Double Electron-electron Resonance Research Articles

Monitoring the Conformation of the Sba1/Hsp90 Complex in the Presence of Nucleotides with Mn(II)-Based Double Electron–Electron Resonance

Hsp90 is an important molecular chaperone that facilitates the maturation of client proteins. It is a homodimer, and its function depends on a conformational cycle controlled by ATP hydrolysis and co-chaperones binding. We explored the binding of co-chaperone Sba1 to yeast Hsp90 (yHsp90) and the associated conformational change of yHsp90 in the pre- and post-ATP hydrolysis states by double electron–electron resonance (DEER) distance measurements. We substituted the Mg(II) cofactor at the ATPase site with paramagnetic Mn(II) and established the binding of Sba1 by measuring the distance between Mn(II) and a nitroxide (NO) spin-label on Sba1. Then, Mn(II)–NO DEER measurements on yHsp90 labeled with NO at the N-terminal domain detected the shift toward the closed conformation for both hydrolysis states. Finally, Mn(II)–Mn(II) DEER showed that Sba1 induced a closed conformation different from those with just bound Mn(II)·nucleotides. Our results provide structural experimental evidence for the binding of Sba1 tuning the closed conformation of yHsp90.

Asymmetric drug binding in an ATP-loaded inward-facing state of an ABC transporter.

Substrate efflux by ATP-binding cassette (ABC) transporters, which play a major role in multidrug resistance, entails the ATP-powered interconversion between transporter intermediates. Despite recent progress in structure elucidation, a number of intermediates have yet to be visualized and mechanistically interpreted. Here, we combine cryogenic-electron microscopy (cryo-EM), double electron-electron resonance spectroscopy and molecular dynamics simulations to profile a previously unobserved intermediate of BmrCD, a heterodimeric multidrug ABC exporter from Bacillus subtilis. In our cryo-EM structure, ATP-bound BmrCD adopts an inward-facing architecture featuring two molecules of the substrate Hoechst-33342 in a striking asymmetric head-to-tail arrangement. Deletion of the extracellular domain capping the substrate-binding chamber or mutation of Hoechst-coordinating residues abrogates cooperative stimulation of ATP hydrolysis. Together, our findings support a mechanistic role for symmetry mismatch between the nucleotide binding and the transmembrane domains in the conformational cycle of ABC transporters and is of notable importance for rational design of molecules for targeted ABC transporter inhibition.

Nanoscale spin detection of copper ions using double electron-electron resonance at room temperature

We report the nanoscale spin detection and electron paramagnetic resonance (EPR) spectrum of copper (Cu$^{2+}$) ions via double electron-electron resonance with single spins in diamond at room temperature and low magnetic fields. We measure unexpectedly narrow EPR resonances with linewidths $\sim 2-3$ MHz from copper-chloride molecules dissolved in poly-lysine. We also observe coherent Rabi oscillations and hyperfine splitting from single Cu$^{2+}$ ions, which could be used for dynamic nuclear spin polarization and higher sensitivity of spin detection. We interpret and analyze these observations using both spin hamiltonian modeling of the copper-chloride molecules and numerical simulations of the predicted DEER response, and obtain a sensing volume $\sim (250 \text{nm})^3$. This work will open the door for copper-labeled EPR measurements under ambient conditions in bio-molecules and nano-materials.

Long-range distance determination in fully deuterated RNA with pulsed EPR spectroscopy

Pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy is powerful in structure and dynamics study of biological macromolecules by providing distance distribution information ranging from 1.8 to 6 nm, providing that the biomolecules are site-specifically labeled with paramagnetic tags. However, long distances up to 16 nm have been measured on perdeuterated and spin-labeled proteins in deuterated solvent by PELDOR. Here we demonstrate long-range distance measurement on a large RNA, the 97-nucleotide 3′SL RNA element of the Dengue virus 2 genome, by combining a posttranscriptional site-directed spin labeling method using an unnatural basepair system with RNA perdeuteration by enzymatic synthesis using deuterated nucleotides. The perdeuteration removes the coupling of the electron spins of the nitroxide spin labels from the proton nuclear spin system of the RNA and does extend the observation time windows of PELDOR up to 50 μs. This enables one to determine long distances up to 14 nm for large RNAs and their conformational flexibility.

Conformational Flexibility of the Protein Insertase BamA in the Native Asymmetric Bilayer Elucidated by ESR Spectroscopy.

The β‐barrel assembly machinery (BAM) consisting of the central β‐barrel BamA and four other lipoproteins mediates the folding of the majority of the outer membrane proteins. BamA is placed in an asymmetric bilayer and its lateral gate is suggested to be the functional hotspot. Here we used in situ pulsed electron‐electron double resonance spectroscopy to characterize BamA in the native outer membrane. In the detergent micelles, the data is consistent with mainly an inward‐open conformation of BamA. The native membrane considerably enhanced the conformational heterogeneity. The lateral gate and the extracellular loop 3 exist in an equilibrium between different conformations. The outer membrane provides a favorable environment for occupying multiple conformational states independent of the lipoproteins. Our results reveal a highly dynamic behavior of the lateral gate and other key structural elements and provide direct evidence for the conformational modulation of a membrane protein in situ.

Location of two Mn2+ affinity sites in photosystem II detected by pulsed electron-electron double resonance.

In this study, we identified two Mn2+ sites in apo-Photosystem II (PSII) using the pulsed electron-electron double resonance (PELDOR). A Mn2+ ion was bound to apo-PSII on the deactivation of the oxygen-evolving complex. The electron-electron magnetic dipole interaction of the Mn2+ to YD· was estimated to be 2.4MHz. The site was assigned at the position between His332 and Glu189 in the D1 polypeptide, which is close to the Mn1 site in mature PS II. Using recent structures observed under electron microscopes (EM), the location of the Mn2+ site on photoactivation was reevaluated. The position between Asp170 and Glu189 in the D1 polypeptide is a good candidate for the initial high-affinity site for photoactivation. Based on a comparison with the PELDOR results, the two EM structures were evaluated.

Clustering of Stearic Acids in Model Phospholipid Membranes Revealed by Double Electron-Electron Resonance.

Free fatty acids play various important roles in biological membranes. Double electron-electron resonance spectroscopy (DEER, also known as PELDOR) of spin-labeled biomolecules is capable of studying magnetic dipole-dipole (d-d) interactions between spin labels at the nanoscale range of distances. Here, DEER is applied to study intermolecular d-d interactions between doxyl-spin-labeled stearic acids (DSA) in gel-phase phospholipid bilayers composed either of an equimolecular mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine or of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. DEER data obtained for different DSA concentrations showed that DSA molecules at their concentration in the bilayer χ larger than 0.5 mol % are assembled into lateral lipid-mediated clusters, with a characteristic intermolecular distance of 2 nm. Some evidences were obtained indicating that clusters may consist of "subclusters", alternatively appearing in two opposite leaflets. Conventional electron paramagnetic resonance (EPR) spectra for the gel-phase bilayers showed that for χ larger than 2 mol % the molecules in the clusters stick together, forming oligomers. Room-temperature EPR spectra for the liquid-crystalline phase were found to change noticeably for χ larger than 0.5 mol %, which may indicate the clustering in a liquid-crystalline phase similar to that observed by DEER in the gel phase.

Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules.

Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole-dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results. These guidelines are substantiated by a multi-laboratory benchmark study and by analysis of data sets with known distance distribution ground truth. The study and the guidelines focus on proteins labeled with nitroxides and on double electron-electron resonance (DEER aka PELDOR) measurements and provide suggestions on how to proceed analogously in other cases.

Integrative structural dynamics probing of the conformational heterogeneity in synaptosomal-associated protein 25

SUMMARYSNAP-25 (synaptosomal-associated protein of 25 kDa) is a prototypical intrinsically disordered protein (IDP) that is unstructured by itself but forms coiled-coil helices in the SNARE complex. With high conformational heterogeneity, detailed structural dynamics of unbound SNAP-25 remain elusive. Here, we report an integrative method to probe the structural dynamics of SNAP-25 by combining replica-exchange discrete molecular dynamics (rxDMD) simulations and label-based experiments at ensemble and single-molecule levels. The rxDMD simulations systematically characterize the coil-to-molten globular transition and reconstruct structural ensemble consistent with prior ensemble experiments. Label-based experiments using Förster resonance energy transfer and double electron-electron resonance further probe the conformational dynamics of SNAP-25. Agreements between simulations and experiments under both ensemble and single-molecule conditions allow us to assign specific helix-coil transitions in SNAP-25 that occur in submillisecond timescales and potentially play a vital role in forming the SNARE complex. We expect that this integrative approach may help further our understanding of IDPs.

Preferential Placement of Aligned Nitrogen Vacancy Centers in Chemical Vapor Deposition Overgrown Diamond Microstructures

The usefulness of nitrogen vacancy (NV) centers in diamond is augmented by a low defect and impurity density in the surrounding host material, and applications benefit from the ability to control the position of the NV centers. Herein, a process to create NV centers on single‐crystalline diamond microstructures by chemical vapor deposition (CVD) is presented. Pyramidal structures with {111} side facets are formed during the intrinsic overgrowth of dry chemically etched cylindrical pillars on a substrate with {100} surface orientation. A thin nitrogen‐doped epitaxial layer is deposited on top of the pyramids resulting in the creation of NV centers exclusively on the {111} pyramid side faces. Optically detected magnetic resonance (ODMR) and spin echo measurements reveal preferential alignment of the NV centers in a single {111} direction and a time of . The time of the NV centers is limited by the surrounding substitutional nitrogen (P1 center) concentration of . A low density of other paramagnetic spin noise is detected by double‐electron electron resonance (DEER) measurements.

Reconstruction of Coupled Intra- and Interdomain Protein Motion from Nuclear and Electron Magnetic Resonance.

Proteins composed of multiple domains allow for structural heterogeneity and interdomain dynamics that may be vital for function. Intradomain structures and dynamics can influence interdomain conformations and vice versa. However, no established structure determination method is currently available that can probe the coupling of these motions. The protein Pin1 contains separate regulatory and catalytic domains that sample "extended" and "compact" states, and ligand binding changes this equilibrium. Ligand binding and interdomain distance have been shown to impact the activity of Pin1, suggesting interdomain allostery. In order to characterize the conformational equilibrium of Pin1, we describe a novel method to model the coupling between intra- and interdomain dynamics at atomic resolution using multistate ensembles. The method uses time-averaged nuclear magnetic resonance (NMR) restraints and double electron-electron resonance (DEER) data that resolve distance distributions. While the intradomain calculation is primarily driven by exact nuclear Overhauser enhancements (eNOEs), J couplings, and residual dipolar couplings (RDCs), the relative domain distribution is driven by paramagnetic relaxation enhancement (PREs), RDCs, interdomain NOEs, and DEER. Our data support a 70:30 population of the compact and extended states in apo Pin1. A multistate ensemble describes these conformations simultaneously, with distinct conformational differences located in the interdomain interface stabilizing the compact or extended states. We also describe correlated conformations between the catalytic site and interdomain interface that may explain allostery driven by interdomain contact.

Determination of local defect density in diamond by double electron-electron resonance

Magnetic impurities in diamond influence the relaxation properties and thus limit the sensitivity of magnetic, electric, strain, and temperature sensors based on nitrogen-vacancy color centers. Diamond samples may exhibit significant spatial variations in the impurity concentrations hindering the quantitative analysis of relaxation pathways. Here, we present a local measurement technique which can be used to determine the concentration of various species of defects by utilizing double electron-electron resonance. This method will help to improve the understanding of the physics underlying spin relaxation and guide the development of diamond samples, as well as offering protocols for optimized sensing.

Unraveling a Ligand-Induced Twist of a Homodimeric Enzyme by Pulsed Electron-Electron Double Resonance.

Mechanistic insights into protein–ligand interactions can yield chemical tools for modulating protein function and enable their use for therapeutic purposes. For the homodimeric enzyme tRNA‐guanine transglycosylase (TGT), a putative virulence target of shigellosis, ligand binding has been shown by crystallography to transform the functional dimer geometry into an incompetent twisted one. However, crystallographic observation of both end states does neither verify the ligand‐induced transformation of one dimer into the other in solution nor does it shed light on the underlying transformation mechanism. We addressed these questions in an approach that combines site‐directed spin labeling (SDSL) with distance measurements based on pulsed electron–electron double resonance (PELDOR or DEER) spectroscopy. We observed an equilibrium between the functional and twisted dimer that depends on the type of ligand, with a pyranose‐substituted ligand being the most potent one in shifting the equilibrium toward the twisted dimer. Our experiments suggest a dissociation–association mechanism for the formation of the twisted dimer upon ligand binding.

Entschlüsselung der ligandeninduzierten Verdrehung eines homodimeren Enzyms mit Hilfe der gepulsten Elektron‐Elektron‐Doppelresonanz‐Spektroskopie

AbstractMechanistische Erkenntnisse über Protein‐Ligand‐Wechselwirkungen können chemische Werkzeuge für die Modulation von Proteinfunktionen liefern und deren Einsatz zu therapeutischen Zwecken ermöglichen. Für das homodimere Enzym tRNA‐Guanin‐Transglycosylase (TGT), eine virulenzrelevante Zielstruktur der Shigellose, wurde kristallographisch gezeigt, dass die Bindung bestimmter Liganden die funktionale Dimergeometrie in eine katalytisch‐inkompetente verdrehte Geometrie umwandelt. Die kristallographische Beobachtung beider Endzustände beweist jedoch weder die ligandeninduzierte Umwandlung zwischen beiden Dimerformen in Lösung noch gibt sie Aufschluss über den zugrunde liegenden Umwandlungsmechanismus. Wir sind diesen Fragen mit einem Ansatz nachgegangen, der ortsspezifische Spinmarkierung (SDSL) mit Abstandsmessungen auf der Grundlage der gepulsten Elektron‐Elektron‐Doppelresonanz‐Spektroskopie (PELDOR oder DEER) kombiniert. Wir beobachteten ein Gleichgewicht zwischen dem funktionalen und dem verdrehten Dimer, das von der chemischen Struktur des Liganden abhängt, wobei ein Pyranose‐substituierter Ligand das Gleichgewicht am stärksten in Richtung des verdrehten Dimers verschiebt. Unsere Experimente deuten auf einen Dissoziations‐Assoziations‐Mechanismus für die Bildung des verdrehten Dimers bei Ligandenbindung hin.

Protein functional dynamics from the rigorous global analysis of DEER data: Conditions, components, and conformations.

The potential of spin labeling to reveal the dynamic dimension of macromolecules has been recognized since the dawn of the methodology in the 1960s. However, it was the development of pulsed electron paramagnetic resonance spectroscopy to detect dipolar coupling between spin labels and the availability of turnkey instrumentation in the 21st century that realized the full promise of spin labeling. Double electron-electron resonance (DEER) spectroscopy has seen widespread applications to channels, transporters, and receptors. In these studies, distance distributions between pairs of spin labels obtained under different biochemical conditions report the conformational states of macromolecules, illuminating the key movements underlying biological function. These experimental studies have spurred the development of methods for the rigorous analysis of DEER spectroscopic data along with methods for integrating these distributions into structural models. In this tutorial, we describe a model-based approach to obtaining a minimum set of components of the distance distribution that correspond to functionally relevant protein conformations with a set of fractional amplitudes that define the equilibrium between these conformations. Importantly, we review and elaborate on the error analysis reflecting the uncertainty in the various parameters, a critical step in rigorous structural interpretation of the spectroscopic data.

Functional basis of electron transport within photosynthetic complex I

Photosynthesis and respiration rely upon a proton gradient to produce ATP. In photosynthesis, the Respiratory Complex I homologue, Photosynthetic Complex I (PS-CI) is proposed to couple ferredoxin oxidation and plastoquinone reduction to proton pumping across thylakoid membranes. However, little is known about the PS-CI molecular mechanism and attempts to understand its function have previously been frustrated by its large size and high lability. Here, we overcome these challenges by pushing the limits in sample size and spectroscopic sensitivity, to determine arguably the most important property of any electron transport enzyme – the reduction potentials of its cofactors, in this case the iron-sulphur clusters of PS-CI (N0, N1 and N2), and unambiguously assign them to the structure using double electron-electron resonance. We have thus determined the bioenergetics of the electron transfer relay and provide insight into the mechanism of PS-CI, laying the foundations for understanding of how this important bioenergetic complex functions.

Pinholin S21 mutations induce structural topology and conformational changes

The bacteriophage infection cycle is terminated at a predefined time to release the progeny virions via a robust lytic system composed of holin, endolysin, and spanin proteins. Holin is the timekeeper of this process. Pinholin S21 is a prototype holin of phage Φ21, which determines the timing of host cell lysis through the coordinated efforts of pinholin and antipinholin. However, mutations in pinholin and antipinholin play a significant role in modulating the timing of lysis depending on adverse or favorable growth conditions. Earlier studies have shown that single point mutations of pinholin S21 alter the cell lysis timing, a proxy for pinholin function as lysis is also dependent on other lytic proteins. In this study, continuous wave electron paramagnetic resonance (CW-EPR) power saturation and double electron-electron resonance (DEER) spectroscopic techniques were used to directly probe the effects of mutations on the structure and conformational changes of pinholin S21 that correlate with pinholin function. DEER and CW-EPR power saturation data clearly demonstrate that increased hydrophilicity induced by residue mutations accelerate the externalization of antipinholin transmembrane domain 1 (TMD1), while increased hydrophobicity prevents the externalization of TMD1. This altered hydrophobicity is potentially accelerating or delaying the activation of pinholin S21. It was also found that mutations can influence intra- or intermolecular interactions in this system, which contribute to the activation of pinholin and modulate the cell lysis timing. This could be a novel approach to analyze the mutational effects on other holin systems, as well as any other membrane protein in which mutation directly leads to structural and conformational changes.

Electron–electron double resonance detected NMR spectroscopy using ensemble NV centers at 230 GHz and 8.3 T

The nitrogen-vacancy (NV) center has enabled widespread study of nanoscale nuclear magnetic resonance (NMR) spectroscopy at low magnetic fields. NMR spectroscopy at high magnetic fields significantly improves the technique’s spectral resolution, enabling clear identification of closely related chemical species. However, NV-detected NMR is typically performed using AC sensing through electron spin echo envelope modulation, a hyperfine spectroscopic technique that is not feasible at high magnetic fields. Within this paper, we have explored an NV-detected NMR technique for applications of high field NMR. We have demonstrated optically detected magnetic resonance with the NV Larmor frequency of 230 GHz at 8.3 T, corresponding to a proton NMR frequency of 350 MHz. We also demonstrated the first measurement of electron–electron double resonance detected NMR using the NV center and successfully detected 13C nuclear bath spins. The described technique is limited by the longitudinal relaxation time (T1), not the transverse relaxation time (T2). Future applications of the method to perform nanoscale NMR of external spins at 8.3 T and even higher magnetic fields are also discussed.

Double Electron-Electron Resonance of Spin-Labeled Cholestane in Model Membranes: Evidence for Substructures inside the Lipid Rafts.

Plasma membranes are assumed to be highly compartmentalized, which is believed to be important for the membrane protein functionality. The liquid ordered-disordered phase segregation in the membranes results in nanoscale liquid-ordered assemblies-lipid rafts. Double electron-electron resonance spectroscopy (DEER, also known as PELDOR) is sensitive to spin-spin dipolar interactions between spin labels at the nanoscale range of distances. Here, DEER is applied to spin-labeled cholestane, 3β-doxyl-5α-cholestane (DChl), diluted in bilayers composed of an equimolar mixture of dioleoyl-glycero-phosphocholine (DOPC) and dipalmitoyl-glycero-phosphocholine (DPPC) phospholipids, with cholesterol (Chol) added. The DEER data allowed us to detect clustering of the DChl molecules. Their lateral distribution in the clusters in the absence of Chol was found to be random, while in the presence of Chol it became quasi-regular. DEER time traces are fairly well simulated within a simple square superlattice model. For the 20 mol % Chol content, for which at physiological temperatures, the lipid rafts are formed, the found superlattice parameter was 3.7 nm. Assuming that lipid rafts are captioned upon shock freezing at the temperature of investigation (80 K), the found regularity of DChl lateral distribution was interpreted by raft substructuring, with the DChl molecules embedded between the substructures.

Copper(II) Complexes of Sulfonated Salan Ligands: Thermodynamic and Spectroscopic Features and Applications for Catalysis of the Henry Reaction.

Copper(II) complexes formed with sulfonated salan ligands (HSS) have been synthesized, and their coordination chemistry has been characterized using pH-potentiometry and spectroscopic methods [UV-vis, electron paramagnetic resonance (EPR), and electron-electron double resonance (ELDOR)-detected NMR (EDNMR)] in aqueous solution. Several bridging moieties between the two salicylamine functions were introduced, e.g., ethyl (HSS), propyl (PrHSS), butyl (BuHSS), cyclohexyl (cis-CyHSS, trans-CyHSS), and diphenyl (dPhHSS). All of the investigated ligands feature excellent copper(II) binding ability via the formation of a (O-,N,N,O-) chelate system. The results indicated that the cyclohexyl moiety significantly enhances the stability of the copper(II) complexes. EPR studies revealed that the arrangement of the coordinated donor atoms is more symmetrical around the copper(II) center and similar for HSS, BuHSS, CyHSS, and dPhHSS, respectively, and a higher rhombicity of the g tensor was detected for PrHSS. The copper(II) complexes of the sulfosalan ligands were isolated in solid form also and showed moderate catalytic activity in the Henry (nitroaldol) reaction of aldehydes and nitromethane. The best yield for nitroaldol production was obtained for copper(II) complexes of PrHSS and BuHSS, although their metal binding ability is moderate compared to that of the cyclohexyl counterparts. However, these complexes possess larger spin density on the nitrogen nuclei than that for the other cases, which alters their catalytic activity.