Light-Induced Pulsed EPR Dipolar Spectroscopy on a Paradigmatic Hemeprotein

Light-induced pulsed EPR dipolar spectroscopic methods allow the determination of nanometer distances between paramagnetic sites. Here we employ orthogonal spin labels, a chromophore triplet state and a stable radical, to carry out distance measurements in singly nitroxide-labeled human neuroglobin. We demonstrate that Zn-substitution of neuroglobin, to populate the Zn(II) protoporphyrin IX triplet state, makes it possible to perform light-induced pulsed dipolar experiments on hemeproteins, extending the use of light-induced dipolar spectroscopy to this large class of metalloproteins. The versatility of the method is ensured by the employment of different techniques: relaxation-induced dipolar modulation enhancement (RIDME) is applied for the first time to the photoexcited triplet state. In addition, an alternative pulse scheme for laser-induced magnetic dipole (LaserIMD) spectroscopy, based on the refocused-echo detection sequence, is proposed for accurate zero-time determination and reliable distance analysis.     

Dynamics of Nucleic Acids at Room Temperature Revealed by Pulsed EPR Spectroscopy

The investigation of the structure and conformational dynamics of biomolecules under physiological conditions is challenging for structural biology. Although pulsed electron paramagnetic resonance (like PELDOR) techniques provide long‐range distance and orientation information with high accuracy, such studies are usually performed at cryogenic temperatures. At room temperature (RT) PELDOR studies are seemingly impossible due to short electronic relaxation times and loss of dipolar interactions through rotational averaging. We incorporated the rigid nitroxide spin label Ç into a DNA duplex and immobilized the sample on a solid support to overcome this limitation. This enabled orientation‐selective PELDOR measurements at RT. A comparison with data recorded at 50 K revealed averaging of internal dynamics, which occur on the ns time range at RT. Thus, our approach adds a new method to study structural and dynamical processes at physiological temperature in the <10 μs time range with atomistic resolution.     

A Comparison of Cysteine-Conjugated Nitroxide Spin Labels for Pulse Dipolar EPR Spectroscopy

The structure-function and materials paradigms drive research on the understanding of structures and structural heterogeneity of molecules and solids from materials science to structural biology. Functional insights into complex architectures are often gained from a suite of complementary physicochemical methods. In the context of biomacromolecular structures, the use of pulse dipolar electron paramagnetic resonance spectroscopy (PDS) has become increasingly popular. The main interest in PDS is providing long-range nanometre distance distributions that allow for identifying macromolecular topologies, validating structural models and conformational transitions as well as docking of quaternary complexes. Most commonly, cysteines are introduced into protein structures by site-directed mutagenesis and modified site-specifically to a spin-labelled side-chain such as a stable nitroxide radical. In this contribution, we investigate labelling by four different commercial labelling agents that react through different sulfur-specific reactions. Further, the distance distributions obtained are between spin-bearing moieties and need to be related to the protein structure via modelling approaches. Here, we compare two different approaches to modelling these distributions for all four side-chains. The results indicate that there are significant differences in the optimum labelling procedure. All four spin-labels show differences in the ease of labelling and purification. Further challenges arise from the different tether lengths and rotamers of spin-labelled side-chains; both influence the modelling and translation into structures. Our comparison indicates that the spin-label with the shortest tether in the spin-labelled side-group, (bis-(2,2,5,5-Tetramethyl-3-imidazoline-1-oxyl-4-yl) disulfide, may be underappreciated and could increase the resolution of structural studies by PDS if labelling conditions are optimised accordingly.     

Erythrosin B as a New Photoswitchable Spin Label for Light-Induced Pulsed EPR Dipolar Spectroscopy

We present a new photoswitchable spin label for light-induced pulsed electron paramagnetic resonance dipolar spectroscopy (LiPDS), the photoexcited triplet state of erythrosin B (EB), which is ideal for biological applications. With this label, we perform an in-depth study of the orientational effects in dipolar traces acquired using the refocused laser-induced magnetic dipole technique to obtain information on the distance and relative orientation between the EB and nitroxide labels in a rigid model peptide, in good agreement with density functional theory predictions. Additionally, we show that these orientational effects can be averaged to enable an orientation-independent analysis to determine the distance distribution. Furthermore, we demonstrate the feasibility of these experiments above liquid nitrogen temperatures, removing the need for expensive liquid helium or cryogen-free cryostats. The variety of choices in photoswitchable spin labels and the affordability of the experiments are critical for LiPDS to become a widespread methodology in structural biology.     

Pulsed EPR Dipolar Spectroscopy on Spin Pairs with one Highly Anisotropic Spin Center: The Low-Spin FeIII Case

Pulsed electron paramagnetic resonance (EPR) dipolar spectroscopy (PDS) offers several methods for measuring dipolar coupling constants and thus the distance between electron spin centers. Up to now, PDS measurements have been mostly applied to spin centers whose g-anisotropies are moderate and therefore have a negligible effect on the dipolar coupling constants. In contrast, spin centers with large g-anisotropy yield dipolar coupling constants that depend on the g-values. In this case, the usual methods of extracting distances from the raw PDS data cannot be applied. Here, the effect of the g-anisotropy on PDS data is studied in detail on the example of the low-spin Fe³⁺ ion. First, this effect is described theoretically, using the work of Bedilo and Maryasov (Appl. Magn. Reson. 2006 , 30, 683–702) as a basis. Then, two known Fe³⁺/nitroxide compounds and one new Fe³⁺/trityl compound were synthesized and PDS measurements were carried out on them using a method called relaxation induced dipolar modulation enhancement (RIDME). Based on the theoretical results, a RIDME data analysis procedure was developed, which facilitated the extraction of the inter-spin distance and the orientation of the inter-spin vector relative to the Fe³⁺ g-tensor frame from the RIDME data. The accuracy of the determined distances and orientations was confirmed by comparison with MD simulations. This method can thus be applied to the highly relevant class of metalloproteins with, for example, low-spin Fe³⁺ ions.     

A Bis‐Manganese(II)–DOTA Complex for Pulsed Dipolar Spectroscopy

High‐spin gadolinium(III) and manganese(II) complexes have emerged as alternatives to standard nitroxide radical spin labels for measuring nanometric distances by using pulsed electron–electron double resonance (PELDOR or DEER) at high fields/frequencies. For certain complexes, particularly those with relatively small zero‐field splitting (ZFS) and short distances between the two metal centers, the pseudosecular term of the dipolar coupling Hamiltonian is non‐negligible. However, in general, the contribution from this term during conventional data analysis is masked by the flexibility of the molecule of interest and/or the long tethers connecting them to the spin labels. The efficient synthesis of a model system consisting of two [Mn(dota)]^2? (MnDOTA; DOTA^4?=1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetate) directly connected to the ends of a central rodlike oligo(phenylene–ethynylene) (OPE) spacer is reported. The rigidity of the OPE is confirmed by Q‐band PELDOR measurements on a bis‐nitroxide analogue. The Mn^II?Mn^II distance distribution profile determined by W‐band PELDOR is in reasonable agreement with one simulated by using a simple rotamer analysis. The small degree of flexibility arising from the linking MnDOTA arm appears to outweigh the contribution from the pseudosecular term at this interspin distance. This study illustrates the potential of MnDOTA‐based spin labels for measuring fairly short nanometer distances, and also presents an interesting candidate for in‐depth studies of pulsed dipolar spectroscopy methods on Mn^II?Mn^II systems.     

A Bioresistant Nitroxide Spin Label for In‐Cell EPR Spectroscopy: In Vitro and In Oocytes Protein Structural Dynamics Studies

Approaching protein structural dynamics and protein–protein interactions in the cellular environment is a fundamental challenge. Owing to its absolute sensitivity and to its selectivity to paramagnetic species, site‐directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) has the potential to evolve into an efficient method to follow conformational changes in proteins directly inside cells. Until now, the use of nitroxide‐based spin labels for in‐cell studies has represented a major hurdle because of their short persistence in the cellular context. The design and synthesis of the first maleimido‐proxyl‐based spin label (M‐TETPO) resistant towards reduction and being efficient to probe protein dynamics by continuous wave and pulsed EPR is presented. In particular, the extended lifetime of M‐TETPO enabled the study of structural features of a chaperone in the absence and presence of its binding partner at endogenous concentration directly inside cells.     

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.     

Probing the Structural Dynamics of a Bacterial Chaperone in Its Native Environment by Nitroxide-Based EPR Spectroscopy

One of the greatest current challenges in structural biology is to study protein dynamics over a wide range of timescales in complex environments, such as the cell. Among magnetic resonances suitable for this approach, electron paramagnetic resonance spectroscopy coupled to site-directed spin labeling (SDSL-EPR) has emerged as a promising tool to study protein local dynamics and conformational ensembles. In this work, we exploit the sensitivity of nitroxide labels to report protein local dynamics at room temperature. We demonstrate that such studies can be performed while preserving both the integrity of the cells and the activity of the protein under investigation. Using this approach, we studied the structural dynamics of the chaperone NarJ in its natural host, Escherichia coli. We established that spin-labeled NarJ is active inside the cell. We showed that the cellular medium affects NarJ structural dynamics in a site-specific way, while the structural flexibility of the protein is maintained. Finally, we present and discuss data on the time-resolved dynamics of NarJ in cellular context.     

Oxidation of quinolones with peracids (an in situ EPR study)

4‐Oxoquinoline derivatives (quinolones) represent heterocyclic compounds with a variety of biological activities, along with interesting chemical reactivity. The quinolone derivatives possessing secondary amino hydrogen at the nitrogen of the enaminone system are oxidized with 3‐chloroperbenzoic acid to nitroxide radicals in the primary step while maintaining their 4‐pyridone ring. Otherwise, N‐methyl substituted quinolones also form nitroxide radicals coupled with the opening of the 4‐pyridone ring in a gradual oxidation of the methyl group via the nitrone–nitroxide spin‐adduct cycle. This was confirmed in an analogous oxidation using N,N‐dimethylaniline as a model compound. N‐Ethyl quinolones in contrast to its N‐methyl analog form only one nitroxide radical without a further degradation. Copyright © 2013 John Wiley & Sons, Ltd.     

Long Distance Measurements up to 160 Å in the GroEL Tetradecamer Using Q‐Band DEER EPR Spectroscopy

Current distance measurements between spin‐labels on multimeric protonated proteins using double electron–electron resonance (DEER) EPR spectroscopy are generally limited to the 15–60 Å range. Here we show how DEER experiments can be extended to dipolar evolution times of ca. 80 μs, permitting distances up to 170 Å to be accessed in multimeric proteins. The method relies on sparse spin‐labeling, supplemented by deuteration of protein and solvent, to minimize the deleterious impact of multispin effects and substantially increase the apparent spin‐label phase memory relaxation time, complemented by high sensitivity afforded by measurements at Q‐band. We demonstrate the approach using the tetradecameric molecular machine GroEL as an example. Two engineered surface‐exposed mutants, R268C and E315C, are used to measure pairwise distance distributions with mean values ranging from 20 to 100 Å and from 30 to 160 Å, respectively, both within and between the two heptameric rings of GroEL. The measured distance distributions are consistent with the known crystal structure of apo GroEL. The methodology presented here should significantly expand the use of DEER for the structural characterization of conformational changes in higher order oligomers.     

Identification and Spectroscopic Characterization of Nonheme Iron(III) Hypochlorite Intermediates

Fe^III–hypohalite complexes have been implicated in a wide range of important enzyme‐catalyzed halogenation reactions including the biosynthesis of natural products and antibiotics and post‐translational modification of proteins. The absence of spectroscopic data on such species precludes their identification. Herein, we report the generation and spectroscopic characterization of nonheme Fe^III–hypohalite intermediates of possible relevance to iron halogenases. We show that Fe^III‐OCl polypyridylamine complexes can be sufficiently stable at room temperature to be characterized by UV/Vis absorption, resonance Raman and EPR spectroscopies, and cryo‐ESIMS. DFT methods rationalize the pathways to the formation of the Fe^III‐OCl, and ultimately Fe^IV?O, species and provide indirect evidence for a short‐lived Fe^II‐OCl intermediate. The species observed and the pathways involved offer insight into and, importantly, a spectroscopic database for the investigation of iron halogenases.     

PELDOR Spectroscopy Distance Fingerprinting of the Octameric Outer‐Membrane Protein Wza from Escherichia coli.

Distance fingerprinting : Pulsed electron–electron double resonance spectroscopy (PELDOR) is applied to the octameric membrane protein complex Wza of E. coli. The data yielded a detailed distance fingerprint of its periplasmic region that compares favorably to the crystal structure. These results provide the foundation to study conformation changes from interaction with partner proteins.

     

Molecular Magnets: The Synthesis and Characterization of High-Spin Nitrenes

Among all C-, N-, and O-centered polyradicals, high-spin nitrenes possess the largest magnetic anisotropy and are of considerable interest as multi-level molecular spin systems for exploration of organic molecular magnetism and quantum information processing. Although the first representatives of quintet and septet nitrenes were obtained almost 50 years ago, the experimental and theoretical studies of these highly reactive species became possible only recently, owing to new achievements in molecular spectroscopy and computational chemistry. Meanwhile, dozens of various quintet dinitrenes and septet trinitrenes were successfully characterized by IR, UV/Vis, and EPR spectroscopy, thus providing important information about the electronic structure, magnetic properties and reactivity of these compounds.