excellent signal-to-noise ratio and relatively high modulation depths. These results have implications for in-cell EPR measurements at naturally occurring biomolecule concentrations.
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Double electron-electron resonance (DEER) spectroscopy applied to orthogonally spin-labeled biomolecular complexes simplifies the assignment of intra- and intermolecular distances, thereby increasing the information content per sample. In fact, various spin labels can be addressed independently in DEER experiments due to spectroscopically nonoverlapping central transitions, distinct relaxation times, and/or transition moments; hence, they are referred to as spectroscopically orthogonal. Molecular complexes which are, for example, orthogonally spin-labeled with nitroxide (NO) and gadolinium (Gd) labels give access to three distinct DEER channels that are optimized to selectively probe NO-NO, NO-Gd, and Gd-Gd distances. Nevertheless, it has been previously recognized that crosstalk signals between individual DEER channels can occur, for example, when a Gd-Gd distance appears in a DEER channel optimized to detect NO-Gd distances. This is caused by residual spectral overlap between NO and Gd spins which, therefore, cannot be considered as perfectly orthogonal. Here, we present a systematic study on how to identify and suppress crosstalk signals that can appear in DEER experiments using mixtures of NO-NO, NO-Gd, and Gd-Gd molecular rulers characterized by distinct, nonoverlapping distance distributions. This study will help to correctly assign the distance peaks in homo- and heterocomplexes of biomolecules carrying not perfectly orthogonal spin labels.
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Distance measurement in the nanometre range is among the most important applications of pulse electron paramagnetic resonance today, especially in biological applications. The longest distance that can be measured by all presently used pulse sequences is determined by the phase memory time Tm of the observed spins. Here we show that one can measure the dipolar coupling during strong microwave irradiation by using an appropriate frequency- or phase-modulation scheme, i.e. by applying pulse sequences in the nutating frame. This decouples the electron spins from the surrounding nuclear spins and thus leads to significantly longer relaxation times of the microwave-dressed spins (i.e. the rotating frame relaxation times T1ρ and T2ρ) compared to Tm. The electron-electron dipolar coupling is not decoupled as long as both spins are excited, which can be implemented for trityl radicals at Q-band frequencies (35â¯GHz, 1.2â¯T). We show results for two bis-trityl rulers with inter-electron distances of about 4.1 and 5.3â¯nm and discuss technical challenges and possible next steps.
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We show that oligo(phenyleneethynylene)s (oligoPEs) are ideal spacers for calibrating dye pairs used for Förster resonance energy transfer (FRET). Ensemble FRET measurements on linear and kinked diads with such spacers show the expected distance and orientation dependence of FRET. Measured FRET efficiencies match excellently with those predicted using a harmonic segmented chain model, which was validated by end-to-end distance distributions obtained from pulsed electron paramagnetic resonance measurements on spin-labeled oligoPEs with comparable label distances.
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Distance determination by Electron Paramagnetic Resonance (EPR) based on measurements of the dipolar coupling are technically challenging for electron spin systems with broad spectra due to comparatively narrow microwave pulse excitation bandwidths. With Na4[{CuII(PyMTA)}-(stiff spacer)-{CuII(PyMTA)}] as a model compound, we compared DEER and RIDME measurements and investigated the use of frequency-swept pulses. We found very large improvements in sensitivity when substituting the monochromatic pump pulse by a frequency-swept one in DEER experiments with monochromatic observer pulses. This effect was especially strong in X band, where nearly the whole spectrum can be included in the experiment. The RIDME experiment is characterised by a trade-off in signal intensity and modulation depth. Optimal parameters are further influenced by varying steepness of the background decay. A simple 2-point optimization experiment was found to serve as good estimate to identify the mixing time of highest sensitivity. Using frequency-swept pulses in the observer sequences resulted in lower SNR in both the RIDME and the DEER experiment. Orientation selectivity was found to vary in both experiments with the detection position as well as with the settings of the pump pulse in DEER. In RIDME, orientation selection by relaxation anisotropy of the inverted spin appeared to be negligible as form factors remain relatively constant with varying mixing time. This reduces the overall observed orientation selection to the one given by the detection position. Field-averaged data from RIDME and DEER with a shaped pump pulse resulted in the same dipolar spectrum. We found that both methods have their advantages and disadvantages for given instrumental limitations and sample properties. Thus the choice of method depends on the situation at hand and we discuss which parameters should be considered for optimization.
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Orthogonal site-directed spin labelling in combination with pulsed EPR spectroscopy is a powerful approach to study biomolecular interactions on a molecular level. Following a surge in pulse EPR method development, it is now possible to access distance distributions in the nanometre range in systems of complex composition. In this article we briefly outline the necessary considerations for measurements of distance distributions in macromolecular systems labelled with two or more different types of paramagnetic centres. We illustrate the approach with two examples: an application of the Double Electron-Electron Resonance (DEER) method on a triple spin-labelled protein dimer labelled with nitroxide and Gd(III), and an optimisation study of the Relaxation Induced Dipolar Modulation Enhancement (RIDME) experiment for the orthogonal spin pair Cu(II)-nitroxide.
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Espectroscopía de Resonancia por Spin del Electrón , Electrones , Sustancias Macromoleculares , Proteínas , Marcadores de SpinRESUMEN
Orientation selection is a challenge in distance determination with double electron electron resonance (DEER) spectroscopy of rigid molecules. The problem is reduced when applying the Relaxation-Induced Dipolar Modulation Enhancement (RIDME) experiment. Here we present an in-depth study on nitroxide-detected RIDME in Cu(ii)-nitroxide spin pairs using two Cu(ii)-nitroxide rulers that are both water soluble and have comparable spin-spin distances. They differ in the type of the ligand (TAHA and PyMTA) for the Cu(ii) ion which results in different contributions of exchange coupling. Both rulers feature substantial orientation correlation between the molecular frames of the Cu(ii) complex and the nitroxide. We discuss how the spin-spin couplings can be accurately measured and how they can be correlated to the nitroxide resonance frequencies. In that, we pay particular attention to the suppression of nuclear modulation and of echo crossing artefacts, to background correction, and to orientation averaging. With a nitroxide observer sequence based on chirp pulses, we achieve wideband detection of all nitroxide orientations. Two-dimensional Fourier transformation of data obtained in this manner affords observer-EPR correlated RIDME spectra that enable visual understanding of the orientation correlation. The syntheses of the Cu(ii)-nitroxide rulers are presented. The synthetic route is considered to be of general use for the preparation of [metal ion complex]-nitroxide rulers, including water soluble ones.
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The relaxation-induced dipolar modulation enhancement (RIDME) technique allows the determination of distances and distance distributions in pairs containing two paramagnetic metal centers, a paramagnetic metal center and an organic radical, and, under some conditions, also in pairs of organic radicals. The strengths of the RIDME technique are its simple setup requirements, and the absence of bandwidth limitations for spin inversion which occurs through relaxation. A strong limitation of the RIDME technique is the background decay, which is often steeper than that in the double electron electron resonance experiment, and the absence of an appropriate description of the intermolecular background signal. Here we address the latter problem and present an analytical calculation of the RIDME background decay in the simple case of two types of randomly distributed spin centers each with total spin S = 1/2. The obtained equations allow the explaination of the key trends in RIDME experiments on frozen chelated metal ion solutions, and singly spin-labeled proteins. At low spin label concentrations, the RIDME background shape is determined by nuclear-driven spectral diffusion processes. This fact opens up a new path for structural characterization of soft matter and biomacromolecules through the determination of the local distribution of protons in the vicinity of the spin-labeled site.
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In-cell distance determination by electron paramagnetic resonance (EPR) spectroscopy reveals essential structural information about biomacromolecules under native conditions. We demonstrate that the pulsed EPR technique RIDME (relaxation induced dipolar modulation enhancement) can be utilized for such distance determination. The performance of in-cell RIDME has been assessed at Q-band using stiff molecular rulers labeled with Gd(III)-PyMTA and microinjected into Xenopus laevis oocytes. The overtone coefficients are determined to be the same for protonated aqueous solutions and inside cells. As compared to in-cell DEER (double electron-electron resonance, also abbreviated as PELDOR), in-cell RIDME features approximately 5 times larger modulation depth and does not show artificial broadening in the distance distributions due to the effect of pseudosecular terms.
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The synthesis and characterization of a chemiluminescent metal-organic framework with high porosity is reported. It consists of Zr6 O6 (OH)4 nodes connected by 4,4'-(anthracene-9,10-diyl)dibenzoate as the linker and luminophore. It shows the topology known for UiO-66 and is therefore denoted PAP-UiO. The MOF was not only obtained as bulk material but also as a thin film. Exposure of PAP-UiO as bulk or film to a mixture of bis-(2,4,6-trichlorophenyl) oxalate, hydrogen peroxide, and sodium salicylate in a mixture of dimethyl and dibutyl phthalate evoked strong and long lasting chemiluminescence of the PAP-UiO crystals. Time dependent fluorescence spectroscopy on bulk PAP-UiO and, for comparison, on dimethyl 4,4'-(anthracene-9,10-diyl)dibenzoate provided evidence that the chemiluminescence originates from luminophores being part of the PAP-UiO, including the luminophores inside the crystals.