Pulsed Electron-electron Double Resonance Spectroscopy Research Articles

Simultaneous Localization of Two High Affinity Divalent Metal Ion Binding Sites in the Tetracycline RNA Aptamer with Mn2+-Based Pulsed Dipolar EPR Spectroscopy.

Mg2+ ions play an essential part in stabilizing the tertiary structure of nucleic acids. While the importance of these ions is well documented, their localization and elucidation of their role in the structure and dynamics of nucleic acids are often challenging. In this work, pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) was used to localize two high affinity divalent metal ion binding sites in the tetracycline RNA aptamer with high accuracy. For this purpose, the aptamer was labeled at different positions with a semirigid nitroxide spin label and diamagnetic Mg2+ was replaced with paramagnetic Mn2+, which did not alter the folding process or ligand binding. Out of the several divalent metal ion binding sites that are known from the crystal structure, two binding sites with high affinity were detected: one that is located at the ligand binding center and another at the J1/2 junction of the RNA.

Gd3+-Trityl-Nitroxide Triple Labeling and Distance Measurements in the Heterooligomeric Cobalamin Transport Complex in the Native Lipid Bilayers.

Increased efforts are being made for observing proteins in their native environments. Pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) is a powerful tool for this purpose. Conventionally, PELDOR employs an identical spin pair, which limits the output to a single distance for monomeric samples. Here, we show that the Gd3+-trityl-nitroxide (NO) three-spin system is a versatile tool to study heterooligomeric membrane protein complexes, even within their native membrane. This allowed for an independent determination of four different distances (Gd3+-trityl, Gd3+-NO, trityl-NO, and Gd3+-Gd3+) within the same sample. We demonstrate the feasibility of this approach by observing sequential ligand binding and the dynamics of complex formation in the cobalamin transport system involving four components (cobalamin, BtuB, TonB, and BtuF). Our results reveal that TonB binding alone is sufficient to release cobalamin from BtuB in the native asymmetric bilayers. This approach provides a potential tool for the structural and quantitative analysis of dynamic protein-protein interactions in oligomeric complexes, even within their native surroundings.

Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET

Pulsed electron-electron double resonance spectroscopy (PELDOR/DEER) and single-molecule Förster resonance energy transfer spectroscopy (smFRET) are frequently used to determine conformational changes, structural heterogeneity, and inter probe distances in biological macromolecules. They provide qualitative information that facilitates mechanistic understanding of biochemical processes and quantitative data for structural modelling. To provide a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET, we use a library of double cysteine variants of four proteins that undergo large-scale conformational changes upon ligand binding. With either method, we use established standard experimental protocols and data analysis routines to determine inter-probe distances in the presence and absence of ligands. The results are compared to distance predictions from structural models. Despite an overall satisfying and similar distance accuracy, some inconsistencies are identified, which we attribute to the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. This large-scale cross-validation of PELDOR/DEER and smFRET highlights the strengths, weaknesses, and synergies of these two important and complementary tools in integrative structural biology.

In situ distance measurements in a membrane transporter using maleimide functionalized orthogonal spin labels and 5-pulse electron-electron double resonance spectroscopy

Spectroscopic investigation of membrane proteins in their native environment is a challenging task. Earlier we demonstrated the feasibility to measure precise distances within outer membrane proteins in E. coli and native membranes using methanethiosulfonate (MTS) functionalized labels combined with pulsed electron-electron double resonance spectroscopy. Here we show the application of maleimide functionalized Gd(III), nitroxide, and trityl labels for in situ distance measurement using the cobalamin transporter BtuB. These labels enabled distance measurements for BtuB in E. coli and native outer membranes and in the membranes maleimide-Gd-DOTA also is effective. Further, we show that the observable dipolar evolution time can be significantly prolonged in the native environments using the Carr-Purcell 5-pulse electron double resonance sequence. For a nitroxide-nitroxide pair, application of sech/tanh inversion pulses substantially suppressed the 4-pulse artifact at the Q-band frequency. In the case of a nitroxide-trityl pair, Gaussian pump pulses of varying amplitude are sufficient to suppress the artifact to the typical noise level. The feasibility of a range of bioresistant spin labels and the 5-pulse electron double resonance offers promising tools for investigating heterooligomeric membrane protein complexes in their native environment.

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.

Spatiotemporal Resolution of Conformational Changes in Biomolecules by Combining Pulsed Electron-Electron Double Resonance Spectroscopy with Microsecond Freeze-Hyperquenching.

The function of proteins is linked to their conformations that can be resolved with several high-resolution methods. However, only a few methods can provide the temporal order of intermediates and conformational changes, with each having its limitations. Here, we combine pulsed electron-electron double resonance spectroscopy with a microsecond freeze-hyperquenching setup to achieve spatiotemporal resolution in the angstrom range and lower microsecond time scale. We show that the conformational change of the Cα-helix in the cyclic nucleotide-binding domain of the Mesorhizobium loti potassium channel occurs within about 150 μs and can be resolved with angstrom precision. Thus, this approach holds great promise for obtaining 4D landscapes of conformational changes in biomolecules.

Hybrid nanophotonic-nanomagnonic SiC-YiG quantum sensor: II/dark spins quantum sensing with V2 spins and fiber based OP-PELDOR/ODMR

First experiments like optically detected (OD) electron paramagnetic resonance (ODMR), photoluminescence detected RABI oscillations, and optical pumping (OP) assisted pulsed EPR measurements of T2 and T1 of V2 spins in bulk SiC, which were previously demonstrated on various home build EPR spectrometers with free space optics, are here all demonstrated for the first time using a commercial X band pulsed EPR spectrometer combined with a single optical fiber and a standard external photoluminescence setup. Quantum sensing of bulk dark spins dipolar coupled to V2 spins in SiC is also demonstrated here for the first time using single fiber based OP assisted pulsed electron electron double resonance spectroscopy (PELDOR). A spin wave resonance study of model permalloy nanostripes is also presented allowing to check the ferromagnetic nanostripes design. These experiments are first key steps towards the fiber-based integration of the recently proposed SiC-YiG quantum sensor device [J. Tribollet, Eur. Phys. J. Appl. Phys. 90, 20102 (2020)], to a commercially available and worldwide used pulsed EPR spectrometer, with important applications expected in structural biology, surface chemistry, and quantum computing.

High-Yield Spin Labeling of Long RNAs for Electron Paramagnetic Resonance Spectroscopy.

Site-directed spin labeling is a powerful tool for investigating the conformation and dynamics of biomacromolecules such as RNA. Here we introduce a spin labeling strategy based on click chemistry in solution that, in combination with enzymatic ligation, allows highly efficient labeling of complex and long RNAs with short reaction times and suppressed RNA degradation. With this approach, a 34-nucleotide aptamer domain of the preQ1 riboswitch and an 81-nucleotide TPP riboswitch aptamer could be labeled with two labels in several positions. We then show that conformations of the preQ1 aptamer and its dynamics can be monitored in the absence and presence of Mg2+ and a preQ1 ligand by continuous wave electron paramagnetic resonance spectroscopy at room temperature and pulsed electron-electron double resonance spectroscopy (PELDOR or DEER) in the frozen state.

Inhibitor-Directed Spin Labelling-A High Precision and Minimally Invasive Technique to Study the Conformation of Proteins in Solution.

Pulsed electron-electron double resonance spectroscopy (known as PELDOR or DEER) has recently become a very popular tool in structural biology. The technique can be used to accurately measure distance distributions within macromolecules or macromolecular complexes, and has become a standard method to validate structural models and to study the conformational flexibility of macromolecules. It can be applied in solution, in lipid environments or even in cells. Because most biological macromolecules are diamagnetic, they are normally invisible for PELDOR spectroscopy. To render a particular target molecule accessible for PELDOR, it can be engineered to contain only one or two surface-exposed cysteine residues, which can be efficiently spin-labelled using thiol-reactive nitroxide compounds. This method has been coined "site-directed spin labelling" (SDSL) and is normally straight-forward. But, SDSL can be very challenging for proteins with many native cysteines, or even a single functionally or structurally important cysteine residue. For such cases, alternative spin labelling techniques are needed. Here we describe the concept of "inhibitor-directed spin labelling" (IDSL) as an approach to spin label suitable cysteine-rich proteins in a site-directed and highly specific manner by employing bespoke spin-labelled inhibitors. Advantages and disadvantages of IDSL are discussed.

Studying structure and function of membrane proteins with PELDOR/DEER spectroscopy - The crystallographers' perspective.

In 1985, the first X-ray structure of a membrane protein was determined. Today, more than 30 years later, many more structures have been solved. Nevertheless, studying the structure of membrane proteins remains a very challenging task. Due to their inherent conformational flexibility, having a single X-ray structure is usually only the first step towards truly understanding the function of these dynamic molecules. For this reason, additional methods are needed that can provide complementary information, especially about conformational flexibility. Pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) is such a method. It can be used to precisely measure nanometer distance distributions between intrinsic or artificially introduced spin-centers in macromolecules and thereby to probe the conformational state of the macromolecule. PELDOR can be applied in solution, in detergent, in lipid bilayers and even within cells. However, PELDOR is an advanced spectroscopy technique and requires specialised equipment and training. This chapter aims to be a starting point for crystallographers and other structural biologists who want to get a better understanding of PELDOR spectroscopy and its application. It gives an insight into the planning stages of the experiment (i.e., which spin labels are possible and where to place them), how a PELDOR experiment is conducted and how the results are interpreted. For this purpose, the substrate binding protein (SBP) from a Vibrio cholerae TRAP transporter is used as a step-by-step example. Further, the chapter gives examples of how PELDOR spectroscopy has previously been applied to overcome known limitations of X-ray crystallography in modern integrative structural biology approaches.

Chain Assembly and Disassembly Processes Differently Affect the Conformational Space of Ubiquitin Chains

Ubiquitination is the most versatile posttranslational modification. The information is encoded by linkage type as well as chain length, which are translated by ubiquitin binding domains into specific signaling events. Chain topology determines the conformational space of a ubiquitin chain and adds an additional regulatory layer to this ubiquitin code. In particular, processes that modify chain length will be affected by chain conformations as they require access to the elongation or cleavage sites. We investigated conformational distributions in the context of chain elongation and disassembly using pulsed electron-electron double resonance spectroscopy in combination with molecular modeling. Analysis of the conformational space of diubiquitin revealed conformational selection or remodeling as mechanisms for chain recognition during elongation or hydrolysis, respectively. Chain elongation to tetraubiquitin increases the sampled conformational space, suggesting that a high intrinsic flexibility of K48-linked chains may contribute to efficient proteasomal degradation.

High-frequency pulsed electron-electron double resonance spectroscopy on DNA duplexes using trityl tags and shaped microwave pulses.

Accurate distances between two trityl paramagnetic tags site-specifically attached to DNA duplexes were measured by pulsed electron-electron double resonance spectroscopy at 180 and 260 GHz microwave frequencies. Up to a threefold increase in the sensitivity of 260 GHz PELDOR measurements was achieved by using shaped broad-band microwave pulses.

Intersubunit distances in full-length, dimeric, bacterial phytochrome Agp1, as measured by pulsed electron-electron double resonance (PELDOR) between different spin label positions, remain unchanged upon photoconversion

Bacterial phytochromes are dimeric light-regulated histidine kinases that convert red light into signaling events. Light absorption by the N-terminal photosensory core module (PCM) causes the proteins to switch between two spectrally distinct forms, Pr and Pfr, thus resulting in a conformational change that modulates the C-terminal histidine kinase region. To provide further insights into structural details of photoactivation, we investigated the full-length Agp1 bacteriophytochrome from the soil bacterium Agrobacterium fabrum using a combined spectroscopic and modeling approach. We generated seven mutants suitable for spin labeling to enable application of pulsed EPR techniques. The distances between attached spin labels were measured using pulsed electron-electron double resonance spectroscopy to probe the arrangement of the subunits within the dimer. We found very good agreement of experimental and calculated distances for the histidine-kinase region when both subunits are in a parallel orientation. However, experimental distance distributions surprisingly showed only limited agreement with either parallel- or antiparallel-arranged dimer structures when spin labels were placed into the PCM region. This observation indicates that the arrangements of the PCM subunits in the full-length protein dimer in solution differ significantly from that in the PCM crystals. The pulsed electron-electron double resonance data presented here revealed either no or only minor changes of distance distributions upon Pr-to-Pfr photoconversion.

Structural Determination of a DNA Oligomer for a Molecular Spin Qubit Lloyd Model of Quantum Computers

Abstract The global molecular and local spin-site structures of a DNA duplex 22-oligomer with site-directed four spin-labeling were simulated by molecular mechanics (MM) calculations combined with Q-band pulsed electron-electron double resonance (PELDOR) spectroscopy. This molecular-spin bearing DNA oligomer is designed to give a complex testing ground for the structural determination of molecular spins incorporated in the DNA duplex, which serves as a platform for 1D periodic arrays of two or three non-equivalent electron spin qubit systems, (AB)n or (ABC)n, respectively, enabling to execute quantum computing or quantum information processing (Lloyd model of electron spin versions): A, B and C designate non-equivalent addressable spin qubits for quantum operations. The non-equivalence originates in difference in the electronic g-tensor. It is not feasible to determine the optimal structures for such DNA oligomers having molecular flexibility only by the MM calculations because there are many local minima in energy for their possible molecular structures. The spin-distance information derived from the PELDOR spectroscopy helps determine the optimal structures out of the possible ones acquired by the MM calculations. Based on the MM searched structures, we suggest the optimal structures for semi-macromolecules having site-directed multi-spin qubits. We emphasize that for our four molecular spins embedded in the DNA oligomer the Fajer’s error analysis in PELDOR-based distance measurements was of essential importance.

Synthesis of a Cytidine Phosphoramidite with Protected Nitroxide Spin Label for EPR Experiments with RNA

Spin labeling of oligonucleotides with nitroxides is hampered by their intrinsic instability under conditions of solid‐phase synthesis and enzymatic ligation. Although nitroxide decomposition can be avoided in some cases by postsynthetic introduction or by special reaction conditions, a more general solution would be reversible protection of the radical. We have recently developed such a method based on photolabile protection groups for DNA oligonucleotides and demonstrated their application in EPR spectroscopy. Here, we extend this method to RNA oligonucleotides. By improving the synthetic procedures, the yield of the coumarin‐protected phosphoramidite could be increased by a factor of 12. Effective recovery of the nitroxides on a duplex RNA enables pulsed EPR experiments to be performed directly after irradiation and air oxidation. Data at Q‐band frequency is shown and distances measured with PELDOR (pulsed electron‐electron double resonance) spectroscopy agree well with the calculated values.

Studies on the X-Ray and Solution Structure of FeoB from Escherichia coli BL21

The ferrous iron transporter FeoB is an important factor in the iron metabolism of many bacteria. Although several structural studies have been performed on its cytosolic GTPase domain (NFeoB), the full-length structure of FeoB remains elusive. Based on a crystal packing analysis that was performed on crystals of NFeoB, a trimeric structure of the FeoB channel was proposed, where the transport pore runs along the trimer axis. Because this trimer has not been observed in some subsequently solved structures of NFeoB homologs, it remains unclear whether or not the trimer is indeed functionally relevant. Here, pulsed electron-electron double resonance spectroscopy, negative stain electron microscopy, and native mass spectrometry are used to analyze the oligomeric state of different soluble and full-length FeoB constructs. The results show that the full-length protein is predominantly monomeric, whereas dimers and trimers are formed to a small percentage. Furthermore, the solution structure of the switch I region is analyzed by pulsed electron-electron double resonance spectroscopy and a new, to our knowledge, crystal structure of NFeoB from Escherichia coli BL21 is presented.

Structural Changes of a Doubly Spin-Labeled Chemically Driven Molecular Shuttle Probed by PELDOR Spectroscopy.

Gaining detailed information on the structural rearrangements associated with stimuli-induced molecular movements is of utmost importance for understanding the operation of molecular machines. Pulsed electron-electron double resonance (PELDOR) was employed to monitor the geometrical changes arising upon chemical switching of a [2]rotaxane that behaves as an acid-base-controlled molecular shuttle. To this aim, the rotaxane was endowed with stable nitroxide radical units in both the ring and axle components. The combination of PELDOR data and molecular dynamic calculations indicates that in the investigated rotaxane, the ring displacement along the axle, caused by the addition of a base, does not alter significantly the distance between the nitroxide labels, but it is accompanied by a profound change in the geometry adopted by the macrocycle.

Conformationally restricted isoindoline-derived spin labels in duplex DNA: distances and rotational flexibility by pulsed electron-electron double resonance spectroscopy.

Three structurally related isoindoline-derived spin labels that have different mobilities were incorporated into duplex DNA to systematically study the effect of motion on orientation-dependent pulsed electron-electron double resonance (PELDOR) measurements. To that end, a new nitroxide spin label, (ExIm)U, was synthesized and incorporated into DNA oligonucleotides. (ExIm)U is the first example of a conformationally unambiguous spin label for nucleic acids, in which the nitroxide N-O bond lies on the same axis as the three single bonds used to attach the otherwise rigid isoindoline-based spin label to a uridine base. Continuous-wave (CW) EPR measurements of (ExIm)U confirm a very high rotational mobility of the spin label in duplex DNA relative to the structurally related spin label (Im)U, which has restricted mobility due to an intramolecular hydrogen bond. The X-band CW-EPR spectra of (ExIm)U can be used to identify mismatches in duplex DNA. PELDOR distance measurements between pairs of the spin labels (Im)U, (Ox)U, and (ExIm)U in duplex DNA showed a strong angular dependence for (Im)U, a medium dependence for (Ox)U, and no orientation effect for (ExIm)U. Thus, precise distances can be extracted from (ExIm)U without having to take orientational effects into account.

A high-frequency electron paramagnetic resonance spectrometer for multi-dimensional, multi-frequency, and multi-phase pulsed measurements.

We describe instrumentation for a high-frequency electron paramagnetic resonance (EPR) and pulsed electron-electron double resonance (PELDOR) spectroscopy. The instrumentation is operated in the frequency range of 107-120 GHz and 215-240 GHz and in the magnetic field range of 0-12.1 T. The spectrometer consisting of a high-frequency high-power solid-state source, a quasioptical system, a phase-sensitive detection system, a cryogenic-free superconducting magnet, and a (4)He cryostat enables multi-frequency continuous-wave EPR spectroscopy as well as pulsed EPR measurements with a few hundred nanosecond pulses. Here we discuss the details of the design and the pulsed EPR sensitivity of the instrumentation. We also present performance of the instrumentation in unique experiments including PELDOR spectroscopy to probe correlations in an insulating electronic spin system and application of dynamical decoupling techniques to extend spin coherence of electron spins in an insulating solid-state system.

Energy Landscapes and Catalysis in Nitric-oxide Synthase

Nitric oxide (NO) plays diverse roles in mammalian physiology. It is involved in blood pressure regulation, neurotransmission, and immune response, and is generated through complex electron transfer reactions catalyzed by NO synthases (NOS). In neuronal NOS (nNOS), protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the heme oxygenase domain, the site of NO generation. Simple models based on crystal structures of nNOS reductase have invoked a role for large scale motions of the FMN-binding domain in shuttling electrons from the FAD-binding domain to the heme oxygenase domain. However, molecular level insight of the dynamic structural transitions in NOS enzymes during enzyme catalysis is lacking. We use pulsed electron-electron double resonance spectroscopy to derive inter-domain distance relationships in multiple conformational states of nNOS. These distance relationships are correlated with enzymatic activity through variable pressure kinetic studies of electron transfer and turnover. The binding of NADPH and calmodulin are shown to influence interdomain distance relationships as well as reaction chemistry. An important effect of calmodulin binding is to suppress adventitious electron transfer from nNOS to molecular oxygen and thereby preventing accumulation of reactive oxygen species. A complex landscape of conformations is required for nNOS catalysis beyond the simple models derived from static crystal structures of nNOS reductase. Detailed understanding of this landscape advances our understanding of nNOS catalysis/electron transfer, and could provide new opportunities for the discovery of small molecule inhibitors that bind at dynamic protein interfaces of this multidimensional energy landscape.