Pulsed Electron-electron Double Resonance Research Articles

PELDOR study on the tyrosyl radicals in the R2 protein of mouse ribonucleotide reductase

Pulse electron-electron double resonance (PELDOR) has been employed to measure the distance between the putative tyrosyl radicals in the two halves of the R2 subunit from mouse ribonucleotide reductase. The results provide experimental evidence that the active, tyrosyl radical containing mouse R2 subunit forms a homodimeric form in solution. The distance between the two tyrosyl radicals present in the dimer was determined to be 3.25 +/- 0.05 nm.

Determination of the Distance between the Two Neutral Flavin Radicals in Augmenter of Liver Regeneration by Pulsed ELDOR

Pulsed electron-electron double resonance (ELDOR) has been used to obtain structural information from a FAD-dependent sulfhydryl oxidase, Augmenter of Liver Regeneration (ALR). ALR is a homodimer with each subunit containing a noncovalently bound FAD cofactor. Both FADs may be converted into the blue neutral radical form by aerobic treatment with DTT. From three-pulse and four-pulse ELDOR experiments, a distance of 26.1 +/- 0.8 A could be determined between the FAD cofactors in human ALR. Taking into account the electron spin density distribution in a neutral flavin radical obtained from density functional theory calculations, a distance of 26.9 A could be estimated for the separation of the spin centers in the X-ray structure of rat ALR. The good agreement confirms that rat ALR may be used as a model for mechanistic discussions of human ALR. The experiments also demonstrate that neutral flavin radicals have the appropriate properties to be used as intrinsic spin labels for distance determinations in proteins.

Membrane-peptide interaction studied by PELDOR and CW ESR: Peptide conformations and cholesterol effect on the spatial peptide distribution in the membrane

Pulsed electron-electron double resonance (PELDOR) combined with continuous-wave electron paramagnetic resonance was used to study inter- and intramolecular dipole-dipole interactions between spin labels for spin-labeled analogs of trichogin GA IV bound to multilamellar membranes of egg L-α-phosphatidylcholine (ePC) and in ePC membranes containing cholesterol. All samples were frozen to 77 K. For mono-labeled peptide concentrations in lipid over the range between 0.5 to 2.2 mol%, it is shown that in these membranes trichogin molecules are distributed homogeneously and are likely to be located on or near the inner and outer membrane surfaces. Addition of cholesterol to a final concentration of 16.5 mol% leads to an increase of the local concentration of trichogin molecules in the membranes. For the double-labeled trichogin, a distribution of the intramolecular distance between the two spin labels was observed. The distribution function is characterized by two main maxima located at distances of 1.3 and 1.8 nm. The distance of 1.3 nm is close to that expected for the α-helix structure of the peptide chain. The distance of 1.8 nm corresponds to a mixed structure in which a 310-helix is combined with a set of even more elongated conformations.

High-frequency 180 GHz PELDOR

For aromatic organic radicals, pulsed electron-electron double resonance (PELDOR) experiments at high magnetic fields offer the possibility to achieve orientation-selective pumping and detection that could allow one not only to determine the distance between paramagnetic species but also their relative orientation with respect to the interconnecting dipolar axis. We present a PELDOR two-frequency setup that was introduced into our homebuilt 180 GHz pulsed electron paramagnetic resonance (EPR) spectrometer and we discuss its technical and experimental features. The capability of 180 GHz PELDOR has been tested using the three-pulse ELDOR sequence on the protein RNR-R2 (ribonucleotide reductase) fromEscherichia coli, which contains two tyrosyl radicals at a distance of 3.3 nm. At 180 GHz, orientation selectivity is observed and the modulation frequency was found in good agreement with theoretical predictions, which take into account the relative orientation of the radicals from X-ray data.

Localization of the N-terminal Domain in Light-harvesting Chlorophyll a/b Protein by EPR Measurements

The conformational distribution of the N-terminal domain of the major light-harvesting chlorophyll a/b protein (LHCIIb) has been characterized by electron-electron double resonance yielding distances between spin labels placed in various domains of the protein. Distance distributions involving residue 3 near the N terminus turned out to be bimodal, revealing that this domain, which is involved in regulatory functions such as balancing the energy flow through photosystems (PS) I and II, exists in at least two conformational states. Models of the conformational sub-ensembles were generated on the basis of experimental distance restraints from measurements on LHCIIb monomers and then checked for consistency with the experimental distance distribution between residues 3 in trimers. Only models where residue 3 is located above the core of the protein and extends into the aqueous phase on the stromal side fit the trimer data. In the other state, which consequently is populated only in monomers, the N-terminal domain extends sideways from the protein core. The two conformational states may correspond to two functional states of LHCIIb, namely trimeric LHCIIb associated with PSII in stacked thylakoid membranes and presumably monomeric LHCIIb associated with PSI in nonstacked thylakoids. The switch between these two is known to be triggered by phosphorylation of Thr-6. A similar phosphorylation-induced conformational change of the N-terminal domain has been observed by others in bovine annexin IV which, due to the conformational switch, also loses its membrane-aggregating property.

Calcium Structural Transition of Human Cardiac Troponin C in Reconstituted Muscle Fibres as Studied by Site-directed Spin Labelling

The in situ structure of human cardiac troponin C (hcTnC) has been studied with site-directed, spin labelling, electron paramagnetic resonance (SDSL-EPR). Analysis of the in situ structures of hcTnC is essential for elucidating the molecular mechanism behind its Ca(2+)-sensitive regulation. We prepared two hcTnC mutants (C35S and C84S) containing one native cysteine residue (84 and 35, respectively) for spin labelling. The mutants were labelled with a methane thiosulfonate spin label (MTSSL) and the TnC was reconstituted into permeabilized muscle fibres. The mobility of Cys84-MTSSL changed markedly after addition of Ca2+, while that of the Cys35 residue did not change in the monomer state or in fibres. The rotational correlation time of Cys84-MTSSL decreased from 32ns to 13ns upon Ca(2+)-binding in the monomer state, whereas in fibres the spectrum of Cys84-MTSSL was resolved into mobile (16ns) and immobile (35ns) components and the addition of Ca2+ increased the immobile component. Moreover, the accessibility of Cys84-MTSSL to molecular oxygen increased slightly in the presence of Ca2+. These data suggest that Cys35 remains in the same location regardless of the addition of Ca2+, whereas Cys84 is located at the position that interacts with B and C helices of hcTnC and interacts with troponin I (TnI) at high concentrations of Ca2+. We determined the distances between Cys35 and Cys84 by measuring pulsed electron-electron double resonance spectra. The distances were 26.0 angstroms and 27.2 angstroms in the monomer state and in fibres, respectively, and the addition of Ca2+ decreased the distance to 23.2 angstroms in fibres but only slightly in the monomer state, showing that Ca2+ binding to the N-domain of hcTnC induced a larger structural change in muscle fibres than in the monomer state.

Structure and spatial distribution of the spin-labelled lipopeptide trichogin GA IV in a phospholipid membrane studied by pulsed electron–electron double resonance (PELDOR)

The method of pulsed electron-electron double resonance (PELDOR) is exploited to study intra- and intermolecular dipole-dipole interactions between the spin labels of trichogin GA IV analogues. This lipopeptaibol antibiotic was studied in multilamellar membranes of dipalmitoylphosphatidylcholine frozen to 77 K. For mono-labelled trichogin analogues, the molecules are shown not to form aggregates in the lipid membranes studied. For the double-labelled trichogin analogues, a function of the distance distribution between the spin labels has been obtained. We determined that the distribution function has two main maxima located at distances of 1.25 nm and 1.75 nm. The value of 1.25 nm is close to the distance between labels of a alpha-helical structure. On the other hand, a distance of 1.75 nm corresponds to a mixed 3D-structure in which a 3(10)-helix is combined with a more elongated conformation.

Pulsed 180-GHz EPR/ENDOR/PELDOR spectroscopy

Within this review, we describe a home-built pulsed electron paramagnetic resonance (EPR) spectrometer operating at 180 GHz as well as the incorporation of two double resonance techniques, electron nuclear double resonance (ENDOR) and pulsed electron double resonance (PELDOR), along with first applications. Hahn-echo decays on a TEMPO/polystyrene sample are presented, demonstrating that the observation of anisotropic librational motions is possible in a very precise manner at high magnetic fields. Bisdiphenylene-phenyl-allyl is used as a model system to illustrate the performance of the setup for 1H-ENDOR using the Mims as well as the Davies sequence. Furthermore, first 1H-Mims and Davies ENDOR spectra on a biological sample, the wild-type Ras*Mn2+*GDP protein, are reported. The capability of the 180-GHz PELDOR setup is demonstrated using the three-pulse ELDOR sequence on the protein ribonucleotide reductase (RNR) subunit R2 from Escherichia coli, which contains two tyrosyl radicals at a 33 angstroms distance. At 180 GHz, orientation selectivity is observed and the modulation frequency is found to be in good agreement with theoretical predictions.

The effect of microwave pulse duration on the distance distribution function between spin labels obtained by PELDOR data analysis

A method is proposed for obtaining a distance distribution function between spin labels in spin-labeled molecules on the basis of the data of pulsed electron-electron double resonance with regard to the finite duration of microwave (mw) pulses. It is shown that taking into account the finite duration of mw pulses makes it possible to extend the range of the studied distances between spin labels to the region of short distance in which the magnitude of the dipole-dipole interaction becomes comparable to or exceeds the spectrum widths of exciting mw pulses. With frozen glassy solutions of biradicals as model systems with a length between spins of less than 2 nm, the shape for the distance distribution function between labels was obtained, and the value and sign of the exchange interaction were estimated. It is demonstrated that the analysis of the dipole-dipole interaction neglecting the duration of mw pulses can lead to substantial distortions in the shape of the distance distribution function.

Visualization of distance distribution from pulsed double electron-electron resonance data

Double electron-electron resonance (DEER), also known as pulsed electron-electron double resonance (PELDOR), is a time-domain electron paramagnetic resonance method that can measure the weak dipole-dipole interactions between unpaired electrons. DEER has been applied to discrete pairs of free radicals in biological macromolecules and to clusters containing small numbers of free radicals in polymers and irradiated materials. The goal of such work is to determine the distance or distribution of distances between radicals, which is an underdetermined problem. That is, the spectrum of dipolar interactions can be readily calculated for any distribution of free radicals, but there are many, quite different distributions of radicals that could produce the same experimental dipolar spectrum. This paper describes two methods that are useful for approximating the distance distributions for the large subset of cases in which the mutual orientations of the free radicals are uncorrelated and the width of the distribution is more than a few percent of its mean. The first method relies on a coordinate transformation and is parameter-free, while the second is based on iterative least-squares with Tikhonov regularization. Both methods are useful in DEER studies of spin-labeled biomolecules containing more than two labels.

Solvent effect on the distance distribution between spin labels in aggregated spin labeled trichogin GA IV dimer peptides as studied by pulsed electron–electron double resonance

Pulsed electron–electron double resonance (PELDOR) was used to study aggregate formation in frozen glassy solutions of mono- and double-spin labeled trichogin GA IV dimers in a toluene–methanol mixture. The modified method proposed and used for the distance distribution function calculation from the PELDOR data. Distance distribution functions between spin labels in the peptide molecules and their aggregates in solution were determined as a function of solvent composition. Double-labeled peptide molecules in aggregates in solutions with low methanol content display two types of structures, i.e. the α-helix with a 2.8 nm distance between labels and the 310-helix with a 3.2 nm distance between labels. As the methanol content of the solvent increases, a part of conformations at 3.2 nm changes. An increase of the methanol content leads to disruption of the aggregates and a change to the peptide conformation as well. In pure methanol peptides fail to form aggregates and a wide distribution of distances between labels centered at 3 nm were observed.

Aggregation of Spin Labeled Trichogin GA IV Dimers: Distance Distribution between Spin Labels in Frozen Solutions by PELDOR Data

Pulsed electron−electron double-resonance (PELDOR) and CW-ESR spectroscopies were used to study the dipole−dipole interaction of spin labels in frozen glassy solutions of mono- and double-labeled analogues of the 22-residue, the head-to-tail covalent dimer of the peptaibol antibiotic trichogin GA IV. The [TOAC-1] and [TOAC-1,19] dimers were studied in methanol and in a chloroform−toluene mixture. It was shown these dimer molecules do not form aggregates in methanol. However, in chloroform−toluene, the PELDOR signal of the mono-labeled dimer shows oscillations, due to the dipole−dipole interaction of the nitroxide spin labels. From the signal decay, the number of molecules per aggregate was estimated to be ∼2−3, depending on the concentration used. The contributions of inter- and intra-aggregate interactions of spin labels to the PELDOR signal decay were separated. The distance distribution functions of the intra-aggregate interactions range from 3.0 to 4.5 nm. The maxima are observed at 3.4 nm. From the P...

Structural analysis of three-spin systems of photosystem II by PELDOR

The pulsed electron electron double resonance (PELDOR) pulse sequence is applied to a three-spin system consisting of three radicals (YD·, YZ· and QA−) generated in spinach PS II. The distance between YZ and QA has been determined to be 3.4 nm with the previously derived distances of the other radical pairs, 2.9 nm for YD·-YZ· and 3.9 nm for YD·-QA−. This distance has been derived from the YZ·-QA− radical pair trapped in YD-less mutants ofChlamydomonas reinhardtii. Furthermore the method was applied to the YD·-QA−-ChlZ+ system to find the unknown distance between QA and ChlZ. The derived distance was 3.4 nm. A triangular configuration was found in the membrane system that gives the relative positions of the electron transfer components.

Applications of pulsed ELDOR-detected NMR measurements to studies of photosystem II: Magnetic characterization of YD tyrosine radical and Mn2+ bound to the high-affinity site

The pulsed electron-electron double resonance (ELDOR)-detected nuclear magnetic resonance (NMR) technique has been applied to the investigation of the hyperfine structure of the oxidized radical of YD tyrosine (Y D · ) and Mn2+ ion bound to the high-affinity site in photosystem II. The resulting ELDOR spectrum of Y D · was found to correspond with the pulsed electron-nuclear double resonance (ENDOR) spectrum except for a slightly larger linewidth (by a factor of 1.7). The spectrum showed marked anisotropy and revealed three peaks which can be assigned to matrix protons, C-3 and −5 protons and one of the β-protons in the tyrosine molecule. The results demonstrate that the pulsed ELDOR-detected NMR technique is applicable to the study of organic radicals in biological systems. The Mn2+ ion bound to the high-affinity site in photosystem II yielded well-resolved ELDOR signals spreading over ±1000 MHz. The magnetic properties of the Mn2+ were characterized on the basis of the calculation of the ENDOR transitions and the anisotropy of the ELDOR spectrum.

Nanometer distance measurements on RNA using PELDOR.

A rapid increase in RNA structural studies has been seen in the past few years, in part due to the interest in the multitude of cellular functions performed by RNA. Electron paramagnetic resonance spectroscopy is a useful technique for studying biopolymer structure under physiologically relevant conditions. We present herein the use of pulsed electron double resonance for the determination of a 35 +/- 2 A distance between two spin label nitroxides that were linked to specific positions within an RNA.

Electron Spin-Lattice Relaxation Processes of Radicals in Irradiated Crystalline Organic Compounds

Electron spin-lattice relaxation times (T1e) for the major radicals in γ-irradiated polycrystalline samples of glycylglycine, l-alanine, 2,4,6-tri-tert-butyl-phenol, and 4-methyl-2,6-di-tert-butyl-phenol were measured as a function of temperature using pulsed EPR. CW-saturation recovery (CW-SR) were obtained at X-band (9.1 GHz) and S-band (3.0 GHz) between about 10 and 295 K. Inversion recovery, echo-detected saturation recovery (ED-SR), and pulsed electron−electron double resonance (ELDOR) curves were obtained at X-band between 77 and about 295 K. For 2,4,6-tri-tert-butyl-phenoxy radical, which has a single-line EPR spectrum, the recovery times obtained by the three methods were in good agreement and were assigned as T1e. For the three radicals with resolved hyperfine splitting, spectral diffusion caused the recovery times observed by inversion recovery or ED-SR to be significantly shorter than T1e obtained by CW-SR or ELDOR. Spectral diffusion processes were observed directly by pulsed ELDOR experiments, and time constants for cross relaxation and nuclear relaxation were obtained by modeling the ELDOR curves. For irradiated l-alanine and for the 4-methyl-2,6-tert-butyl-phenoxy radical at some temperatures, the effects of rapid cross relaxation on CW-SR curves could not be fully mitigated even by long saturating pulses, and T1e could only be determined by ELDOR. For the radicals in γ-irradiated l-alanine, 2,4,6-tri-tert-butyl-phenol, and 4-methyl-2,6-di-tert-butyl-phenol, methyl group rotation makes significant contributions to T1e at temperatures where the rate of rotation of a methyl group is comparable to the microwave frequency. Activation energies for methyl rotation were determined by modeling the temperature dependence of T1e at X-band and S-band. In temperature ranges where methyl rotation did not dominate, T1e was dominated by Raman, direct, or local mode processes.

Electron spin–lattice relaxation in radicals containing two methyl groups, generated by γ-irradiation of polycrystalline solids

The effects of methyl rotation on electron spin–lattice relaxation times were examined by pulsed electron paramagnetic resonance for the major radicals in γ-irradiated polycrystalline α-amino isobutyric acid, dimethyl-malonic acid, and l-valine. The dominant radical is the same in irradiated dimethyl-malonic acid and α-amino isobutyric acid. Continuous wave saturation recovery was measured between 10 and 295 K at S-band and X-band. Inversion recovery, echo-detected saturation recovery, and pulsed electron–electron double resonance (ELDOR) data were obtained between 77 and 295 K. For the radicals in the three solids, recovery time constants measured by the various techniques were not the same, because spectral diffusion processes contribute differently for each measurement. Hyperfine splitting due to the protons of two methyl groups is resolved in the EPR spectra for each of the samples. Pulsed ELDOR data were obtained to characterize the spectral diffusion processes that transfer magnetization between hyperfine lines. Time constants were obtained for electron spin–lattice relaxation ( T 1e), nuclear spin relaxation ( T 1n), cross-relaxation ( T x1), and spin diffusion ( T s). Between 77 and 295 K rapid cross-relaxation (Δ M s=±1, Δ M I=∓1) was observed for each sample, which is attributed to methyl rotation at a rate that is approximately equal to the electron Larmor frequency. The large temperature range over which cross-relaxation was observed suggests that methyl groups in the radical and in the lattice, with different activation energies for rotation, contribute to the rapid cross-relaxation. Activation energies for methyl and amino group rotation between 160 and 1900 K (1.3–16 kJ/mol) were obtained by analysis of the temperature dependence of 1/ T 1e at S-band and X-band in the temperature intervals where the dynamic process dominates T 1e.

Pulsed electron-electron double resonance on multinuclear metal clusters: assignment of spin projection factors based on the dipolar interaction.

The interaction between two paramagnetic metal centers, a [3Fe-4S](+) cluster and a [NiFe] center, is investigated in the hydrogenase from Desulfovibrio vulgaris Miyazaki F by pulsed ELDOR (electron-electron double resonance). The distance between the metal centers is known from X-ray crystallography. The experimental dipolar spin-spin interaction deviates from the value expected for two point-dipoles located at the centers of the metal clusters. An extended spin-coupling model accounting for the spin coupling in the [3Fe-4S](+) cluster yields the observed interaction under the assumption of a particular magnetic coupling scheme for the three Fe ions. These results demonstrate that pulsed ELDOR can be used to gain insight into the inner structure of a multinuclear metal cluster.

Submicrosecond field-jump device for pulsed high-field ELDOR

A field-jump device for fast stepping the electron paramagnetic resonance magnetic field around 3.4 T during pulsed electron-electron double resonance experiments at W-band (95 GHz) is described. Field jumps up to ±160 G and submicrosecond times for the full field-jump cycle allow precession frequency transfer experiments to be made for the determination of the nanometer distance and the orientation of nitroxide spin-label pairs in disordered samples.

PELDOR at S- and X-Band Frequencies and the Separation of Exchange Coupling from Dipolar Coupling

A pulsed electron double resonance (PELDOR) setup working at S-band frequencies is introduced and its performance compared with an X-band setup. Furthermore, to verify experimentally that it is possible to disentangle the dipolar coupling ν Dip from the exchange coupling J by PELDOR we synthesized and investigated four bisnitroxide radicals. They exhibit in pairs the same distances r AB between the nitroxide moieties but only one of each pair possesses a non-zero J. The experimental values for r AB match the ones from molecular modeling very well for the molecules without exchange coupling. For one bisnitroxide it was possible to separate ν Dip from J and to ascertain the magnitude and sign of J to +11 MHz (antiferromagnetic spin–spin coupling).