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.

Rational Design of Dinitroxide Polarizing Agents for Dynamic Nuclear Polarization to Enhance Overall NMR Sensitivity.

We evaluate the overall sensitivity gains provided by a series of eighteen nitroxide biradicals for dynamic nuclear polarization (DNP) solid-state NMR at 9.4 T and 100 K, including eight new biradicals. We find that in the best performing group the factors contributing to the overall sensitivity gains, namely the DNP enhancement, the build-up time, and the contribution factor, often compete with each other leading to very similar overall sensitivity across a range of biradicals. NaphPol and HydroPol are found to provide the best overall sensitivity factors, in organic and aqueous solvents respectively. One of the new biradicals, AMUPolCbm, provides high sensitivity for all three solvent formulations measured here, and can be considered to be a "universal" polarizing agent.

Ensemble structure of the N-terminal domain (1-267) of FUS in a biomolecular condensate.

Solutions of some proteins phase separate into a condensed state of high protein concentration and a dispersed state of low concentration. Such behavior is observed in living cells for a number of RNA-binding proteins that feature intrinsically disordered domains. It is relevant for cell function via the formation of membraneless organelles and transcriptional condensates. On a basic level, the process can be studied in vitro on protein domains that are necessary and sufficient for liquid-liquid phase separation (LLPS). We have performed distance distribution measurements by electron paramagnetic resonance for 13 sections in an N-terminal domain (NTD) construct of the protein fused in sarcoma (FUS), consisting of the QGSY-rich domain and the RGG1 domain, in the denatured, dispersed, and condensed state. Using 10 distance distribution restraints for ensemble modeling and three such restraints for model validation, we have found that FUS NTD behaves as a random-coil polymer under good-solvent conditions in both the dispersed and condensed state. Conformation distribution in the biomolecular condensate is virtually indistinguishable from the one in an unrestrained ensemble, with the latter one being based on only residue-specific Ramachandran angle distributions. Over its whole length, FUS NTD is slightly more compact in the condensed than in the dispersed state, which is in line with the theory for random coils in good solvent proposed by de Gennes, Daoud, and Jannink. The estimated concentration in the condensate exceeds the overlap concentration resulting from this theory. The QGSY-rich domain is slightly more extended, slightly more hydrated, and has slightly higher propensity for LLPS than the RGG1 domain. Our results support previous suggestions that LLPS of FUS is driven by multiple transient nonspecific hydrogen bonding and π-sp2 interactions.

Integrative solution structure of PTBP1-IRES complex reveals strong compaction and ordering with residual conformational flexibility.

RNA-binding proteins (RBPs) are crucial regulators of gene expression, often composed of defined domains interspersed with flexible, intrinsically disordered regions. Determining the structure of ribonucleoprotein (RNP) complexes involving such RBPs necessitates integrative structural modeling due to their lack of a single stable state. In this study, we integrate magnetic resonance, mass spectrometry, and small-angle scattering data to determine the solution structure of the polypyrimidine-tract binding protein 1 (PTBP1/hnRNP I) bound to an RNA fragment from the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). This binding, essential for enhancing the translation of viral RNA, leads to a complex structure that demonstrates RNA and protein compaction, while maintaining pronounced conformational flexibility. Acting as an RNA chaperone, PTBP1 orchestrates the IRES RNA into a few distinct conformations, exposing the RNA stems outward. This conformational diversity is likely common among RNP structures and functionally important. Our approach enables atomic-level characterization of heterogeneous RNP structures.

Pulse EPR spectroscopy and molecular modeling reveal the origins of the local heterogeneity of dietary fibers.

Optimizing human diet by including dietary fibers would be more efficient when the fibers' chain interactions with other molecules are understood in depth. Thereby, it is important to develop methods for characterizing the fiber chain to be able to monitor its structural alterations upon intermolecular interactions. Here, we demonstrate the utility of the electron paramagnetic resonance (EPR) spectroscopy, complemented by simulations in probing the atomistic details of the chain conformations for spin-labeled fibers. Barley ß-glucan, a native polysaccharide with linear chain, was utilized as a test fiber system to demonstrate the technique's capabilities. Pulse dipolar EPR data show good agreement with results of the fiber chain modeling, revealing sinuous chain conformations and providing polymer shape descriptors: the gyration tensor, spin-spin distance distribution function, and information about proton density near the spin probe. Results from EPR measurements point to the fiber aggregation in aqueous solution, which agrees with the results of the dynamic light scattering. We propose that the combination of pulse EPR measurements with modeling can be a perfect experimental tool for in-depth structural investigation of dietary fibers and their interaction under such conditions, and that the presented methodology can be extended to other weakly ordered or disordered macromolecules.

Deuterated TEKPol Biradicals and the Spin-Diffusion Barrier in MAS DNP.

The sensitivity of NMR spectroscopy is considerably enhanced by dynamic nuclear polarization (DNP). In DNP polarization is transferred from unpaired electrons of a polarizing agent to nearby proton spins. In solids, this transfer is followed by the transport of hyperpolarization to the bulk via 1 H-1 H spin diffusion. The efficiency of these steps is critical to obtain high sensitivity gains, but the pathways for polarization transfer in the region near the unpaired electron spins are unclear. Here we report a series of seven deuterated and one fluorinated TEKPol biradicals to probe the effect of deprotonation on MAS DNP at 9.4 T. The experimental results are interpreted with numerical simulations, and our findings support that strong hyperfine couplings to nearby protons determine high transfer rates across the spin diffusion barrier to achieve short build-up times and high enhancements. Specifically, 1 H DNP build-up times increase substantially with TEKPol isotopologues that have fewer hydrogen atoms in the phenyl rings, suggesting that these protons play a crucial role transferring the polarization to the bulk. Based on this new understanding, we have designed a new biradical, NaphPol, which yields significantly increased NMR sensitivity, making it the best performing DNP polarizing agent in organic solvents to date.

Ferric Pyrophosphate Forms Soluble Iron Coordination Complexes with Zinc Compounds and Solubilizing Agents in Extruded Rice and Predicts Increased Iron Solubility and Bioavailability in Young Women.

BACKGROUND: Co-extrusion of ferric pyrophosphate (FePP) with solubilizers, citric acid/trisodium citrate (CA/TSC), or ethylenediaminetetraacetic acid (EDTA) sharply increases iron absorption. Whether this can protect against the inhibition of iron absorption by phytic acid (PA) is unclear. Sodium pyrophosphate (NaPP) may be a new enhancer of iron absorption from FePP. OBJECTIVES: Our objectives were to 1) investigate the ligand coordination of iron, zinc, and solubilizers in extruded rice and test associations with iron solubility and absorption, 2) assess whether co-extrusion of FePP + CA/TSC rice can protect against inhibition of iron absorption by PA; 3) determine the effect of zinc oxide (ZnO) compared with zinc sulfate (ZnSO4), and 4) quantify iron absorption from FePP + NaPP rice. METHODS: We produced labeled 57FePP rice cofortified with ZnSO4 and EDTA, CA/TSC or NaPP, and FePP + EDTA rice with ZnO. We used electron paramagnetic resonance (EPR) to characterize iron-ligand complexes. We measured in vitro iron solubility and fractional iron absorption (FIA) in young women (n = 21, age: 22 ± 2 y, BMI: 21.3 ± 1.5 kg/m2 geometric mean plasma ferritin, 28.5 µg/L) compared with ferrous sulfate (58FeSO4). FIA was compared by linear mixed-effect model analysis. RESULTS: The addition of zinc and solubilizers created new iron coordination complexes of Fe(III) species with a weak ligand field at a high-spin state that correlated with solubility (r2 = 0.50, P = 0.02) and absorption (r2 = 0.72, P = 0.02). Phytic acid reduced FIA from FePP + CA/TSC rice by 50% (P < 0.001), to the same extent as FeSO4. FIA from FePP + EDTA + ZnO and FePP + EDTA + ZnSO4 rice did not significantly differ. Mean FIAs from FePP + EDTA + ZnSO4, FePP + CA/TSC + ZnSO4, and FePP + NaPP + ZnSO4 rice were 9% to 11% and did not significantly differ from each other or from FeSO4. CONCLUSION: Rice extrusion of FePP with solubilizers resulted in bioavailable iron coordination complexes. In the case of FePP + CA/TSC, PA exerted similar inhibition of FIA as with FeSO4. FePP + NaPP could be a further viable solubilizing agent for rice fortification. This study was registered at clinicaltrials.gov as NCT03703739.

Interaction of barley ß-glucan with food dye molecules - An insight from pulse dipolar EPR spectroscopy.

The interactions between dietary fibers (DFs) and small molecules are of great interest to food chemistry and nutrition science. However, the corresponding interaction mechanisms and structural rearrangements of DFs at the molecular level are still opaque due to the usually weak binding and the lack of appropriate techniques to determine details of conformational distributions in such weakly organized systems. By combining our previously established methodology on stochastic spin-labelling of DFs with the appropriately revised set of pulse electron paramagnetic resonance techniques, we present here a toolkit to determine the interactions between DFs and small molecules, using barley ß-glucan as an example for neutral DF and a selection of food dye molecules as examples for small molecules. The proposed here methodology allowed us to observe subtle conformational changes of ß-glucan by detecting multiple details of the local environment of the spin labels. Substantial variations of binding propensities were detected for different food dyes.

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.

Intermolecular contributions, filtration effects and signal composition of SIFTER (single-frequency technique for refocusing).

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.

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.

Site-selective and stochastic spin labelling of neutral water-soluble dietary fibers optimized for electron paramagnetic resonance spectroscopy.

Use of spin labels to study structures of polymers has been widely spread in polymer science. However, for the studies of neutral water-soluble dietary fibers (DFs), labelling efficiencies in past studies have only been sufficient for application of continuous wave electron paramagnetic resonance spectroscopy (CW-EPR), but still insufficient for some advanced methods such as pulse EPR. Thus, in this paper, two spin labelling strategies, namely, site-selective mono-spin-labelling and stochastic multi-spin-labelling, were examined on linear cereal ß-glucan, as well as linearly branched arabinoxylan and galactomannan. The effects of both methods in DF properties were evaluated. For the mono-labelling pathway, labelling efficiency could reach up to 46 %. In the stochastic labelling strategy, a degree of substitution (DS) up to 150 % could be reached, whereas optimized conditions for this strategy were achieved at DS = 3 % to obtain DFs whose bioactivity properties were still preserved while spin labelling efficiency was still sufficient for CW and pulse EPR experiments.

The Cu(ii) - dietary fibre interactions at molecular level unveiled via EPR spectroscopy.

While dietary fibres have a reputation of a healthy food component, the interaction between nutrients and neutral fibers is non-covalent, and its characterization is challenging for most analytical techniques. Here, on the example of barley ß-glucan (BBG) and paramagnetic Cu(ii) ions we demonstrate the performance of different Electron Paramagnetic Resonance (EPR) methods in the fibre studies. EPR techniques were tested on two spin probe systems with different affinity in the interaction with dietary fibres - Cu(OAc)2 salt, which weakly dissociates under physiological conditions and CuSO4 salt, which easily dissociates, so that in the latter case Cu(ii) can be considered as a 'free' ion, only chelated by water molecules. The Cu(ii)-BBG interaction was determined by pulse EPR relaxation measurements, but this interaction appears not strong enough for continuous wave EPR detection. The capability of the fibres for Cu(ii) absorption was successfully analyzed by comparison of the results from the pulse dipolar spectroscopy with numerical simulations. The local distribution of sugar hydrogen atoms around the Cu(ii) ion has been determined by electron spin echo envelope modulation (ESEEM) and electron-nuclei double resonance (ENDOR) techniques.

Design Principles for the Development of Gd(III) Polarizing Agents for Magic Angle Spinning Dynamic Nuclear Polarization.

Nuclear magnetic resonance suffers from an intrinsically low sensitivity, which can be overcome by dynamic nuclear polarization (DNP). Gd(III) complexes are attractive exogenous polarizing agents for magic angle spinning (MAS) DNP due to their high chemical stability in contrast to nitroxide-based radicals. However, even the state-of-the-art Gd(III) complexes have so far provided relatively low DNP signal enhancements of ca. 36 in comparison to standard DNP biradicals, which show enhancements of over 200. Here, we report a series of new Gd(III) complexes for DNP and show that the observed DNP enhancements of the new and existing Gd(III) complexes are inversely proportional to the square of the zero-field splitting (ZFS) parameter D, which is in turn determined by the ligand-type and the local coordination environment. The experimental DNP enhancements at 9.4 T and the ZFS parameters measured with pulsed electron paramagnetic resonance (EPR) spectroscopy agree with the above model, paving the way for the development of more efficient Gd(III) polarizing agents.

Phase Separation of Heterogeneous Nuclear Ribonucleoprotein A1 upon Specific RNA-Binding Observed by Magnetic Resonance.

Interaction of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) with specific single-stranded RNA and its relation to liquid-liquid phase separation (LLPS) were studied in vitro by magnetic resonance based on site-directed spin labelling. An ensemble model of dispersed hnRNP A1 in the absence of RNA was derived from distance distributions between spin labelled sites and small angle X-ray scattering. This model revealed a compact state of the low-complexity domain and its interaction with the RNA recognition motifs. Paramagnetic relaxation enhancement NMR spectroscopy confirmed this interaction. Addition of RNA to dispersed hnRNP A1 induced liquid-droplet formation. Such LLPS depended on RNA concentration and sequence, with continuous wave EPR spectroscopy showing an influence of RNA point mutations on local protein dynamics. We propose that an interplay of sequence-specific RNA binding and LLPS contributes to regulation of specific RNA segregation during stress response.

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.

Efficient Dynamic Nuclear Polarization up to 230 K with Hybrid BDPA-Nitroxide Radicals at a High Magnetic Field.

Pairing the spectral resolution provided by high magnetic fields at ambient temperature with the enhanced sensitivity offered by dynamic nuclear polarization (DNP) is a major goal of modern solid-state NMR spectroscopy, which will allow one to unlock ever-challenging applications. This study demonstrates that, by combining HyTEK2, a hybrid BDPA-nitroxide biradical polarizing agent, with ortho-terphenyl (OTP), a rigid DNP matrix, enhancement factors as high as 65 can be obtained at 230 K, 40 kHz magic angle spinning (MAS), and 18.8 T. The temperature dependence of the DNP enhancement and its behavior around the glass transition temperature (Tg) of the matrix is investigated by variable-temperature EPR measurements of the electron relaxation properties and numerical simulations. A correlation is suggested between the decrease in enhancement at the passage of the Tg and the concomitant drop of both transverse electron relaxation times in the biradical.

Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR.

Labeling of biomolecules with a paramagnetic probe for nuclear magnetic resonance (NMR) spectroscopy enables determining long-range distance restraints, which are otherwise not accessible by classically used dipolar coupling-based NMR approaches. Distance restraints derived from paramagnetic relaxation enhancements (PREs) can facilitate the structure determination of large proteins and protein complexes. We herein present the site-directed labeling of the large oligomeric bacterial DnaB helicase from Helicobacter pylori with cysteine-reactive maleimide tags carrying either a nitroxide radical or a lanthanide ion. The success of the labeling reaction was followed by quantitative continuous-wave electron paramagnetic resonance (EPR) experiments performed on the nitroxide-labeled protein. PREs were extracted site-specifically from 2D and 3D solid-state NMR spectra. A good agreement with predicted PRE values, derived by computational modeling of nitroxide and Gd3+ tags in the low-resolution DnaB crystal structure, was found. Comparison of experimental PREs and model-predicted spin label-nucleus distances indicated that the size of the "blind sphere" around the paramagnetic center, in which NMR resonances are not detected, is slightly larger for Gd3+ (∼14 Å) than for nitroxide (∼11 Å) in 13C-detected 2D spectra of DnaB. We also present Gd3+-Gd3+ dipolar electron-electron resonance EPR experiments on DnaB supporting the conclusion that DnaB was present as a hexameric assembly.

Resolving distance variations by single-molecule FRET and EPR spectroscopy using rotamer libraries.

Förster resonance energy transfer (FRET) and electron paramagnetic resonance (EPR) spectroscopy are complementary techniques for quantifying distances in the nanometer range. Both approaches are commonly employed for probing the conformations and conformational changes of biological macromolecules based on site-directed fluorescent or paramagnetic labeling. FRET can be applied in solution at ambient temperature and thus provides direct access to dynamics, especially if used at the single-molecule level, whereas EPR requires immobilization or work at cryogenic temperatures but provides data that can be more reliably used to extract distance distributions. However, a combined analysis of the complementary data from the two techniques has been complicated by the lack of a common modeling framework. Here, we demonstrate a systematic analysis approach based on rotamer libraries for both FRET and EPR labels to predict distance distributions between two labels from a structural model. Dynamics of the fluorophores within these distance distributions are taken into account by diffusional averaging, which improves the agreement with experiment. Benchmarking this methodology with a series of surface-exposed pairs of sites in a structured protein domain reveals that the lowest resolved distance differences can be as small as ∼0.25 nm for both techniques, with quantitative agreement between experimental and simulated transfer efficiencies within a range of ±0.045. Rotamer library analysis thus establishes a coherent way of treating experimental data from EPR and FRET and provides a basis for integrative structural modeling, including studies of conformational distributions and dynamics of biological macromolecules using both techniques.