Pulsed Electron Paramagnetic Resonance Measurements Research Articles

Nanometer-Scale Distance Measurements in Proteins Using Gd3+ Spin Labeling

Methods for measuring nanometer-scale distances between specific sites in proteins are essential for analysis of their structure and function. In this work we introduce Gd(3+) spin labeling for nanometer-range distance measurements in proteins by high-field pulse electron paramagnetic resonance (EPR). To evaluate the performance of such measurements, we carried out four-pulse double-electron electron resonance (DEER) measurements on two proteins, p75ICD and tau(C)14, labeled at strategically selected sites with either two nitroxides or two Gd(3+) spin labels. In analogy to conventional site-directed spin labeling using nitroxides, Gd(3+) tags that are derivatives of dipicolinic acid were covalently attached to cysteine thiol groups. Measurements were carried out on X-band (approximately 9.5 GHz, 0.35 T) and W-band (95 GHz, 3.5 T) spectrometers for the nitroxide-labeled proteins and at W-band for the Gd(3+)-labeled proteins. In the protein p75ICD, the orientations of the two nitroxides were found to be practically uncorrelated, and therefore the distance distribution could as readily be obtained at W-band as at X-band. The measured Gd(3+)-Gd(3+) distance distribution had a maximum at 2.9 nm, as compared to 2.5 nm for the nitroxides. In the protein tau(C)14, however, the orientations of the nitroxides were correlated, and the W-band measurements exhibited strong orientation selection that prevented a straightforward extraction of the distance distribution. The X-band measurements gave a nitroxide-nitroxide distance distribution with a maximum at 2.5 nm, and the W-band measurements gave a Gd(3+)-Gd(3+) distance distribution with a maximum at 3.4 nm. The Gd(3+)-Gd(3+) distance distributions obtained are in good agreement with expectations from structural models that take into account the flexibility of the tags and their tethers to the cysteine residues. These results show that Gd(3+) labeling is a viable technique for distance measurements at high fields that features an order of magnitude sensitivity improvement, in terms of protein quantity, over X-band pulse EPR measurements using nitroxide spin labels. Its advantage over W-band distance measurements using nitroxides stems from an intrinsic absence of orientation selection.

Analysis of the Spatial Distribution of Free Radicals in Ammonium Tartrate by Pulse EPR Techniques

Using pulse electron paramagnetic resonance (EPR) on a series of l(+)-ammonium tartrate (AT) dosimeters exposed to radiations with different linear energy transfer (LET), we assessed the ability of pulse EPR spectroscopy to discriminate the quality of various radiation beams such as (60)Co gamma-ray photons, protons and thermal neutrons at various doses by analyzing the local radical distributions produced by the different beams. We performed two types of pulse EPR investigations: two-pulse electron spin echo decay obtained by varying the microwave power, and a double electron-electron resonance (DEER) study. Both methods provide information about the dipolar interactions among the free radicals and about their spatial distributions. The first method provided information on the instantaneous diffusion and hence the microscopic concentration of the radicals that is compared with the macroscopic one obtained by CW-EPR. The DEER spectra yielded the distributions of distances between pairs of radicals two to five crystal cells apart produced by the same radiation event, a result reported here for the first time. The inter-radical distributions given by the DEER results have been simulated by modeling the radical distributions according to the details of the matter-radiation interactions for the various beams. The results of both types of pulse experiments are strongly dependent on the radiation quality. This was also observed for samples giving indistinguishable CW-EPR spectral profiles. We conclude that the pulse EPR measurements can be valuable tools for distinguishing the LET of the radiation beams, an important parameter for radiobiological considerations.

Electron spin relaxation and quantum localization in carbon nanoparticle: Electron spin echo studies

We present pulsed electron spin resonance measurements of activated carbon fibers (ACFs) for pristine samples and after adsorption of nitrobenzene in the fibers' porous structure. Two-level tunneling state (TLS) model is discussed in order to define the nature of paramagnetic centers and to explain the electron spin-lattice relaxation mechanism in the studied system. Electron spin echo decay is dominated by spin diffusion at low temperatures (below $7\phantom{\rule{0.3em}{0ex}}\mathrm{K}$) and for higher temperatures the echo dephasing rate is governed by thermal excitations of the TLS model. The model enables us to treat the nanographitic units being the basic structural units of ACF, as quantum wells (dots) separated by potential barriers of the order of $25(2)\phantom{\rule{0.3em}{0ex}}{\mathrm{cm}}^{\ensuremath{-}1}$ determined from temperature dependence of the spin-lattice relaxation rate. Stimulation of these barriers leads to spin localization within nanographitic units. A possible control of the potential barriers is the crucial point for the problem of nanographitic unit system treated as a quantum dot matrix.

Electron spin coherence in Si

We discuss pulsed electron spin resonance measurements of electrons in Si and determine the spin coherence from the decay of the spin echo signals. Tightly bound donor electrons in isotopically enriched 28Si are found to have exceptionally long spin coherence. Placing the donors near a surface or interface is found to decrease the spin coherence time, but it is still in the range of milliseconds. Unbound two-dimensional electrons have shorter coherence times of a few microseconds, though still long compared to the Zeeman frequency or the typical time to manipulate a spin with microwave pulses. Longer spin coherence is expected in two-dimensional systems patterned into quantum dots, but relatively small dots will be required. Data from dots with a lithographic size of 400 nm do not yet show longer spin coherence.

Triradical Cation ofp-Phenylenediamine Having Two Nitroxide Radical Groups: Spin Alignment Mediated by Delocalized Spin

The cationic state of a p-phenylenediamine (PDA) molecule having two nitroxide radical groups was prepared and characterized using electrochemical, electron spin resonance (ESR) spectroscopic, and absorption spectroscopic methods. The delocalized intervalence state of the p-phenylendiamine (PDA) moiety was detected in the cationic state. From the pulsed ESR measurements, it was confirmed that the delocalized spin induces parallel spin alignment between the localized two nitroxide groups which are magnetically weakly coupled in the neutral state. It was found that the resulting high-spin alignment does not seriously affect the delocalized intervalence state of the PDA radical cation.

N@C60 and P@C60 as quantum bits

Due to their long electron spin relaxation times, the endohedral fullerenes N@C60 and P@C60 are good candidates for the implementation of qubits in an electron spin quantum computer. A central operation in this context is the rotation of the spin direction by an arbitrary angle. In the present experiment, this nutation behavior was studied in pulsed electron spin resonance measurements. We show that, even at room temperature, about 50 Rabi oscillations (about 100 qubit operations) can be performed without refocusing the spin system, although inhomogeneities are present. A special feature of the group V endohedral fullerenes is the electron spinS=3/2, which complicates the nutation behavior. The zero-field splitting at low temperature gives rise to different nutation frequencies for the (1/2,−1/2) transition and the (±3/2, ±1/2) transitions. The frequency ratio is 2/31/2.

Electron spin relaxation times of phosphorus donors in silicon

Pulsed electron paramagnetic resonance measurements of donor electron spins in natural phosphorus-doped silicon (Si:P) and isotopically-purified 28Si:P show a strongly temperature-dependent longitudinal relaxation time, T1, due to an Orbach process with DeltaE = 126 K. The 2-pulse echo decay is exponential in 28Si:P, with the transverse relaxation (decoherence) time, T2, controlled by the Orbach process above ~12 K and by instantaneous diffusion at lower temperatures. Spin echo experiments with varying pulse turning angles show that the intrinsic T2 of an isolated spin in 28Si:P is ~60 ms at 7 K.

Molecular reorientation in pyrene hexafluoroarsenate salts

The quasi-one dimensional pyrene (PY) organic conductor (PY)7(PY)4(AsF6)4 . 4CH2Cl2shows parallel as well as 60° rotated PY radical cations in its stacks, but crystallizes in two different modifications I and II. One of the seven intra-stack pyrene molecules is susceptible to a reorientation by 60°, that is stable already at room temperature for modification II, but occurs at a hysteretic first order transition between 170 K and 240 K for modification I. Crystal structure, microwave conductivity and static magnetic susceptibility are typical for a quasi-one dimensional organic conductor with Peierls transition at TP = 73 K (mod. I) or TP = 105 K (mod. II). The pyrene radical packing is analysed by continuous wave and pulsed electron spin resonance measurements, using 9.45 GHz as well as 425 MHz as measuring frequency. Anisotropy of the conduction electron diffusion constant exceeds 1000 in the metallic phase.

Electron spin relaxation in solids-an application to spontaneously occurring radical centre in phenol-formaldehyde resins

An outline of electron spin-relaxation is briefly presented with mechanisms and processes of spin-lattice relaxation in ionic and amorphous solids. Electron spin resonance (EPR) and pulsed EPR measurements were performed in the temperature range 4-300 K on a radical occurring in a dried resol phenol-formaldehyde resin. The radical appears in a small concentration of about 1/spl times/10/sup 16/ radicals/g in 30/spl deg/C dried samples and the number of the radicals increases continuously with time up to 2/spl times/10/sup 17/ radicals/g after 100 days. Computer simulations of the EPR spectrum show that breaking of the dimethylene ether linkage probably forms the radical during synthesis reactions. Electron spin-lattice relaxation rate increases relatively slowly on heating and at <80 K is determined by interactions with two-level tunnelling systems in the polymer structure. For higher temperatures the relaxation is governed by interactions with centres having energy levels split by 298 cm/sup -1/. Phase relaxation is described by the phase memory time with rigid lattice limit T/sub M//sup 0/=2 /spl mu/s. This time is shortened above 100 K by thermally activated radical molecule motions with activation energy 85 cm/sup -1/. The electron spin echo envelope modulation (ESEEM) spectrum shows unresolved peaks at /sup 1/H indicating strong molecular dynamics and delocalisation of the unpaired electron.

New materials for ESR dosimetry

New materials for electron spin resonance (ESR) dosimetry have been investigated with the aim to find systems more sensitive than l-alanine accepted as a standard for high dose determinations. Among the investigated systems ammonium tartrate, 2-methylalanine, salts of formic acids and dithionates have been found to be more sensitive than alanine by a factor 2–10. The lower limit applies to tissue equivalent materials, while much higher sensitivities were obtained with formates and dithionates containing heavier atoms. The increased sensitivity was mainly attributed to suitable ESR properties of the room temperature stable radicals as regards spectral shape (narrow lines, little or no hyperfine structure) and microwave saturation properties (short relaxation times). The radical structures have when necessary been clarified by ENDOR spectroscopy, while the saturation properties have been screened by pulsed ESR measurements.

Continuous wave and pulsed EPR measurements of chromium-doped fluoro-chlorozirconate and fluoro-bromozirconate glasses

A continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) investigation of chromium-doped fluoro-chlorozirconate and fluoro-bromozirconate glasses with different contents of the heaviest halogens is presented. The CW EPR spectra are interpreted by assuming a preferential substitution of the fluoride ions in the first co-ordination shell of the ions by chloride or bromide ions, which is in agreement with previous optical data. Values for the spin Hamiltonian parameters are discussed for different amounts of halide substitution. Pulsed EPR studies are mainly focused on the ESEEM (electron spin echo envelope modulation) signal. It shows the frequency peaks due to fluoride ions and to the corresponding alkali metal cation (Na or Li), which provides information about the bulk structure of the glass and strongly suggests that it is essentially the same as that of the fluorozirconate glasses. ESEEM results indicate that centres are mainly isolated.

Design of a multicoupled loop-gap resonator used for pulsed electron paramagnetic resonance measurements

The purpose of the present paper is to establish a method of design for a multicoupled loop-gap resonator used for pulsed electron paramagnetic resonance measurements. For the design of resonator characteristics, the method has an advantage of a systematic approach without iterative calculations. In this design, the number of loop-gap resonators used is first determined from the pass band required as the specifications of the resonator. To satisfy the specifications, electrical parameters of an equivalent circuit and the dimensions of the resonator are estimated. By the proposed method, a prototype resonator which has the operation frequency of 1.3 GHz is designed and fabricated. For the prototype resonator, the characteristics of the return loss agree with the required ones. As a result, the validity of the design method is experimentally confirmed.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>

Applications of pulsed EPR spectroscopy to the study of mitochondrial iron-sulphur clusters and Escherichia coli fumarate reductase

Pulsed methods of electron paramagnetic resonance (EPR) spectroscopy open up new possibilities for studying the structure and properties of paramagnetic centres in biological systems 11 I . The electron spin-echo envelope modulation (ESEEM) technique offers improved resolution of hyperfine interactions between an EPR-detectable species and nuclei of ligand atoms. I t has been shown to be particularly sensitive to weak interactions with quadrupolar nuclei, and is therefore suited for the detection of nuclei, such as 14N, in the vicinity of a paramagnetic centre 121. The spin-echo method allows the accurate determination of time-domain properties, such as the hptn-lattice (T,) and the spin-spin (Tz) relaxation times. Spinspin interactions between the various mitochondrial centres may be observed, and the distances between the centres can then, in principle, be determined. These pulsed methods have so far mainly been applied to the study of transition metal complexes and purified metalloproteins, such as ferredoxins. Our experiments were undertaken to assess the feasibility of pulsed EPR measurements on the more complex proteins of unpurified electron-transferring membranes. The spectrometer used was a Bruker ESP380, fitted with a liquid helium flow cryostat (Oxford Instruments, CS935). Preliminary experiments were done on the, previously studied [3], soluble, 2-subunit form of fumarate reductase (FRD) from Escherichia coli , a membrane protein analogous to the mitochondrial succinate dehydrogenase (SDH). In mitochondria the major EPR-detectable species at low temperatures are the iron-sulphur clusters 141 o f Complex I (Centres Nl-N4), Complex I1 (Centres S 1 S 3 ) and Complex 111 (the Rieske iron-sulphur protein). Submitochondrial particles (SMP) were prepared by sonication of bovine heart mitochondria. The high protein concentration required for pulsed measurements was achieved by centrifuging the SMP suspension inside quartz EPR tubes. Two samples were prepared, and NADH was then added to one tube. Continuous-wave (c.w) EPR spectra were recorded at ISK, with a Bruker ESP300 spectrometer, and verified that each tube contained the expected spectral species; that is, the sample as prepared (oxidized) gave a signal at g=2.01, typical of SDH (a subunit of Complex 11) centre S3, and the NADHreduced sample gave rise to various signals typical of centres N 1 , N2, N3 and N4 of Complex 1. Note that centres S1 and S2 of SDH are probably superimposed on the g=1.94 signal from centre N 1. ESEEM data were collected at 4K (by the Bruker Pulsespel