Double Resonance Spectroscopy Research Articles

High‐Frequency Electron Paramagnetic Resonance and Electron‐Nuclear Double Resonance Spectroscopy Study of the Ga Vacancy in β‐Ga2O3

The Ga vacancy (VGa) defect in β‐Ga2O3, generated by proton irradiation, is studied using high‐frequency electron paramagnetic resonance (EPR) and electron‐nuclear double resonance spectroscopy. The previous X‐band EPR studies of this defect, attributed to VGa2−, are extended to higher frequencies (240 GHz) and lower temperatures (T = 6 K). The spin Hamiltonian parameters of the VGa2− center are determined: electron spin S = 1/2, g‐tensor: gb = 2.0313, gc = 2.0079, and ga = 2.0025; the hyperfine interaction parameters with 2 equivalent Ga neighbors: Ab = 14.0 G, Ac = 14.6 G, and Aa* = 12.8 G for 69Ga; the superhyperfine interaction with distant Ga neighbors ASHF(69Ga) = 11 MHz; and the quadrupole interaction Qb(69Ga) = 0.32 MHz and Qb(71Ga) = 0.22 MHz. These results shall allow to refine the assignment of this center to a split vacancy or an unrelaxed VGa2− defect.

Bayesian Probabilistic Inference of Nonparametric Distance Distributions in DEER Spectroscopy.

Double electron-electron resonance (DEER) spectroscopy measures distance distributions between spin labels in proteins, yielding important structural and energetic information about conformational landscapes. Analysis of an experimental DEER signal in terms of a distance distribution is a nontrivial task due to the ill-posed nature of the underlying mathematical inversion problem. This work introduces a Bayesian probabilistic inference approach to analyze DEER data, assuming a nonparametric distance distribution with a Tikhonov smoothness prior. The method uses Markov Chain Monte Carlo sampling with a compositional Gibbs sampler to determine a posterior probability distribution over the entire parameter space, including the distance distribution, given an experimental data set. This posterior contains all of the information available from the data, including a full quantification of the uncertainty about the model parameters. The corresponding uncertainty about the distance distribution is visually captured via an ensemble of posterior predictive distributions. Several examples are presented to illustrate the method. Compared with bootstrapping, it performs faster and provides slightly larger uncertainty intervals.

Measurement and assignment of J = 5 to 9 rotational energy levels in the 9070-9370cm-1 range of methane using optical frequency comb double-resonance spectroscopy.

We use optical-optical double-resonance spectroscopy with a continuous wave (CW) pump and a cavity-enhanced frequency comb probe to measure the energy levels of methane in the upper part of the triacontad polyad (P6) with higher rotational quantum numbers than previously assigned. A high-power CW optical parametric oscillator, tunable around 3000 cm-1, is consecutively locked to the P(7, A2), Q(7, A2), R(7, A2), and Q(6, F2) transitions in the ν3 band, and a comb covering the 5800-6100cm-1 range probes sub-Doppler ladder-type transitions from the pumped levels with J' = 6 to 8, respectively. We report 118 probe transitions in the 3ν3 ← ν3 spectral range with uncertainties down to 300kHz (1 × 10-5cm-1), reaching 84 unique final states in the 9070-9370cm-1 range with rotational quantum numbers J between 5 and 9. We assign these states using combination differences and by comparison with theoretical predictions from a new abinitio-based effective Hamiltonian and dipole moment operator. This is the first line-by-line experimental verification of theoretical predictions for these hot-band transitions, and we find a better agreement of transition wavenumbers with the new calculations compared to the TheoReTS/HITEMP and ExoMol databases. We also compare the relative intensities and find an overall good agreement with all three sets of predictions. Finally, we report the wavenumbers of 27 transitions in the 2ν3 spectral range, observed as V-type transitions from the ground state, and compare them to the new Hamiltonian, HITRAN2020, ExoMol, and the WKMLC line lists.

In-Cell DEER Spectroscopy of Nanodisc-Delivered Membrane Proteins in Living Cell Membranes.

Membrane proteins are integral to numerous cellular processes, yet their conformational dynamics in native environments remains difficult to study. This study introduces a nanodelivery method using nanodiscs to transport spin-labeled membrane proteins into the membranes of living cells, enabling direct in-cell double electron-electron resonance (DEER) spectroscopy measurements. We investigated the membrane protein BsYetJ, incorporating spin labels at key positions to monitor conformational changes. Our findings demonstrate successful delivery and high-quality DEER data for BsYetJ in both Gram-negative E. coli and Gram-positive B. subtilis membranes. The delivered BsYetJ retains its ability to transport calcium ions. DEER analysis reveals distinct conformational states of BsYetJ in different membrane environments, highlighting the influence of lipid composition on the protein structure. This nanodelivery method overcomes traditional limitations, enabling the study of membrane proteins in more physiologically relevant conditions.

Alternating access of a bacterial homolog of neurotransmitter: sodium symporters determined from AlphaFold2 ensembles and DEER spectroscopy

Neurotransmitter:sodium symporters (NSSs) play critical roles in neural signaling by regulating neurotransmitter uptake into cells powered by sodium electrochemical gradients. Bacterial NSSs orthologs, including MhsT from Bacillus halodurans, have emerged as model systems to understand the structural motifs of alternating access in NSSs and the extent of conservation of these motifs across the family. Here, we apply a computational/experimental methodology to illuminate the conformational landscape of MhsT alternating access. Capitalizing on our recently developed method, Sampling Protein Ensembles and Conformational Heterogeneity with AlphaFold2 (SPEACH_AF), we derived clusters of MhsT models spanning the transition from inward-facing to outward-facing conformations. Systematic application of double electron-electron resonance (DEER) spectroscopy revealed ligand-dependent movements of multiple structural motifs that underpin MhsT's conformational cycle. Remarkably, comparative DEER analysis in detergent micelles and lipid nanodiscs highlights the profound effect of the environment on the energetics of conformational changes. Through experimentally derived selection of collective variables, we present a model of ion and substrate-powered transport by MhsT consistent with the conformational cycle derived from DEER. Our findings not only advance the understanding of MhsT's function but also uncover motifs of conformational dynamics conserved within the broader context of the NSS family and within the LeuT-fold class of transporters. Importantly, our methodological blueprint introduces an approach that can be applied across a diverse spectrum of transporters to describe their conformational landscapes.

Role of ammonia-lyases in the synthesis of the dithiomethylamine ligand during [FeFe]-hydrogenase maturation

The generation of an active [FeFe]-hydrogenase requires the synthesis of a complex metal center, the H-cluster, by three dedicated maturases: the radical S-adenosyl-l-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. A key step of [FeFe]-hydrogenase maturation is the synthesis of the dithiomethylamine (DTMA) bridging ligand, a process recently shown to involve the aminomethyl-lipoyl-H-protein from the glycine cleavage system, whose methylamine group originates from serine and ammonium. Here we use functional assays together with electron paramagnetic resonance and electron-nuclear double resonance spectroscopies to show that serine or aspartate together with their respective ammonia-lyase enzymes can provide the nitrogen for DTMA biosynthesis during in vitro [FeFe]-hydrogenase maturation. We also report bioinformatic analysis of the hyd operon, revealing a strong association with genes encoding ammonia-lyases, suggesting important biochemical and metabolic connections. Together, our results provide evidence that ammonia-lyases play an important role in [FeFe]-hydrogenase maturation by delivering the ammonium required for dithiomethylamine ligand synthesis.

Charge and Spin Transfer Dynamics in a Weakly Coupled Porphyrin Dimer.

The dynamics of electron and spin transfer in the radical cation and photogenerated triplet states of a tetramethylbiphenyl-linked zinc-porphyrin dimer were investigated, so as to test the relevant parameters for the design of a single-molecule spin valve and the creation of a novel platform for the photogeneration of high-multiplicity spin states. We used a combination of multiple techniques, including variable-temperature continuous wave EPR, pulsed proton electron-nuclear double resonance (ENDOR), transient EPR, and optical spectroscopy. The conclusions are further supported by density functional theory (DFT) calculations and comparison to reference compounds. The low-temperature cw-EPR and room-temperature near-IR spectra of the dimer monocation demonstrate that the radical cation is spatially localized on one side of the dimer at any point in time, not coherently delocalized over both porphyrin units. The EPR spectra at 298 K reveal rapid hopping of the radical spin density between both sites of the dimer via reversible intramolecular electron transfer. The hyperfine interactions are modulated by electron transfer and can be quantified using ENDOR spectroscopy. This allowed simulation of the variable-temperature cw-EPR spectra with a two-site exchange model and provided information on the temperature-dependence of the electron transfer rate. The electron transfer rates range from about 10.0 MHz at 200 K to about 53.9 MHz at 298 K. The activation enthalpies Δ‡H of the electron transfer were determined as Δ‡H = 9.55 kJ mol-1 and Δ‡H = 5.67 kJ mol-1 in a 1:1:1 solvent mixture of CD2Cl2/toluene-d8/THF-d8 and in 2-methyltetrahydrofuran, respectively, consistent with a Robin-Day class II mixed valence compound. These results indicate that the interporphyrin electronic coupling in a tetramethylbiphenyl-linked porphyrin dimer is suitable for the backbone of a single-molecule spin valve. Investigation of the spin density distribution of the photogenerated triplet state of the Zn-porphyrin dimer reveals localization of the triplet spin density on a nanosecond time scale on one-half of the dimer at 20 K in 2-methyltetrahydrofuran and at 250 K in a polyvinylcarbazole film. This establishes the porphyrin dimer as a molecular platform for the formation of a localized, photogenerated triplet state on one porphyrin unit that is coupled to a second redox-active, ground-state porphyrin unit, which can be explored for the formation of high-multiplicity spin states.

Pulsed Dipolar EPR for Self-Limited Complexes of Oligonucleotides Studies.

Pulsed electron-electron double resonance (PELDOR) spectroscopy is a powerful method for determining nucleic acid (NA) structure and conformational dynamics. PELDOR with molecular dynamics (MD) simulations opens up unique possibilities for defining the conformational ensembles of flexible, three-dimensional, self-assembled complexes of NA. Understanding the diversity and structure of these complexes is vital for uncovering matrix and regulative biological processes in the human body and artificially influencing them for therapeutic purposes. To explore the reliability of PELDOR and MD simulations, we site-specifically attached nitroxide spin labels to oligonucleotides, which form self-assembled complexes between NA chains and exhibit significant conformational flexibility. The DNA complexes assembled from a pair of oligonucleotides with different linker sizes showed excellent agreement between the distance distributions obtained from PELDOR and calculated from MD simulations, both for the mean inter-spin distance and the distance distribution width. These results prove that PELDOR with MD simulations has significant potential for studying the structure and dynamics of conformational flexible complexes of NA.

Partial wrapping of single-stranded DNA by replication protein A and modulation through phosphorylation.

Single-stranded DNA (ssDNA) intermediates which emerge during DNA metabolic processes are shielded by replication protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper to direct the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent functional specificity requires knowledge of the structural conformation of ssDNA when RPA-bound. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and double electron-electron resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.

Conformations of influenza A M2 protein in DOPC/DOPS and E. coli native lipids and proteins

We compared the conformations of the transmembrane domain (TMD) of influenza A M2 (IM2) protein reconstituted in 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPC/DOPS) bilayers to those in isolated Escherichia coli (E. coli) membranes, having preserved its native proteins and lipids. IM2 is a single-pass transmembrane protein known to assemble into a homo-tetrameric proton channel. To represent this channel, we made a construct containing the IM2’s TMD region flanked by the juxtamembrane residues. The single cysteine substitution, L43C, of leucine located in the bilayer polar region was paramagnetically tagged with a methanethiosulfonate nitroxide label for the electron spin resonance (ESR) study. For this particular residue, we probed the conformations of the spin-labeled IM2 reconstituted in DOPC/DOPS and isolated E. coli membranes using continuous-wave ESR and double electron-electron resonance (DEER) spectroscopy. The total protein-to-lipid molar ratio spanned the range from 1:230 to 1:10,400. The continuous-wave ESR spectra corresponded to very slow spin-label motion in both environments. In all cases, the DEER data were reconstructed into distance distributions with well-resolved peaks at 1.68 and 2.37 nm in distance and amplitude ratios of 1.41 ± 0.2 and 2:1, respectively. This suggests four nitroxide spin labels located at the corners of a square, indicative of an axially symmetric tetramer. The distance modeling of DEER data with molecular modeling software applied to the NMR molecular structures (PDB: 2L0J) confirmed the symmetry and closed state of the C-terminal exit pore of the IM2 TMD tetramer in agreement with the model. Thus, we can conclude that, under conditions of pH 7.4 used in this study, IM2 TMD has similar conformations in model lipid bilayers and membranes made of native E. coli lipids and proteins of comparable thickness and fluidity, notwithstanding the complexity of the E. coli membranes caused by their lipid diversity and the abundance of integral and peripheral membrane proteins.

Hyperfine interactions of paramagnetic radiation-induced defect centres in advanced ceramic breeder pebbles

Advanced ceramic breeder (ACB) pebbles consisting of 65 mol% lithium orthosilicate (Li4SiO4) and 35 mol% lithium metatitanate (Li2TiO3) are developed for tritium breeding in the European Union’s (EU) helium cooled pebble bed (HCPB) breeder blanket concept of the demonstration fusion power plant (DEMO). Electron-nuclear double resonance (ENDOR) spectroscopy is a powerful and widely used magnetic resonance technique that combines aspects of electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR). In the present work, ENDOR spectroscopy was applied to investigate hyperfine (HF) interactions of paramagnetic radiation-induced defect centres (containing unpaired electrons) in the biphasic ACB pebbles after irradiation with 10 MeV accelerated electrons. To separate individual EPR signals of the formed and accumulated paramagnetic centres, isochronal annealing of the irradiated ACB pebbles followed by EPR spectra measurements at X- and Q-microwave frequency bands at room and low temperature were performed. Afterwards, X-band ENDOR spectra at low temperature were recorded for selected EPR signals to reveal HF interactions between the unpaired electron spins with the spins of magnetic 7Li and 1H isotope nuclei. The g-factor and HF coupling values determined from EPR and ENDOR spectra simulations provide novel insights into the local structure of paramagnetic hole- and electron-type centres in the irradiated ACB pebbles.

Impact of N-Glycosylation on Protein Structure and Dynamics Linked to Enzymatic C-H Activation in the M. oryzae Lipoxygenase.

Lipoxygenases (LOXs) from pathogenic fungi are potential therapeutic targets for defense against plant and select human diseases. In contrast to the canonical LOXs in plants and animals, fungal LOXs are unique in having appended N-linked glycans. Such important post-translational modifications (PTMs) endow proteins with altered structure, stability, and/or function. In this study, we present the structural and functional outcomes of removing or altering these surface carbohydrates on the LOX from the devastating rice blast fungus, M. oryzae, MoLOX. Alteration of the PTMs did notinfluence the active site enzyme-substrate ground state structures as visualized by electron-nuclear double resonance (ENDOR) spectroscopy. However, removal of the eight N-linked glycans by asparagine-to-glutamine mutagenesis nonetheless led to a change in substrate selectivity and an elevated activation energy for the reaction with substrate linoleic acid, as determined by kinetic measurements. Comparative hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of wild-type and Asn-to-Gln MoLOX variants revealed a regionally defined impact on the dynamics of the arched helix that covers the active site. Guided by these HDX results, a single glycan sequon knockout was generated at position 72, and its comparative substrate selectivity from kinetics nearly matched that of the Asn-to-Gln variant. The cumulative data from model glyco-enzyme MoLOX showcase how the presence, alteration, or removal of even a single N-linked glycan can influence the structural integrity and dynamics of the protein that are linked to an enzyme's catalytic proficiency, while indicating that extensive glycosylation protects the enzyme during pathogenesis by protecting it from protease degradation.

Selective Tautomer Production and Cryogenic Ion Spectroscopy of Radical Cations: The Uracil and Thymine Cases.

The vibrational and electronic spectroscopy of the radical cations of two nucleobases (NB) (uracil and thymine) was studied by cryogenic ion photodissociation spectroscopy. The radical cations have been generated from the photodissociation of NB-Ag+ complexes. A charge transfer process from the NB to Ag+ governs the deactivation mechanism, leading to the formation of the radical cation without further tautomerization. Single- and double-resonance spectroscopy allows for structural assignments of both the silver complexes and the radical cations by comparison with DFT-based calculations. Interestingly, a tautomer-dependent fragmentation is observed in the thymine enol form that involves the loss of NCO, a fragment which was never reported before for this NB. This selective photodissociation of silver complexes containing aromatic chromophore greatly expands the current technique to produce isomer-selected radical cations in the gas phase providing benchmark experimental data to assess calculations of open-shell species.

Self-assembly of spin-labeled antimicrobial peptides magainin 2 and PGLa in lipid bilayers

The cationic antimicrobial peptides PGLa and magainin 2 (Mag2) are known for their antimicrobial activity and synergistic enhancement in antimicrobial and membrane leakage assays. Further use of peptides in combinatory therapy requires knowledge of the mechanisms of action of both individual peptides and their mixtures. Here, electron paramagnetic resonance (EPR), double electron-electron resonance (DEER, also known as PELDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies were applied to study self-assembly and localization of spin-labeled PGLa and Mag2 in POPE/POPG membranes with a wide range of peptide/lipid ratios (P/L) from ∼1/1500 to 1/50. EPR and DEER data showed that both peptides tend to organize in clusters, which occurs already at the lowest peptide/lipid molar ratio of 1/1500 (0.067 mol%). For individual peptides, these clusters are quite dense with intermolecular distances of the order of ∼2 nm. In the presence of a synergistic peptide partner, these homo-clusters are transformed into lipid-diluted hetero-clusters. These clusters are characterized by a local surface density that is several times higher than expected from a random distribution. ESEEM data indicate a slightly different insertion depth of peptides in hetero-clusters when compared to homo-clusters.

Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC.

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.

The crystal structure of bacteriophage λ RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins.

RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on Escherichia coli λ phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the λ bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here, we present the 2.05-Å crystal structure of the λ RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron-electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.

Mid-infrared-near-infrared double-resonance spectroscopy of molecules with kilohertz accuracy.

Precision measurements of molecular transitions to highly excited states are needed in potential energy surface modeling, state-resolved chemical dynamics studies, and astrophysical spectra analysis. Selective pumping and probing of molecules are often challenging due to the high state density and weak transition moments. We present a mid-infrared and near-infrared double-resonance spectroscopy method for precision measurements. As a demonstration, Doppler-free stepwise two-photon absorption spectra of 13CO2 were recorded by pumping the fundamental transition of R14 (00011)-(00001) and probing the P15 (00041)-(00011) transition enhanced by a high-finesse optical cavity, and the transition frequencies were determined with an accuracy of a few kilohertz.

Hyperfine-resolved rotational spectroscopy of HCNH.

The rotational spectrum of the molecular ion HCNH+ is revisited using double-resonance spectroscopy in an ion trap apparatus, with six transitions measured between 74 and 445GHz. Due to the cryogenic temperature of the trap, the hyperfine splittings caused by the 14N quadrupolar nucleus were resolved for transitions up to J = 4 ← 3, allowing for a refinement of the spectroscopic parameters previously reported, especially the quadrupole coupling constant eQq.

Sub-Doppler optical-optical double-resonance spectroscopy using a cavity-enhanced frequency comb probe

Accurate parameters of molecular hot-band transitions, i.e., those starting from vibrationally excited levels, are needed to accurately model high-temperature spectra in astrophysics and combustion, yet laboratory spectra measured at high temperatures are often unresolved and difficult to assign. Optical-optical double-resonance (OODR) spectroscopy allows the measurement and assignment of individual hot-band transitions from selectively pumped energy levels without the need to heat the sample. However, previous demonstrations lacked either sufficient resolution, spectral coverage, absorption sensitivity, or frequency accuracy. Here we demonstrate OODR spectroscopy using a cavity-enhanced frequency comb probe that combines all these advantages. We detect and assign sub-Doppler transitions in the spectral range of the 3ν3 ← ν3 resonance of methane with frequency precision and sensitivity more than an order of magnitude better than before. This technique will provide high-accuracy data about excited states of a wide range of molecules that is urgently needed for theoretical modeling of high-temperature data and cannot be obtained using other methods.

Structural Flexibility of Tau in Its Interaction with Microtubules as Viewed by Site-Directed Spin Labeling EPR Spectroscopy.

Tau is a microtubule-associated protein that belongs to the Intrinsically Disordered Proteins (IDPs) family. IDPs or Intrinsically Disordered Regions (IDRs) play key roles in protein interaction networks and their dysfunctions are often related to severe diseases. Defined by their lack of stable secondary and tertiary structures in physiological conditions while being functional, these proteins use their inherent structural flexibility to adapt to and interact with various binding partners. Knowledges on the structural dynamics of IDPs and their different conformers are crucial to finely decipher fundamental biological processes controlled by mechanisms such as conformational adaptations or switches, induced fit, or conformational selection events. Different mechanisms of binding have been proposed: among them, the so-called folding-upon-binding in which the IDP adopts a certain conformation upon interacting with a partner protein, or the formation of a "fuzzy" complex in which the IDP partly keeps its dynamical character at the surface of its partner. The dynamical nature and physicochemical properties of unbound as well as bound IDPs make this class of proteins particularly difficult to characterize by classical bio-structural techniques and require specific approaches for the fine description of their inherent dynamics.Among other techniques, Site-Directed Spin Labeling combined with Electron Paramagnetic Resonance (SDSL-EPR) spectroscopy has gained much interest in this last decade for the study of IDPs. SDSL-EPR consists in grafting a paramagnetic label (mainly a nitroxide radical) at selected site(s) of the macromolecule under interest followed by its observation using and/or combining different EPR strategies. These nitroxide spin labels detected by continuous wave (cw) EPR spectroscopy are used as perfect reporters or "spy spins" of their local environment, being able to reveal structural transitions, folding/unfolding events, etc. Another approach is based on the measurement of inter-label distance distributions in the 1.5-8.0nm range using pulsed dipolar EPR experiments, such as Double Electron-Electron Resonance (DEER) spectroscopy. The technique is then particularly well suited to study the behavior of Tau in its interaction with its physiological partner: microtubules (MTs). In this chapter we provide a detailed experimental protocol for the labeling of Tau protein and its EPR study while interacting with preformed (Paclitaxel-stabilized) MTs, or using Tau as MT inducer. We show how the choice of nitroxide label can be crucial to obtain functional information on Tau/tubulin complexes.