Relaxation-induced Dipolar Modulation Enhancement Research Articles

Quantification of Distributions of Local Proton Concentrations in Heterogeneous Soft Matter and Non-Anfinsen Biomacromolecules.

A new method to quantitatively analyze heterogeneous distributions of local proton densities around paramagnetic centers in unstructured and weakly structured biomacromolecules and soft matter is introduced, and its feasibility is demonstrated on aqueous solutions of stochastically spin-labeled polysaccharides. This method is based on the pulse EPR experiment ih-RIDME (intermolecular hyperfine relaxation-induced dipolar modulation enhancement). Global analysis of a series of RIDME traces allows for a mathematically stable transformation of the time-domain data to the distribution of local proton concentrations. Two pulse sequences are proposed and tested, which combine the ih-RIDME block and the double-electron-electron resonance (DEER) experiment. Such experiments can be potentially used to correlate the local proton concentration with the macromolecular chain conformation. We anticipate an application of this approach in studies of intrinsically disordered proteins, biomolecular aggregates, and biomolecular condensates.

Evaluation of Copper(II) Transfer between Amyloid-beta Peptides by Relaxation-Induced Dipolar Modulation Enhancement (RIDME).

In the brains of Alzheimer's disease patients, fibrillar aggregates containing amyloid-beta (Aβ) peptides are found, along with elevated concentrations of Cu(II) ions. The aggregation pathways of Aβ peptides can be modulated by Cu(II) ions and is determined by the formation and nature of the Cu(II)-Aβ complex. If spin-labeled, the Cu(II)-Aβ 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)-Aβ complex, the aggregation-inert Component 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.

Pulse Dipolar Electron Paramagnetic Resonance Spectroscopy Distance Measurements at Low Nanomolar Concentrations: The CuII-Trityl Case.

Recent sensitivity enhancements in pulse dipolar electron paramagnetic resonance spectroscopy (PDS) have afforded distance measurements at submicromolar spin concentrations. This development opens the path for new science as more biomolecular systems can be investigated at their respective physiological concentrations. Here, we demonstrate that the combination of orthogonal spin-labeling using CuII ions and trityl yields a >3-fold increase in sensitivity compared to that of the established CuII-nitroxide labeling strategy. Application of the recently developed variable-time relaxation-induced dipolar modulation enhancement (RIDME) method yields a further ∼2.5-fold increase compared to the commonly used constant-time RIDME. This overall increase in sensitivity of almost an order of magnitude makes distance measurements in the range of 3 nm with protein concentrations as low as 10 nM feasible, >2 times lower than the previously reported concentration. We expect that experiments at single-digit nanomolar concentrations are imminent, which have the potential to transform biological PDS applications.

PDSFit: PDS data analysis in the presence of orientation selectivity, g-anisotropy, and exchange coupling.

Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS), encompassing techniques such as pulsed electron-electron double resonance (PELDOR or DEER) and relaxation-induced dipolar modulation enhancement (RIDME), is a valuable method in structural biology and materials science for obtaining nanometer-scale distance distributions between electron spin centers. An important aspect of PDS is the extraction of distance distributions from the measured time traces. Most software used for this PDS data analysis relies on simplifying assumptions, such as assuming isotropic g-factors of ~2 and neglecting orientation selectivity and exchange coupling. Here, the program PDSFit is introduced, which enables the analysis of PELDOR and RIDME time traces with or without orientation selectivity. It can be applied to spin systems consisting of up to two spin centers with anisotropic g-factors and to spin systems with exchange coupling. It employs a model-based fitting of the time traces using parametrized distance and angular distributions, and parametrized PDS background functions. The fitting procedure is followed by an error analysis for the optimized parameters of the distributions and backgrounds. Using five different experimental data sets published previously, the performance of PDSFit is tested and found to provide reliable solutions.

Differentiating between Label and Protein Conformers in Pulsed Dipolar EPR Spectroscopy with the dHis-Cu2+ (NTA) Motif.

Pulsed dipolar EPR spectroscopy (PDS) in combination with site-directed spin labeling is a powerful tool in structural biology. However, the commonly used spin labels are conjugated to biomolecules via rather long and flexible linkers, which hampers the translation of distance distributions into biomolecular conformations. In contrast, the spin label copper(II)-nitrilotriacetic acid [Cu2+ (NTA)] bound to two histidines (dHis) is rigid and yields narrow distance distributions, which can be more easily translated into biomolecular conformations. Here, we use this label on the 71 kDa Yersinia outer protein O (YopO) to decipher whether a previously experimentally observed bimodal distance distribution is due to two conformations of the biomolecule or of the flexible spin labels. Two different PDS experiments, that is, pulsed electron-electron double resonance (PELDOR aka DEER) and relaxation-induced dipolar modulation enhancement (RIDME), yield unimodal distance distribution with the dHis-Cu2+ (NTA) motif; this result suggests that the α-helical backbone of YopO adopts a single conformation in frozen solution. In addition, we show that the Cu2+ (NTA) label preferentially binds to the target double histidine (dHis) sites even in the presence of 22 competing native histidine residues. Our results therefore suggest that the generation of a His-null background is not required for this spin labeling methodology. Together these results highlight the value of the dHis-Cu2+ (NTA) motif in PDS experiments.

A new 13C trityl-based spin label enables the use of DEER for distance measurements

Triarylmethyl (TAM)-based labels, while still underutilized, are a powerful class of labels for pulsed-Electron Spin Resonance (ESR) distance measurements. They feature slow relaxation rates for long-lasting signals, high stability for cellular experiments, and narrow spectral features for efficient excitation of the spins. However, the typical narrow line shape limits the available distance measurements to only single-frequency experiments, such as Double Quantum Coherence (DQC) and Relaxation Induced Dipolar Modulation Enhancement (RIDME), which can be complicated to perform or hard to process. Therefore, widespread usage of TAM labels can be enhanced by the use of Double Electron-Electron Resonance (DEER) distance measurements. In this work, we developed a new spin label, 13C1-mOX063-d24, with a 13C isotope as the radical center. Due to the resolved hyperfine splitting, the spectrum is sufficiently broadened to permit DEER-based experiments at Q-band spectrometers. Additionally, this new label can be incorporated orthogonally with Cu(II)-based protein label. The orthogonal labeling scheme enables DEER distance measurement at X-band frequencies. Overall, the new trityl label allows for DEER-based distance measurements that complement existing TAM-label DQC and RIDME experiments.

A GdIII-Based Spin Label at the Limits for Linewidth Reduction through Zero-Field Splitting Optimization.

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.

Modelling Conformational Flexibility in a Spectrally Addressable Molecular Multi-Qubit Model System.

Dipolar coupled multi-spin systems have the potential to be used as molecular qubits. Herein we report the synthesis of a molecular multi-qubit model system with three individually addressable, weakly interacting, spin centres of differing g-values. We use pulsed Electron Paramagnetic Resonance (EPR) techniques to characterise and separately address the individual electron spin qubits; CuII , Cr7 Ni ring and a nitroxide, to determine the strength of the inter-qubit dipolar interaction. Orientation selective Relaxation-Induced Dipolar Modulation Enhancement (os-RIDME) detecting across the CuII spectrum revealed a strongly correlated CuII -Cr7 Ni ring relationship; detecting on the nitroxide resonance measured both the nitroxide and CuII or nitroxide and Cr7 Ni ring correlations, with switchability of the interaction based on differing relaxation dynamics, indicating a handle for implementing EPR-based quantum information processing (QIP) algorithms.

Pulse Dipolar Electron Paramagnetic Resonance Spectroscopy Reveals Buffer-Modulated Cooperativity of Metal-Templated Protein Dimerization.

Self-assembly of protein monomers directed by metal ion coordination constitutes a promising strategy for designing supramolecular architectures complicated by the noncovalent interaction between monomers. Herein, two pulse dipolar electron paramagnetic resonance spectroscopy (PDS) techniques, pulse electron–electron double resonance and relaxation-induced dipolar modulation enhancement, were simultaneously employed to study the CuII-templated dimerization behavior of a model protein (Streptococcus sp. group G, protein G B1 domain) in both phosphate and Tris-HCl buffers. A cooperative binding model could simultaneously fit all data and demonstrate that the cooperativity of protein dimerization across α-helical double-histidine motifs in the presence of CuII is strongly modulated by the buffer, representing a platform for highly tunable buffer-switchable templated dimerization. Hence, PDS enriches the family of techniques for monitoring binding processes, supporting the development of novel strategies for bioengineering structures and stable architectures assembled by an initial metal-templated dimerization.

A Low-Spin CoII/Nitroxide Complex for Distance Measurements at Q-Band Frequencies

Pulse dipolar electron paramagnetic resonance spectroscopy (PDS) is continuously furthering the understanding of chemical and biological assemblies through distance measurements in the nanometer range. New paramagnets and pulse sequences can provide structural insights not accessible through other techniques. In the pursuit of alternative spin centers for PDS, we synthesized a low-spin CoII complex bearing a nitroxide (NO) moiety, where both the CoII and NO have an electron spin S of 1/2. We measured CoII-NO distances with the well-established double electron–electron resonance (DEER aka PELDOR) experiment, as well as with the five- and six-pulse relaxation-induced dipolar modulation enhancement (RIDME) spectroscopies at Q-band frequencies (34 GHz). We first identified challenges related to the stability of the complex in solution via DEER and X-ray crystallography and showed that even in cases where complex disproportionation is unavoidable, CoII-NO PDS measurements are feasible and give good signal-to-noise (SNR) ratios. Specifically, DEER and five-pulse RIDME exhibited an SNR of ~100, and while the six-pulse RIDME exhibited compromised SNR, it helped us minimize unwanted signals from the RIDME traces. Last, we demonstrated RIDME at a 10 μM sample concentration. Our results demonstrate paramagnetic CoII to be a feasible spin center in medium magnetic fields with opportunities for PDS studies involving CoII ions.

How accurately defined are the overtone coefficients in Gd(III)-Gd(III) RIDME?

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.

Neural networks in pulsed dipolar spectroscopy: A practical guide

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.

Fullerene-based triplet spin labels: methodology aspects for pulsed dipolar EPR spectroscopy.

Triplet states of photoexcited organic molecules are promising spin labels with advanced spectroscopic properties for pulsed dipolar electron paramagnetic resonance (PD EPR) spectroscopy. Recently proposed triplet fullerene labels have shown great potential for double electron-electron resonance (DEER) distance measurements as "observer spins" due to a high quantum yield of the triplet state, hyperpolarization and relatively narrow EPR spectra. Here, we demonstrate the applicability of fullerene labels to other PD EPR techniques, such as relaxation induced dipolar modulation enhancement (RIDME) and laser induced magnetic dipolar spectroscopy (LaserIMD). In particular, a specific contaminating signal in LaserIMD experiments was observed, explained and mitigated. Comparative analyses of the signal-to-noise (SNR) ratios were performed for all employed methods. DEER on the fullerene-triarylmethyl pair shows the best performance, which allows state-of-the-art DEER acquisition at 100 nM with a SNR of ∼35 within reasonable 42 hours.

Diffusion equation for the longitudinal spectral diffusion: the case of the RIDME experiment

Relaxation-induced dipolar modulation enhancement (RIDME) time trace shapes reveal linear scaling with the proton concentration in homogeneous glassy samples. We describe here an approximate diffusion equation-based analysis of such data, which uses only two fit parameters and allows for global data fitting with good accuracy. By construction, the approach should be transferable to other pulse EPR experiments with longitudinal mixing block(s) present. The two fit parameters appear to be sensitive to the type of the glassy matrix and can be thus used for sample characterisation. The estimates suggest that the presented technique should be sensitive to protons at distances up to 3 nm from the electron spin at a 90% matrix deuteration level. We propose that a structural method might be developed based on such an intermolecular hyperfine (ih-)RIDME technique, which would be useful, for instance, in structural biology or dynamic nuclear polarisation experiments.

Six-Pulse RIDME Sequence to Avoid Background Artifacts

Relaxation induced dipolar modulation enhancement (RIDME) is a valuable method for measuring nanometer-scale distances between electron spin centers. Such distances are widely used in structural biology to study biomolecular structures and track their conformational changes. Despite significant improvements of RIDME in recent years, the background analysis of primary RIDME signals remains to be challenging. In particular, it was recently shown that the five-pulse RIDME signals contain an artifact which can hinder the accurate extraction of distance distributions from RIDME time traces [as reported by Ritsch et al. (Phys Chem Chem Phys 21: 9810, 2019)]. Here, this artifact, as well as one additionally identified artifact, are systematically studied on several model compounds and the possible origins of both artifacts are discussed. In addition, a new six-pulse RIDME sequence is proposed that eliminates the artifact with the biggest impact on the extracted distance distributions. The efficiency of this pulse sequence is confirmed on several examples.

DEER and RIDME Measurements of the Nitroxide-Spin Labelled Copper-Bound Amine Oxidase Homodimer from Arthrobacter Globiformis

In the study of biological structures, pulse dipolar spectroscopy (PDS) is used to elucidate spin–spin distances at nanometre-scale by measuring dipole–dipole interactions between paramagnetic centres. The PDS methods of Double Electron Electron Resonance (DEER) and Relaxation Induced Dipolar Modulation Enhancement (RIDME) are employed, and their results compared, for the measurement of the dipolar coupling between nitroxide spin labels and copper-II (Cu(II)) paramagnetic centres within the copper amine oxidase from Arthrobacter globiformis (AGAO). The distance distribution results obtained indicate that two distinct distances can be measured, with the longer of these at c.a. 5 nm. Conditions for optimising the RIDME experiment such that it may outperform DEER for these long distances are discussed. Modelling methods are used to show that the distances obtained after data analysis are consistent with the structure of AGAO.

Pulse Dipolar EPR Reveals Double-Histidine Motif CuII-NTA Spin-Labeling Robustness against Competitor Ions.

Pulse-dipolar EPR is an appealing strategy for structural characterization of complex systems in solution that complements other biophysical techniques. Significantly, the emergence of genetically encoded self-assembling spin labels exploiting exogenously introduced double-histidine motifs in conjunction with CuII-chelates offers high precision distance determination in systems nonpermissive to thiol-directed spin labeling. However, the noncovalency of this interaction exposes potential vulnerabilities to competition from adventitious divalent metal ions, and pH sensitivity. Herein, a combination of room-temperature isothermal titration calorimetry (ITC) and cryogenic relaxation-induced dipolar modulation enhancement (RIDME) measurements are applied to the model protein Streptococcus sp. group G. protein G, B1 domain (GB1). Results demonstrate double-histidine motif spin labeling using CuII-nitrilotriacetic acid (CuII–NTA) is robust against the competitor ligand ZnII–NTA at >1000-fold molar excess, and high nM binding affinity is surprisingly retained under acidic and basic conditions even though room temperature affinity shows a stronger pH dependence. This indicates the strategy is well-suited for diverse biological applications, with the requirement of other metal ion cofactors or slightly acidic pH not necessarily being prohibitive.

A general model to optimise CuII labelling efficiency of double-histidine motifs for pulse dipolar EPR applications.

Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to studies of biomolecules underpinning health and disease by providing highly accurate and precise geometric constraints. Combining double-histidine (dH) motifs with CuII spin labels shows promise for further increasing the precision of distance measurements, and for investigating subtle conformational changes. However, non-covalent coordination-based spin labelling is vulnerable to low binding affinity. Dissociation constants of dH motifs for CuII-nitrilotriacetic acid were previously investigated via relaxation induced dipolar modulation enhancement (RIDME), and demonstrated the feasibility of exploiting the dH motif for EPR applications at sub-μM protein concentrations. Herein, the feasibility of using modulation depth quantitation in CuII-CuII RIDME to simultaneously estimate a pair of non-identical independent KD values in such a tetra-histidine model protein is addressed. Furthermore, we develop a general speciation model to optimise CuII labelling efficiency, depending upon pairs of identical or disparate KD values and total CuII label concentration. We find the dissociation constant estimates are in excellent agreement with previously determined values, and empirical modulation depths support the proposed model.

Reaction: Molecular Spins as Qubits

Floriana Tuna is a Leverhulme Trust Senior Research Fellow at the University of Manchester (UoM). She holds MSc and PhD degrees in chemistry from the University of Bucharest (1989) and National Academy of Romania (1997), respectively. She worked as a visiting DAAD fellow at the University of Heidelberg (Germany), a CNRS postdoctoral fellow at ICMCB Bordeaux (France), and a Marie Curie fellow at the University of Warwick (UK) before joining UoM in 2003. She received the Romanian Academy “Ilie Murgulescu” Prize for excellence in research in 2005 and has published over 300 high-impact papers. Her major interests are in coordination chemistry, molecular magnetism, and quantum information science.

Pulsed EPR Dipolar Spectroscopy on Spin Pairs with one Highly Anisotropic Spin Center: The Low-Spin FeIII Case.

Pulsed electron paramagnetic resonance (EPR) dipolar spectroscopy (PDS) offers several methods for measuring dipolar coupling constants and thus the distance between electron spin centers. Up to now, PDS measurements have been mostly applied to spin centers whose g‐anisotropies are moderate and therefore have a negligible effect on the dipolar coupling constants. In contrast, spin centers with large g‐anisotropy yield dipolar coupling constants that depend on the g‐values. In this case, the usual methods of extracting distances from the raw PDS data cannot be applied. Here, the effect of the g‐anisotropy on PDS data is studied in detail on the example of the low‐spin Fe3+ ion. First, this effect is described theoretically, using the work of Bedilo and Maryasov (Appl. Magn. Reson. 2006, 30, 683–702) as a basis. Then, two known Fe3+/nitroxide compounds and one new Fe3+/trityl compound were synthesized and PDS measurements were carried out on them using a method called relaxation induced dipolar modulation enhancement (RIDME). Based on the theoretical results, a RIDME data analysis procedure was developed, which facilitated the extraction of the inter‐spin distance and the orientation of the inter‐spin vector relative to the Fe3+ g‐tensor frame from the RIDME data. The accuracy of the determined distances and orientations was confirmed by comparison with MD simulations. This method can thus be applied to the highly relevant class of metalloproteins with, for example, low‐spin Fe3+ ions.