High-field EPR-detected shifts of magnetic tensor components of spin label side chains reveal protein conformational changes: The proton entrance channel of bacteriorhodopsin

Continuous-wave high-field electron paramagnetic resonance (95 GHz, 3.4 T) is performed on a spin label side chain located at residue position 171 in the proton entrance channel of bacterior-hodopsin The conformational differences of three bacteriorhodopsin mutants, the single mutant F171C, the double mutant D96G/F171C, and the triple mutant D96G/F171C/F219L, are reflected in different g_xx and A_zz tensor component shifts of the nitroxide side chain. The most polar microenvironment is found in the single mutant, whereas the open proton entrance channel reported for the triple mutant allows a reorientation of the nitroxide group towards a microenvironment of lower polarity and/or reduced hydrogen bonding. The experimental data of the double mutant are explained by a light-independent equilibrium of two nitroxide orientations with different polarities of the local microenvironment. Upon illumination the spectrum of the single mutant revealsg _xx andA _zz tensor component shifts which resemble those determined for the triple mutant in the dark. This result provides strong evidence for a light-induced opening of the proton entrance channel of the single mutant similar to that found in the unilluminated triple mutant, in agreement with electron diffraction data.     

Angle-selective measurements of spin soliton in ladder polydiacetylene by pulsed 94 GHz EPR

Angle-selection experiments of a spin soliton in randomly oriented ladder polydiacetylene were carried out by pulsed electron paramagnetic resonance (EPR) at W-band. EPR measurement using 94 GHz microwaves increased the difference in the resonance field due tog anisotropy of the spin soliton to allow the orientation dependence of transient nutation, electron nuclear double resonance (ENDOR) and spin relaxations to be investigated. The shape of theg anisotropy-resolved nutation spectrum was discussed on the basis of the EPR transition moments and the differences between spin relaxation times. Reliable assignments of hyperfine couplings to the β protons (H_β) of the alkyl side chains were achieved with the support of W-band ENDOR measurements. No significant orientational dependence in theT ₁ andT ₂ processes was found in terms of isotropy of the H_β-hyperfine interaction.     

Comparative analysis of sensory rhodopsin II structures in complex with a transducer and without it

Sensory rhodopsin II (NpSRII) is a heptahelical transmembrane protein containing the retinal chromophore. In complex with another membrane protein, i.e., NpHtrII transducer, NpSRII transmits a signal into a cell, initiating negative phototaxis of Natronobacterium pharaonis. In the absence of a transducer, rhodopsin II can work as a proton pump similar to bacteriorhodopsin. The main objective of this study is to comparatively analyze receptor structures in complex with the transducer and without it to understand the mechanism of the protein switching function. Diffraction data for sensory rhodopsin II crystals grown in the lipidic cubic phase were obtained at the synchrotron x-ray source. In this paper, we present a new NpSRII structure with a resolution of 2.1 Å and the results of a comparative analysis of the obtained NpSRII structure with our previously published data on the NpSRII/NpHtrII complex structure and with two NpSRII structures without a transducer, previously published in the literature.     

Resolving domains of interdigitated phospholipid membranes with 95 GHz spin labeling EPR

High-field W-band (95 GHz) electron paramagnetic resonance (EPR) study of partitioning of a small nitroxide TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) in multilamellar liposomes composed from 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) is described. The high-resolution spectra with a high signal-to-noise ratio were combined with automated least-squares simulation analysis to derive accurate partitioning coefficients of TEMPO in the membrane lipid phase and to follow the membrane phase transitions. The isotropic magnetic parameters, g_iso and A_iso were used to characterize the average polarity the spin label is experiencing in the membrane. We also report an empirical correlation between g_iso and A_iso for a set of protic and aprotic solvents and use this correlation to assign domains formed by interdigitation of DPPC bilayer under a high ethanol concentration of 1.2 M.     

High-field (275 GHz) spin-label EPR for high-resolution polarity determination in proteins

The polarity of protein surfaces is one of the factors driving protein-protein interactions. High-field, spin-label EPR at 95 GHz, i.e., 10 times higher than conventional EPR, is an upcoming technique to determine polarity parameters of the inside of proteins. Here we show that by 275 GHz EPR even the small polarity differences of sites at the protein surface can be discriminated. To do so, four single cysteine mutations were introduced at surface sites (positions 12, 27, 42, and 118) of azurin and spin labeled. By 275 GHz EPR in frozen solution, polarity/proticity differences between all four sites can be resolved, which is impossible by 95 GHz EPR. In addition, by 275 GHz EPR, two spectral components are observed for all mutants. The difference between them corresponds to one additional hydrogen bond.     

Molecular orbital study of polarity and hydrogen bonding effects on the g and hyperfine tensors of site directed NO spin labelled bacteriorhodopsin

Semiempirical molecular orbital methods (PM3, INDO, ZINDO/S) have been used to calculate the effects of local electric fields and of hydrogen bonding on the g and hyperfine tensors of a nitroxide spin label model system. The results yield a linear correlation between the two principal tensor components g _xx and A ^N _zz at label sites of varying polarity. Hydrogen bonding with a single water molecule produces a constant shift of Δg _xx ? ?4 × 10^?4. These theoretical results are used to interpret recent high field (3.4 T, 95 GHz) electron paramagnetic resonance investigations on site-directed spin labelled bacteriorhodopsin. This protein reveals a close correlation between proticity and polarity at the various label sites. The slope of the g _xx versus A ^N _zz dependence is affected strongly by polarity induced structural strains of the spin label.     

Saturation recovery EPR and ELDOR at W-band for spin labels

A reference arm W-band (94 GHz) microwave bridge with two sample-irradiation arms for saturation recovery (SR) EPR and ELDOR experiments is described. Frequencies in each arm are derived from 2 GHz synthesizers that have a common time-base and are translated to 94 GHz in steps of 33 and 59 GHz. Intended applications are to nitroxide radical spin labels and spin probes in the liquid phase. An enabling technology is the use of a W-band loop-gap resonator (LGR) [J.W. Sidabras, R.R. Mett, W. Froncisz, T.G. Camenisch, J.R. Anderson, J.S. Hyde, Multipurpose EPR loop-gap resonator and cylindrical TE₀₁₁ cavity for aqueous samples at 94 GHz, Rev. Sci. Instrum. 78 (2007) 034701]. The high efficiency parameter (8.2 GW^−1/2 with sample) permits the saturating pump pulse level to be just 5 mW or less. Applications of SR EPR and ELDOR to the hydrophilic spin labels 3-carbamoyl-2,2,5,5-tetra-methyl-3-pyrroline-1-yloxyl (CTPO) and 2,2,6,6,-tetramethyl-4-piperidone-1-oxyl (TEMPONE) are described in detail. In the SR ELDOR experiment, nitrogen nuclear relaxation as well as Heisenberg exchange transfer saturation from pumped to observed hyperfine transitions. SR ELDOR was found to be an essential method for measurements of saturation transfer rates for small molecules such as TEMPONE. Free induction decay (FID) signals for small nitroxides at W-band are also reported. Results are compared with multifrequency measurements of T_1e previously reported for these molecules in the range of 2–35 GHz [J.S. Hyde, J.-J. Yin, W.K. Subczynski, T.G. Camenisch, J.J. Ratke, W. Froncisz, Spin label EPR T₁ values using saturation recovery from 2 to 35 GHz. J. Phys. Chem. B 108 (2004) 9524–9529]. The values of T_1e decrease at 94 GHz relative to values at 35 GHz.     

Spin relaxation studies on conducting poly(tetrathiafulvalene)

Magnetic parameters and the relaxation behavior of paramagnetic centers in an iodine-doped poly(tetrathiafulvalene) semiconductor with a d.c. conductivity of 10^?5 S·cm^?1 have been studied using mainly the 2 mm waveband EPR technique in the temperature range of 110–270 K. The EPR line shape analysis confirms the existence of immobile radicals pinne on short polymer chains and mobile polarons with different relaxation parameters in slightly doped poly(tetrathiafulvalene). The temperature dependences of electron spin-lattice and spin-spin relaxation times of paramagnetic centers of both types have been determined independently using the saturation method at the operation frequency ν _e = 140 GHz. An anisotropic slow libration of immobile polarons with an activation energy of 0.02 eV have been registered for the first time using the saturation transfer EPR method. The temperature dependences of intrachain diffusion and interchain hopping rates in poly(tetrathiafulvalene) are determined from theT ₁ andT ₂ EPR data. The interchain spin dynamics is shown to correlate with libration of polarons trapped on polymer chains and is in good agreement with a hopping charge transport mechanism.     

Anisotropic exchange interactions in CuTe2O5

Electron paramagnetic resonance (EPR) was used to study CuTe₂O₅ single crystals at frequencies of 9.4 and 160 GHz. Analytic expressions for the second and fourth moments of the EPR line are deduced with inclusion of the difference between the exchange couplings of the copper spin with its different neighbors. From comparing the calculated and measured EPR linewidths, the positions of copper ions with the strongest exchange interactions are identified. The parameters of the anisotropic exchange interaction between copper ions in a pair are found. The parameter of the exchange interaction between magnetically nonequivalent copper centers is determined from the frequency dependence of the EPR linewidth. The directions of the principal axes of the g tensors are established. The data obtained count in favor of a quasi-one-dimensional model of magnetism in CuTe₂O₅.     

EPR spectroscopy and imaging of TEMPO-labeled magnetite nanoparticles

The article presents results of a study of TEMPO-labeled polymer coated superparamagnetic iron(II,III) oxide nanoparticles using both Electron Paramagnetic Resonance (EPR) spectroscopy and Electron Paramagnetic Resonance imaging technique (EPRI). The X-band (9.4 GHz) EPR spectroscopy was used to investigate the behavior of TEMPO-labeled polymer coated magnetite nanoparticles in different conditions (temperature and orientation in magnetic field). The broad line, which comes from the core of Fe₃O₄ nanoparticles, shows anisotropy. This signal broadens with decreasing temperature, its intensity increases with increasing temperature and the g factor decreases with increasing temperature. The shape of the signal from nitroxide radical strongly depends on temperature. When temperature is higher than 200 K, a narrow triplet appears, but when it is lower than 200 K the signal consists of broad asymmetric lines. Analysis of the signal allowed characterization of the motion of the spin label attached to nanoparticles. Values of anisotropy parameter ɛ and rotational correlation time τ_c were calculated for TEMPO in the fast rotation regime.The ability of TEMPO-labeled PEG coated magnetite nanoparticles to diffuse within the hydrogel medium was also investigated. The EPR imaging of nanoparticles diffusion in hydrogel was made at room temperature using an EPR L-band (1 GHz) spectrometer. EPRI has been proved effective for evaluation of changes in the spatial distribution of nanoparticles in the sample.     

Biochemical EPR imaging of skin

EPR Imaging (EPRI) of spin labels is a powerful method for investigating skin and can give information about biochemical processes which are involved in numerous skin diseases. Furthermore it enables the non invasive investigation of the liberation, penetration and distribution of spin labelled drugs. The basis of these measurements is spectral spatial EPR imaging employing modulated field gradients and simultaneous field scans (MOSS). A skin region (?=6 mm) was treated with a 10 μl spin label solution (1 mM). EPR spectra of 128 points were recorded in 128 spatial planes resulting in a 128×128 image matrix. A spatial resolution of better than 10 μm can be obtained for a spectral line width of 0.1 mT and a gradient of 4 Tm^?1.In vivo imaging on mammalian skin can be performed by employing surface coils at S-band frequencies, 3 GHz.     

A Low Temperature (10 K) High-Frequency (208 GHz) EPR Study of the Non-Kramers Ion Mn3+ in a MnMo6Se8 Single Crystal

A high-frequency (208 GHz) electron paramagnetic resonance (EPR) study on Mn³⁺ (3d⁴, S = 2) ions embedded in a MnMo₆Se₈ single crystal has been performed at 10 K. The experimental spectra reveal the presence of only one set of EPR lines from Mn³⁺ ions, whose magnetic axes are oriented along the crystal axes. The spin-Hamiltonian parameters are evaluated by the method of least-squares, fitting all the observed line positions simultaneously, for the three orthogonal orientations of the external magnetic field. The symmetry of the spin Hamiltonian at the site of the Mn³⁺ ions has been deduced from the EPR spectra.     

Paramagnetic resonance linewidth of EuO near the curie temperature

Measurements of the EPR linewidth ΔH of EuO at 9 GHz are reported in the temperature range of 66–300° K, with particular attention to the region near T_c (69.6° K). Comparison with the earlier data of Eastman at 25 GHz in the critical region shows considerable suppression of ΔH at 25 GHz. The temperature-dependent behavior of ΔH in EuO at 9 GHz is similar to the observations in CrBr₃ and it is in qualitative agreement with the zero-field predictions of Huber and Maleev. The quantitative discrepancies are believed to be due to the effect of the resonance magnetic field on EPR spin dynamics near T_c and an inadequate decoupling of the four-spin correlation functions used in the theories.     

EPR of Yb3+ ions in Ba1−xLaxF2+x mixed crystals

Electron paramagnetic resonance (EPR) spectra of impurity Yb³⁺ ions (about 0.1 at.%) in mixed crystals BaF₂(1-x) plus LaF₃(x) have been investigated for different values of the concentrationx at a frequency of about 9.5 GHz by both continuous-wave (CW) EPR and electron spin echo methods. A spectrum of trigonal symmetry with a complex hyperfine structure is observed in “pure” BaF₂:Yb³⁺ (x=0). Upon admixture of small amounts of LaF₃ (x=0.001), additional EPR lines arise with intensities increasing with the increase ofx up to 0.005. These lines are attributed to trigonal centers including two rare-earth ions and two compensating fluorine ions. A further increase ofx results in a decrease of the total EPR spectrum intensity, and atx≥0.05 the CW resonance becomes practically unobservable. This may be due to the formation of rare-earth ion clusters with paramagnetic Yb³⁺ ions occurring in domains with a disordered structure of surroundings resulting in very broad EPR lines, which cannot be registered by CW EPR. Indeed, very broad (not less than 1 KG) EPR lines were observed by the electron spin echo method for concentrationsx<-0.02.     

Spin-labeled photosynthetic reaction centers fromRhodobacter sphaeroides studied by electron paramagnetic resonance spectroscopy and molecular dynamics simulations

A new strategy has been applied that combines molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy to study the structure and conformational dynamics of the spin-labeled photosynthetic reaction center (RC) ofRhodobacter sphaeroides. This protein serves here as a model system to demonstrate the applicability of this new methodology. The RC contains five native cysteines and EPR experiments show that only one cysteine, located on the H subunit, is accessible for spin labeling. The EPR spectra calculated from MD simulation trajectories of spin labels bound to the native cysteines C156 and C234 in subunit H reveal that only the spin label side chain at position 156 provides a spectrum which agrees with the experimental EPR spectrum.     

A New Q-Band EPR Probe for Quantitative Studies of Even Electron Metalloproteins

Existing Q-band (35 GHz) EPR spectrometers employ cylindrical cavities for more intense microwave magnetic fields B₁, but are so constructed that only one orientation between the external field B and B₁is allowed, namely the B B₁orientation, thus limiting the use of the spectrometer to measurements on Kramers spin systems (odd electron systems). We have designed and built a Q-band microwave probe to detect EPR signals in even electron systems, which operates in the range 2 K ≤ T ≤ 300 K for studies of metalloprotein samples. The cylindrical microwave cavity operates in the TE₀₁₁mode with cylindrical wall coupling to the waveguide, thus allowing all orientations of the external magnetic field B relative to the microwave field B₁. Such orientations allow observation of EPR transitions in non-Kramers ions (even electron) which are either forbidden or significantly weaker for B B₁. Rotation of the external magnetic field also permits easy differentiation between spin systems from even and odd electron oxidation states. The cavity consists of a metallic helix and thin metallic end walls mounted on epoxy supports, which allows efficient penetration of the modulation field. The first quantitative EPR measurements from a metalloprotein (Hemerythrin) at 35 GHz with B₁ B are presented.     

Spin-Label EPR for Determining Polarity and Proticity in Biomolecular Assemblies: Transmembrane Profiles

Hyperfine couplings and g-values of nitroxyl spin labels are sensitive to polarity and hydrogen bonding in the environment probed. The dependences of these electronic paramagnetic resonance (EPR) properties on environmental dielectric permittivity and proticity are reviewed. Calibrations are given, in terms of the Block–Walker reaction field and local proton donor concentration, for the nitroxides that are commonly used in spin labeling of lipids and proteins. Applications to studies of the transverse polarity profiles in lipid bilayers, which constitute the permeability barrier of biological membranes, are reviewed. Emphasis is given to parallels with the permeation profiles of oxygen and nitric oxide that are determined from spin-label relaxation enhancements by using nonlinear continuous-wave EPR and saturation recovery EPR, and with permeation profiles of D₂O that are determined by using ²H electron spin echo envelope modulation spectroscopy.     

A short note on orientation selection in the DEER experiments on a native cofactor and a spin label in the reaction center ofRhodobacter sphaeroides

The analysis of the two-frequency pulsed electron paramagnetic resonance (EPR) (double electron-electron spin resonance, DEER) investigation on the coupling between the semiquinone anion state of the primary acceptor (Q_A) and the spin label at the cysteine 156 in the H-subunit in the photosynthetic reaction center (RC) fromRhodobacter sphaerodes (R26) (I. V. Borovykh, S. Ceola, P. Gajula, P. Gast, H. J. Steinhoff, M. Huber: J. Magn. Reson. 180, 178–185, 2006) is reinvestigated to include orientation selection. The combination of the EPR properties of the two radicals and the pump and observer frequencies suggests that such an effect could play a role even at the X-band (9 GHz) EPR fields and frequencies employed. The magnitude of the effect is estimated from the structures obtained from the molecular-dynamics (MD) simulations from the previous study: the distance change is small (around 2%) and the distance of 3.05 nm obtained then is possibly underestimated by 0.06 nm. Thus, the difference of at least 0.2 nm between the measured distance and the average distance of 2.8 nm found by the MD simulation remains, suggesting a significant difference between the measurement and the X-ray structure of the RC, as discussed previously.     

Estimation of higher-order magnetic spin interactions of Fe(III) and Gd(III) ions doped in α-alumina powder with multifrequency EPR

Fe(III) and Gd(III) ions in α-alumina (A1₂O₃) exhibit spin states ofS = 5/2 andS = 7/2 respectively. The magnitude of the zero-field interaction (ZFI) (D = 0.10?0.15 cm^?1) gives rise to an inter Kramers doublet splitting in the same order of magnitude as the X-band electron paramagnetic resonance (EPR) quantum (0.3 cm^?1). It is demonstrated that through a careful step-by-step analysis and spectral simulation of EPR spectra taken at D-band (130 GHz), Q-band (35 GHz), and X-band (9 GHz) at room temperature, the (relative) sign and magnitude of the ZFI parameters, b ₂ ⁰ , b ₄ ⁰ , and b ₄ ³ , can be reliably estimated.     

Single-crystal EPR studies of transition-metal ions in inorganic crystals at very high frequency

Electron paramagnetic resonance (EPR) single-crystal rotation studies at very high frequency (249.9 GHz) of transition metal ions with electron spins greater than one-half are reported. At 249.9 GHz, the spectra are in the high-field limit despite large zero-field splittings. This leads to a considerable simplification of the spectra, and aids in their interpretation. Single-crystal 249.9 GHz EPR spectra of Ni²⁺ in Ni₂CdCl₆· 12H₂O, Mn²⁺ (0.2%) in ZnV₂O₇, and Fe³⁺ (2%) in CaYA10₄ were recorded at 253 K in an external magnetic field of up to 9.2 T, along with those at X-band and Q-band frequencies at 295 K and lower temperatures. The goniometer used at 249.9 GHz for single-crystal rotation is based on a quasi-optical design and is an integral part of a special Fabry-Pérot resonator. The values of the spin-Hamiltonian parameters were estimated from a simultaneous fitting of all of the observed line positions at several microwave frequencies recorded at various orientations of each crystal with respect to the external magnetic field with least-squares fitting in conjunction with matrix diagonalization. Estimates of zero-field splitting parameterD at room temperature are: for Ni²⁺, about ?31 GHz (site I) and about ?7 GHz (site II); for Mn²⁺, about 6 GHz; and for Fe³⁺, about 29 GHz.