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.     

Determination of End-to-End Distances in a Series of TEMPO Diradicals of up to 2.8 nm Length with a New Four-Pulse Double Electron Electron Resonance Experiment

A four-pulse version of the pulsed double electron electron resonance (DEER) experiment has been applied to a series of TEMPO diradicals with well-defined interradical distances ranging from 1.4 to 2.8 nm (see picture). The new pulse sequence allows broad distributions of electron–electron distances to be measured without dead-time artifacts.     

Photochemical Reactions and Photoinduced Electron‐Transfer Processes in Liquids,Frozen Solutions,and Proteins as Studied by Multifrequency Time‐Resolved EPR Spectroscopy

In this overview, modern multifrequency EPR spectroscopy, in particular at high magnetic fields, is shown to provide detailed information about structure, motional dynamics, and spin chemistry of transient radicals and radical pairs occurring in photochemical reactions. Examples discussed comprise photochemical reactions in liquid solution and light‐initiated electron transfer processes both in biomimetic donor–acceptor model systems in frozen solution or liquid crystals and in natural photosynthetic‐reaction‐center protein complexes. The transient paramagnetic states exhibit characteristic electron polarization (CIDEP) effects. They contain valuable information about structure and dynamics of the transient reaction intermediates. Moreover, they are exploited for signal enhancement. Continuous‐wave (cw) and pulsed versions of time‐resolved high‐field EPR spectroscopy, such as cw‐transient‐EPR (TREPR) and pulsed‐electron‐spin‐echo (ESE) experiments, are compared with respect to their advantages and limitations for the specific system under study. For example, W‐band (95‐GHz) TREPR spectroscopy in conjunction with a continuous‐flow system for light‐generated short‐lived transient spin‐polarized radicals of organic photoinitiators in solution was performed with a time resolution of 10 ns. The increased Boltzmann polarization at high fields even allows detection of transient radicals without CIDEP effects. This enables one to determine initial radical polarization contributions as well as radical‐addition reaction constants. Another example of the power of combined X‐band and W‐band TREPR spectroscopy is given for the complex electron‐transfer and spin dynamics of covalently linked porphyrin–quinone as well as Watson–Crick base‐paired porphyrin–dinitrobenzene donor–acceptor biomimetic model systems. Furthermore, W‐band ESE experiments on the spin‐correlated coupled radical pair in reaction centers of the purple photosynthetic bacterium Rb. sphaeroides reveal details of distance and orientation of the pair partners in their charge‐separated transient state. The results are compared with those of the ground‐state P₈₆₅Q_A. The high orientation selectivity of high‐field EPR provides single‐crystal‐like information even from disordered frozen‐solution samples. The examples given demonstrate that high‐field EPR adds substantially to the capability of ‘classical’ spectroscopic and diffraction techniques for determining structure–dynamics–function relations of biochemical systems, since transient intermediates can be observed in real time in their working states on biologically relevant time scales.     

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.     

Light‐Induced Porphyrin‐Based Spectroscopic Ruler for Nanometer Distance Measurements

We present a novel pulsed electron paramagnetic resonance (EPR) spectroscopic ruler to test the performance of a recently developed spin‐labeling method based on the photoexcited triplet state (S=1). Four‐pulse electron double resonance (PELDOR) experiments are carried out on a series of helical peptides, labeled at the N‐terminal end with the porphyrin moiety, which can be excited to the triplet state, and with the nitroxide at various sequence positions, spanning distances in the range 1.8–8 nm. The PELDOR traces provide accurate distance measurements for all the ruler series, showing deep envelope modulations at frequencies varying in a progressive way according to the increasing distance between the spin labels. The upper limit is evaluated and found to be around 8 nm. The PELDOR‐derived distances are in excellent agreement with theoretical predictions. We demonstrate that high sensitivity is acquired using the triplet state as a spin label by comparison with Cu(II)–porphyrin analogues. The new labeling approach has a high potential for measuring nanometer distances in more complex biological systems due to the properties of the porphyrin triplet state.     

The Double‐Histidine Cu2+‐Binding Motif: A Highly Rigid,Site‐Specific Spin Probe for Electron Spin Resonance Distance Measurements

The development of ESR methods that measure long‐range distance distributions has advanced biophysical research. However, the spin labels commonly employed are highly flexible, which leads to ambiguity in relating ESR measurements to protein‐backbone structure. Herein we present the double‐histidine (dHis) Cu²⁺‐binding motif as a rigid spin probe for double electron–electron resonance (DEER) distance measurements. The spin label is assembled in situ from natural amino acid residues and a metal salt, requires no postexpression synthetic modification, and provides distance distributions that are dramatically narrower than those found with the commonly used protein spin label. Simple molecular modeling based on an X‐ray crystal structure of an unlabeled protein led to a predicted most probable distance within 0.5 Å of the experimental value. Cu²⁺ DEER with the dHis motif shows great promise for the resolution of precise, unambiguous distance constraints that relate directly to protein‐backbone structure and flexibility.     

Co‐Conformational Distribution of Nanosized [2]Catenanes Determined by Pulse EPR Measurements

The co‐conformational ensembles of three differently sized [2]catenanes were studied by measuring pair correlation functions corresponding to the separation of nitroxide spin labels—one attached to each of the two macrocycles—with the double electron–electron resonance (DEER) experiment. A geometric model for the [2]catenanes was derived that approximates the macrocycles by circles and takes into account the topological constraint. Comparison of the experimental to the theoretically predicted pair correlation functions gives insight into the co‐conformational distribution and the size of the macrocycles. It was found that the macrocycles of the medium‐ and large‐sized catenanes in chloroform are close to fully expanded, while they are partially collapsed in glassy o‐terphenyl. For the small‐sized catenane, moderate interaction between the unsaturated sections of the macrocycles in chloroform is indicated by a slight overrepresentation of short label‐to‐label separations in the pair correlation function.     

Radiochemical aging of ethylene–propylene–diene monomer elastomers. I. Mechanism of degradation under inert atmosphere

This article is devoted to the study of electron‐beam‐induced degradation under argon atmosphere of an ethylene–propylene–diene monomer (EPDM, based on 5‐ethylidene 2‐norbornene) and an ethylene–propylene rubber (EPR) containing the same molar ratio of ethylene/propylene. The chemical structure modifications of polymeric samples were analyzed by ultraviolet–visible and IR spectroscopies. Crosslinking reactions were deduced by measuring the changes in gel fraction and the degree of swelling in n‐heptane. Irradiation of EPDM and EPR created trans‐vinylene, vinyl, vinylidene, and dienic‐type unsaturations. The radiochemical yields for unsaturation formations in EPDM and EPR were similar. Degradation also involved crosslinking and the production of molecular hydrogen. The comparison between EPDM and EPR showed that the diene (in which a double bond is consumed with a high radiochemical yield) contributes to the increase in rate and intermolecular bridges density. Mechanisms are proposed to account for the main routes of EPDM degradation. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 1239–1248, 2004     

Electron paramagnetic resonance spin probe study of carbon dioxide‐induced polymer plasticization

Steady‐state electron paramagnetic resonance (EPR) spectroscopy using nitroxide spin probes has been used to investigate the plasticization of poly(vinyl acetate) and poly(ethyl methacrylate) by carbon dioxide. By varying the CO₂ pressure at constant ambient temperature, the glass transition for each polymer could be depressed to 25 °C. This effect has been quantified by a parameter P_50G, obtained by plotting the EPR spectral width as a function of CO₂ pressure. Certain spin probes showed free volume distribution effects, manifested in the EPR spectra as “double peaks.” Possible reasons for this phenomenon are presented and discussed, and the efficacy of CO₂ as a plasticizer is clearly demonstrated by direct comparison with di‐n‐butyl phthalate. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 2097–2108, 2005     

Multi‐frequency (S,X, Q and W‐band) EPR and ENDOR Study of Vanadium(IV) Incorporation in the Aluminium Metal–Organic Framework MIL‐53

Doping the well‐known metal–organic framework MIL‐53(Al) with vanadium(IV) ions leads to significant changes in the breathing behaviour and might have repercussions on the catalytic behaviour as well. To understand the properties of such a doped framework, it is necessary to determine where dopant ions are actually incorporated. Electron paramagnetic resonance (EPR) and electron–nuclear double resonance (ENDOR) are applied to reveal the nearest environment of the paramagnetic vanadium(IV) dopant ions. EPR spectra of as‐synthesised vanadium‐doped MIL‐53 are recorded at S‐, X‐, Q‐ and W‐band microwave frequencies. The EPR spectra suggest that at low dopant concentrations (1.0–2.6 mol %) the vanadium(IV) ions are well dispersed in the matrix. Varying the vanadium dopant concentration within this range or the dopant salt leads to the same dominant EPR component. In the ENDOR spectra, hyperfine (HF) interactions with ¹H, ²⁷Al and ⁵¹V nuclei are observed. The HF parameters extracted from simulations strongly suggest that the vanadium(IV) ions substitute Al in the framework.     

A Reactive,Rigid GdIII Labeling Tag for In‐Cell EPR Distance Measurements in Proteins

The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in‐cell distance measurements by the double electron–electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed Gd^III spin label, a thiol‐specific DOTA‐derivative (DO3MA‐3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in‐cell distance measurements by the DEER method carried out at W‐band frequencies. The high performance of DO3MA‐3BrPy‐Gd^III is demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High‐quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in‐cell protein concentrations as low as 5–10 μm .     

Selective High‐Resolution Detection of Membrane Protein–Ligand Interaction in Native Membranes Using Trityl–Nitroxide PELDOR

The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero‐oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro–electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter‐subunit interaction and the intra‐subunit conformational changes in hetero‐oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein–ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment.     

Tetrathiafulvalene‐Based Mixed‐Valence Acceptor–Donor–Acceptor Triads: A Joint Theoretical and Experimental Approach

This work presents a joint theoretical and experimental characterisation of the structural and electronic properties of two tetrathiafulvalene (TTF)‐based acceptor–donor–acceptor triads (BQ–TTF–BQ and BTCNQ–TTF—BTCNQ; BQ is naphthoquinone and BTCNQ is benzotetracyano‐p‐quinodimethane) in their neutral and reduced states. The study is performed with the use of electrochemical, electron paramagnetic resonance (EPR), and UV/Vis/NIR spectroelectrochemical techniques guided by quantum‐chemical calculations. Emphasis is placed on the mixed‐valence properties of both triads in their radical anion states. The electrochemical and EPR results reveal that both BQ–TTF–BQ and BTCNQ–TTF–BTCNQ triads in their radical anion states behave as class‐II mixed‐valence compounds with significant electronic communication between the acceptor moieties. Density functional theory calculations (BLYP35/cc‐pVTZ), taking into account the solvent effects, predict charge‐localised species (BQ ^{. ?}–TTF–BQ and BTCNQ ^{. ?}–TTF–BTCNQ) as the most stable structures for the radical anion states of both triads. A stronger localisation is found both experimentally and theoretically for the BTCNQ–TTF–BTCNQ anion, in accordance with the more electron‐withdrawing character of the BTCNQ acceptor. CASSCF/CASPT2 calculations suggest that the low‐energy, broad absorption bands observed experimentally for the BQ–TTF–BQ and BTCNQ–TTF–BTCNQ radical anions are associated with the intervalence charge transfer (IV‐CT) electronic transition and two nearby donor‐to‐acceptor CT excitations. The study highlights the molecular efficiency of the electron‐donor TTF unit as a molecular wire connecting two acceptor redox centres.     

Accurate Extraction of Nanometer Distances in Multimers by Pulse EPR

Pulse electron paramagnetic resonance (EPR) is gaining increasing importance in structural biology. The PELDOR (pulsed electron–electron double resonance) method allows extracting distance information on the nanometer scale. Here, we demonstrate the efficient extraction of distances from multimeric systems such as membrane‐embedded ion channels where data analysis is commonly hindered by multi‐spin effects.     

The Double‐Histidine Cu2+‐Binding Motif: A Highly Rigid,Site‐Specific Spin Probe for Electron Spin Resonance Distance Measurements

The development of ESR methods that measure long‐range distance distributions has advanced biophysical research. However, the spin labels commonly employed are highly flexible, which leads to ambiguity in relating ESR measurements to protein‐backbone structure. Herein we present the double‐histidine (dHis) Cu²⁺‐binding motif as a rigid spin probe for double electron–electron resonance (DEER) distance measurements. The spin label is assembled in situ from natural amino acid residues and a metal salt, requires no postexpression synthetic modification, and provides distance distributions that are dramatically narrower than those found with the commonly used protein spin label. Simple molecular modeling based on an X‐ray crystal structure of an unlabeled protein led to a predicted most probable distance within 0.5 Å of the experimental value. Cu²⁺ DEER with the dHis motif shows great promise for the resolution of precise, unambiguous distance constraints that relate directly to protein‐backbone structure and flexibility.     

Sub‐Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing CuII‐labelling of Double‐Histidine Motifs with Lower Temperature

Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints. Combining double‐histidine motifs with Cu^II spin labels can further increase the precision of distance measurements. It is also useful for proteins containing essential cysteines that can interfere with thiol‐specific labelling. However, the non‐covalent Cu^II coordination approach is vulnerable to low binding‐affinity. Herein, dissociation constants (K_D) are investigated directly from the modulation depths of relaxation‐induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low‐ to sub‐μm Cu^II K_Ds under EPR distance measurement conditions at cryogenic temperatures. We show the feasibility of exploiting the double‐histidine motif for EPR applications even at sub‐μm protein concentrations in orthogonally labelled Cu^II–nitroxide systems using a commercial Q‐band EPR instrument.     

Interaction between Molecular Oxygen and Nitroxide Radicals: A Search for a Reversible Complex

At high concentrations of oxygen, the EPR spectrum of the nitroxide radical 4‐oxo‐TEMPO (= 4‐oxo‐2,2,6,6‐tetramethylpiperidin‐1‐yloxy) is found to broaden significantly. In addition to the expected broadening, double integration of the EPR signals indicates that a significant fraction of the nitroxide spins has ‘disappeared’. In perfluoro(2‐butyltetrahydrofuran) at 273 K, the extent of diminution of the EPR signal intensity is ca. 20%. The results are analyzed in terms of collision and supramolecular complexes between oxygen and 4‐oxo‐TEMPO. It is concluded that a supramolecular complex is responsible for the observed phenomenon.     

W-Band pulse EPR distance measurements in peptides using Gd(3+)-dipicolinic acid derivatives as spin labels

We present high field DEER (double electron-electron resonance) distance measurements using Gd(3+) (S = 7/2) spin labels for probing peptides' conformations in solution. The motivation for using Gd(3+) spin labels as an alternative for the standard nitroxide spin labels is the sensitivity improvement they offer because of their very intense EPR signal at high magnetic fields. Gd(3+) was coordinated by dipicolinic acid derivative (4MMDPA) tags that were covalently attached to two cysteine thiol groups. Cysteines were introduced in positions 15 and 27 of the peptide melittin and then two types of spin labeled melittins were prepared, one labeled with two nitroxide spin labels and the other with two 4MMDPA-Gd(3+) labels. Both types were subjected to W-band (95 GHz, 3.5 T) DEER measurements. For the Gd(3+) labeled peptide we explored the effect of the solution molar ratio of Gd(3+) and the labeled peptide, the temperature, and the maximum dipolar evolution time T on the DEER modulation depth. We found that the optimization of the [Gd(3+)]/[Tag] ratio is crucial because excess Gd(3+) masked the DEER effect and too little Gd(3+) resulted in the formation of Gd(3+)-tag(2) complexes, generating peptide dimers. In addition, we observed that the DEER modulation depth is sensitive to spectral diffusion processes even at Gd(3+) concentrations as low as 0.2 mM and therefore experimental conditions should be chosen to minimize it as it decreases the DEER effect. Finally, the distance between the two Gd(3+) ions, 3.4 nm, was found to be longer by 1.2 nm than the distance between the two nitroxides. The origin and implications of this difference are discussed.     

Synthesis and Properties of Isoindoline Nitroxide‐containing Porphyrins

Isoindoline nitroxide‐containing porphyrins were synthesized by the reaction of 5‐phenyldipyrromethane and 5‐(4′‐nitrophenyl)‐dipyrromethane with 5‐formyl‐1,1,3,3‐tetramethylisoindolin‐2‐yloxyl using the Lindsey method. These spin‐labeled porphyrins were further characterized by MS, UV, FTIR, ¹H‐NMR, cyclic voltammetry, electron paramagnetic resonance (EPR), and fluorescence spectroscopy. The electrochemical assay demonstrated that these isoindoline nitroxides‐containing porphyrins had similar electrochemical and redox properties as 5‐carboxy‐1,1,3,3‐tetramethylisoindolin‐2‐yloxyl. Electron paramagnetic resonance test exhibited these porphyrins possessed the hyperfine splittings and characteristic spectra of isoindoline nitroxides, with typical nitroxide g‐values and nitrogen isotropic hyperfine coupling constants. Fluorescence spectroscopy revealed that these porphyrins indicated fluorescence suppression characteristic of nitroxide–fluorophore systems. Moreover, their reduced isoindoline nitroxide‐containing porphyrins eliminated the fluorescence suppression and displayed strong fluorescence. Thus, these isoindoline nitroxide‐containing porphyrins may be considered as the potential fluorescent and EPR probes.