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Dipolar electron paramagnetic resonance (EPR) experiments, such as double electron-electron resonance (DEER), measure distributions of nanometer-scale distances between unpaired electrons, which provide valuable information for structural characterization of proteins and other macromolecular systems. We present an extension to our previously published general model based on dipolar pathways valid for multi-dimensional dipolar EPR experiments with more than two spin-1/2 labels. We examine the 4-pulse DEER and TRIER experiments in terms of dipolar pathways and show experimental results confirming the theoretical predictions. This extension to the dipolar pathways model allows the analysis of previously challenging datasets and the extraction of multivariate distance distributions.
Asunto(s)
Proteínas , Espectroscopía de Resonancia por Spin del Electrón/métodos , Sustancias Macromoleculares , Proteínas/química , Marcadores de SpinRESUMEN
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.
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Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Sitios de Unión , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/química , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Espectroscopía de Resonancia Magnética , ARN/metabolismoRESUMEN
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 â¼0.25 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.
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Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , Difusión , Espectroscopía de Resonancia por Spin del Electrón , Conformación MolecularRESUMEN
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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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
We describe a broadband light scattering setup for the characterization of size and refractive index of single submicron-to-micron sized aerosol particles. Individual particles are isolated in air by a quadruple Bessel beam optical trap or a counter-propagating optical tweezer. The use of very broadband radiation in the wavelength range from 320 to 700 nm covering the ultraviolet region allows to size submicron particles. We show that a broad wavelength range is required to determine the particle radius and the refractive index with an uncertainty of several nanometers and â¼ 0.01, respectively. The smallest particle radius that can be accurately determined lies around 300 nm. Wavelength-dependent refractive index data over a broad range are obtained, including the ultraviolet region where corresponding data are rare. Four different applications are discussed: (1) the sizing of submicron polystyrene latex spheres, (2) the evaporation of binary glycerol water droplets, (3) hydration/dehydration cycling of aqueous potassium carbonate droplets, and (4) photochemical reactions of oleic acid droplets.
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Function of intrinsically disordered proteins may depend on deviation of their conformational ensemble from that of a random coil. Such deviation may be hard to characterize and quantify, if it is weak. We explored the potential of distance distributions between spin labels, as they can be measured by electron paramagnetic resonance techniques, for aiding such characterization. On the example of the intrinsically disordered N-terminal domain 1-267 of fused in sarcoma (FUS) we examined what such distance distributions can and cannot reveal on the random-coil reference state. On the example of the glycine-rich domain 188-320 of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) we studied whether deviation from a random-coil ensemble can be robustly detected with 19 distance distribution restraints. We discuss limitations imposed by ill-posedness of the conversion of primary data to distance distributions and propose overlap of distance distributions as a fit criterion that can tackle this problem. For testing consistency and size sufficiency of the restraint set, we propose jack-knife resampling. At current desktop computers, our approach is expected to be viable for domains up to 150 residues and for between 10 and 50 distance distribution restraints.
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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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The availability of bioresistant spin labels is crucial for the optimization of site-directed spin labeling protocols for EPR structural studies of biomolecules in a cellular context. As labeling can affect proteins' fold and/or function, having the possibility to choose between different spin labels will increase the probability to produce spin-labeled functional proteins. Here, we report the synthesis and characterization of iodoacetamide- and maleimide-functionalized spin labels based on the gem-diethyl pyrroline structure. The two nitroxide labels are compared to conventional gem-dimethyl analogs by site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, using two water soluble proteins: T4 lysozyme and Bid. To foster their use for structural studies, we also present rotamer libraries for these labels, compatible with the MMM software. Finally, we investigate the "true" biocompatibility of the gem-diethyl probes comparing the resistance towards chemical reduction of the NO group in ascorbate solutions and E. coli cytosol at different spin concentrations.
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Invited for this month's cover picture is the group of Professor Enrica Bordignon at the Ruhr University Bochum. The cover picture shows an artistic view of E. coli cells and two spin-labeled recombinantly produced proteins, which can be inserted into the cells for EPR studies. The primary sequence of the proteins is schematically shown with the one-letter amino acid code, and cysteine residues are functionalized with the two new gem diethyl nitroxide spin labels designed to better sustain the reducing cellular environment. Read the full text of their Full Paper at 10.1002/open.201900119.