Self-Aggregation and Orientation of the Ion Channel-Forming Zervamicin IIA in the Membranes of ePC Vesicles Studied by cw EPR and ESEEM Spectroscopy

The ion channel-forming peptide antibiotic zervamicin A was studied in egg phosphocholin lipid membranes of large multilamellar vesicles (LMV) at 77 K. Continuous wave electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) methods combined with site-specific electron spin labeling were used to study the aggregation and immersion depth of two analog molecules, i.e., each monolabeled either at the N- or C-terminal end of the helical molecule. Analysis of the shape of the EPR spectra indicates that zervamicin molecules form aggregates in which the dipolar interaction between the spin labels at the N-terminus is substantially larger than that between the labels at the C-terminus. The ESEEM method was used to study the interaction between the nitroxide radical spin labels of the zervamicin molecules and deuterium nuclei in LMV, which were prepared using a D₂O buffer. It is established that the largest amplitude of deuterium modulation of the unpaired electron is observed for zervamicin molecules labeled at the N-terminus. Based on the analysis of the Fourier parameters of the deuterium modulated spectrum, a model of the immersion depth of the terminal ends of the zervamicin molecule in a lipid bilayer is formulated. All of the spin labels at the N-terminus are grouped at the lipid–water interface, whereas 60% of labels at the C-terminus are located at the lipid–water interface and 40% are more deeply inserted into the lipid bilayer.     

Procain interaction with DPPC multilayers: an ESR spin label investigation

A spin label electron spin resonance (ESR) study has been carried out on the interaction between the local anesthetic procaine (PRC) and multilamellar dispersions of di-palmitoylphosphatidylcholine (DPPC). The investigation has been performed either in the gel or in the liquid crystal phase of DPPC varying the anesthetic/DPPC molar ratio as well as the pH of the dispersion medium and using spin labeled DPPC molecules (5- and 16-PCSL) and the di-tert-butyl nitroxide (DTBN) spin probe. On increasing the anesthetic concentration in the dispersion medium up to PRC/DPPC molar ratio of 33 mol.%, a lyotropic transition of the bilayer from the lamellar gel phase L_β, to the interdigitated L_βi one is observed either at pH 5.0 or at pH 10.0. At intermediate pH values, about pH 7.0, such a PRC concentration dependent transition is absent. A decrease of the partition of the DTBN molecules between the dispersion medium and the fluid hydrophobic phase of DPPC multilayers is observed at pH 5.0 with anesthetic concentration, while an opposite behaviour is evidenced at pH 10.0. The pre- and main-phase transition temperatures of the DPPC multilayers are also affected although to different extent. These results are interpreted in terms of adsorption of the PRC molecules at the surface of the model membrane at the extreme pH values investigated, while at about neutral pH values they intercalate among the DPPC molecules. The protonation state of procaine plays an important role as it determines the way of surface adsorption of the PRC molecules in the neutral form at the high pH value and in the charged cationic form at pH 5.0.     

TR-EPR of single and double spin-labeled C60 derivatives in frozen matrices

Time-resolved electron paramagnetic resonance spectra of two single spin-labeled and two double spin-labeled C₆₀ derivatives in frozen solution are recorded with pulsed laser excitation and 100 ns time resolution. Quartet and quintet excited species are detected which arise from the electron spin coupling of the triplet excited fullerene moiety with the unpaired spin(s) of the nitroxide label(s). Despite the similar molecular structure, in both series of single and double labeled derivatives a different behavior was found, which is due to substantial difference of the energy of exchange coupling.     

Comparison of CW-EPR and ESEEM technique for determination of water permeability profile in liposome membranes

The hydrophobicity profile across a membrane is an important parameter of biological membranes as it defines the barrier for permeation of polar molecules into the cell interior and of the stability of transmembrane proteins. In this work, the hydrophobicity profile was measured by conventional continuos-wave electron paramagnetic resonance (CW-EPR) and by electron spin echo envelope modulation (ESEEM) in two liposome suspensions with different ionic strengths in order to compare the sensitivity of both methods for the determination of the water penetration depth into the liposome membrane. Multilamellar liposomes from egg-phosphatidylcholine, cholesterol and dicetyl phosphate were prepared in deuterium oxide, buffered with phosphate buffer with or without NaCl at the phosphate concentrations 0.1 or 0.01 M. Liposomes were spin labeled with stearic acids spin probes with the nitroxide group at different positions on the acyl chain. The deuterium penetration depth was measured by the ESEEM technique and the signal was analyzed in the frequency domain (Fourier transform [FT]-ESEEM). The ratio of the intensities of FT-ESEEM lines at deuterium and hydrogen Larmor frequencies was used as a measures of the water penetration depth. It was also indirectly determined from the hyperfine splitting constants in frozen and in fluid state, measured by CW-EPR, as they depend on the polarity of the nitroxide group environment. The comparison of the two techniques demonstrated that FT-ESEEM is more sensitive to the changes on the surface of the membrane and thus more appropriate for the determination of the hydrophobic barrier than the conventional CW-EPR method.     

Permethyl-β-Cyclodextrin Spin-Labeled with Nitronyl Nitroxide: Synthesis and EPR Study

A method for synthesis of new covalently linked spin-labeled cyclodextrin (CD) via the attachment of nitronyl nitroxide 2-(4-hydroxyphenyl)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazole-3-oxide-1-oxyl to permethylated β-cyclodextrin is described. Electron spin echo envelope modulation (ESEEM) studies demonstrate that the new spin-labeled CD exhibits dynamic equilibrium between conformations with radical fragment capping the cavity of CD and radical fragment located outside the cavity. In solution, nitronyl nitroxide attached to CD retains its sensitivity to nitric oxide (NO), as reaction with NO leads to formation of iminonitroxide fragment evidenced by continuous-wave (CW) electron paramagnetic resonance (EPR). At the same time, CW EPR study of the reaction with ascorbic acid shows that the described binding of nitronyl nitroxide to CD does not provide higher stability of radical towards the reduction, and the corresponding rate constants are close to those obtained for free nitronyl nitroxide. Plausible explanations of these observations are discussed.     

Polyelectrolyte Multilayers Studied by Electron Paramagnetic Resonance (EPR) Spin-Label Technique

A nitroxide spin label has been covalently attached to the polyelectrolyte poly(ethylene-alt-maleic acid) (P(E-alt-MA)) to study the interaction between this weak polyanion, the oppositely charged strong polycation poly(diallyldimethylammonium chloride) (PDADMAC) and water in swollen polyelectrolyte multilayers (PEM) by electron paramagnetic resonance (EPR) spectroscopy. If the spin-labeled polyanion has been used for the preparation of every double layer, the growth of the PEM film can be monitored by quantitative EPR. On the other hand, if the spin-labeled polyanion has been selectively placed in different layers in the PEM film the influence of the environment such as pH of the swelling medium of the mobility of the polyelectrolyte molecules positioned in the selected layer can be investigated.     

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.     

First-order phase transition in protein dynamics of ferritin

Detailed Mössbauer spectra of⁵⁷Fe in the iron storage protein, ferritin, in the temperature range between 250 and 280 K reveal a first-order phase transition with a thermal hysteresis loop of 7 K width. While the temperature is raised from 90 K to 271 K, Mössbauer spectra composed of a narrow line quadrupole doublet, typical for solids, are observed. Above this temperature, each spectrum is composed of the narrow line subspectrum and a broad line subspectrum whose relative intensity increases with temperature. The intensity of the narrow line subspectrum decreases by a factor of five at the critical temperature and thus shows a large increase in the mean square displacements atT _up=271 K. While decreasing the temperature, the bounded diffusive motions, expressed in the spectra by the coexistence of the narrow and broad lines, survive down toT _down=264 K, where again the spectral shapes and areas undergo a discontinuous jump. The narrow line subspectrum increases in intensity and the broad line subspectrum disappears. These phenomena may be understood in terms of supercooling of the water in the free channels and in the cavity of the ferritin molecule.     

The Temperature Dependence of Nitroxide Spin–Label Interaction Parameters: a High-Field EPR Study of Intramolecular Motional Contributions

High-field W-band electron paramagnetic resonance (EPR) spectroscopy was utilized to study the temperature dependence of the magnetic interaction parameters (g-, hyperfine-, quadrupole tensors) of two types of doublet-state nitroxide spin probes in glass-forming ortho-terphenyl solution: a five-membered ring system of pyrroline type (model for the commonly used methane thiosulfonate spin label) and a six-membered ring system of piperidine type (model for the commonly used TOAC spin label). The analysis of the g- and hyperfine tensors in terms of their isotropic and anisotropic parts reveals at least two mechanisms of motion that are responsible for the temperature dependence of the interaction parameters. The first mechanism is attributed to the overall small-angle motion of the nitroxide molecule in the glassy matrix; it leads to an averaging of the anisotropies of the EPR parameters. The second mechanism originates in an intramolecular out-of-plane motion of oxygen in the nitroxide group. This type of motion is evidenced by comparing the experimental findings for the spin-interaction parameters with the results of density functional theory calculations. The harmonic oxygen out-of-plane vibrations result in a variation of both the isotropic and anisotropic parts of the g- and hyperfine tensors. In contrast, the quadrupole tensor is not influenced by this vibration mechanism in the temperature range under study (90–240 K). Consequences of the applicability of such typical nitroxide radicals for probing details of their protein environment and for studying librational dynamics in frozen solutions are discussed.     

Magnetic properties of a quasi-one-dimensional NaFeGe<Subscript>2</Subscript>O<Subscript>6</Subscript> polycrystal

The magnetic properties of a synthesized dielectric NaFeGe₂O₆. polycrystal have been studied. The antiferromagnetic ordering of this compound below 15 K has been established. The Mössbauer spectrum at 300 K is a quadrupole doublet; it is characterized by an isomeric shift typical of the high-spin Fe³⁺ ion in the octahedral coordination and quadrupole splitting, which indicates distortion of the oxygen octahedron around the iron cation. Quasi-one-dimensionality of the sample magnetic structure is proved.     

Electron spin-echo envelope modulation at S-band. 3: Analysis of nuclear quadrupole interaction effects

In the present paper the nuclear modulation of electron spin echo signals at S-band is investigated in the case of interacting nuclei with a quadrupole moment high enough to cause nuclear quadrupole couplings not negligible with respect to the nuclear Zeeman and dipolar hyperfine couplings. Both the two-pulse and three-pulse electron spin echo envelope modulation (ESEEM) due to²⁷Al and¹⁴N are simulated at different values of the nuclear quadrupole coupling by numerical diagonalization of the nuclear Hamiltonians. The behavior of their amplitude and periods is discussed on the basis of the ratios between the strengths of the nuclear quadrupole interaction and the nuclear Zeeman and the dipolar hyperfine interactions. The interpretation of their trends in terms of the eigenfunctions and eigenvectors of the nuclear Hamiltonians is carried out by using analytical equations obtained by perturbation approaches. First order perturbation treatments for integer and half-integer nuclear spin quantum numbers are developed when the nuclear quadrupole coupling is the main interaction. A discussion on the limits of the interpretation based on the perturbation approach is also given by comparing the magnitude Fourier transform of the patterns calculated by exact diagonalization and analytical equations.     

Magnetic excitations in antiferromagnetic Bi<Subscript>2</Subscript>CuO<Subscript>4</Subscript>

Magnetic excitations in the antiferromagnetic Bi₂CuO₄ (T _N =42K) are investigated on the basis of anisotropic exchange interaction between spins of Cu²⁺ ions. We calculate the dispersion curves and evaluate the intensity of the inelastic neutron scattering by spin wave excitations. Spin contraction at OK and the effect of spin wave interaction are studied.     

Synthesis and properties of NaFeGe<Subscript>2</Subscript>O<Subscript>6</Subscript> polycrystals

NaFeGe₂O₆ polycrystals were synthesized and their x-ray diffraction, magnetic, electrical, and Mössbauer characteristics were measured. It is established that this monoclinic compound is a dielectric with a temperature of antiferromagnetic ordering of 15 K. The Mössbauer spectrum at 300 K is a quadrupole doublet. The isomer shift is 0.40 mm/s, which is characteristic of the high-spin Fe³⁺ ion in the octahedral coordination. The quadrupole splitting is 0.34 mm/s, which indicates that the oxygen octahedron around the iron cation is distorted. The exchange interactions are estimated, and the crystal magnetic structure is discussed.     

Nitroxide Side-Chain Dynamics in a Spin-Labeled Helix-Forming Peptide Revealed by High-Frequency (139.5-GHz) EPR Spectroscopy

High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxide spin-labeled peptide in fluid aqueous solution. The peptide, which follows the single letter sequence, was reacted with the methanethiosulfonate spin label at the cysteine sulfur. The spin sensitivity of high-frequency EPR is excellent with less than 20 pmol of sample required to obtain spectra with good signal-to-noise ratios. Simulation of the temperature-dependent spectral lineshapes reveals the existence of local anisotropic motion about the nitroxide N-O bond with a motional anisotropy tau( perpendicular)/tau( parallel) ( identical with N) approaching 2.6 at 306 K. Comparison with previous work on rigidly labeled peptides suggests that the spin label is reorienting about its side-chain tether. This study demonstrates the feasibility of performing 140-GHz EPR on biological samples in fluid aqueous solution.     

<Superscript>209</Superscript>Bi NMR spectrum of BiFeO<Subscript>3</Subscript> in the presence of spatial modulation of hyperfine fields

A complicated spin-echo spectrum was observed for ²⁰⁹Bi nuclei in a ferroelectric antiferromagnet BiFeO₃ in zero external field. This spectrum is the first example of nuclear quadrupole resonance of a system of diamagnetic atoms in a hyperfine magnetic field produced by the spatially modulated Fe³⁺ spin system and varying in orientation and magnitude. An attempt is undertaken to theoretically simulate the spectrum.     

Nonperturbative simulation of two-pulse electron spin-echo-envelope modulation spectra. Comparison with experimental single-crystal spectra

The direct method of calculating magnetic observables provided by the Liouville formalism is utilized for a nonperturbative numerical treatment of electron spin-echo-envelope modulation (ESEEM). Calculated time-domain signals are compared with experimental ones. The particular system studied is the C0₂ anion trapped in NaHC₂O₄·H₂O single crystals. The experimental ESEEM spectra show modulations due to both proton and sodium nuclei. The hyperfine and quadrupole tensors, determined independently by ENDOR spectroscopy, are used as input for calculating the ESEEM spectra.     

Nuclear spin relaxation in free radicals as revealed in a stimulated electron spin echo experiment

Electron spin echo envelope modulation (ESEEM) in a three-pulse stimulated echo experiment, when the time interval between the first and second pulses τ is varied, is attributed to a spontaneous change of the electron spin Larmor frequency in the time intervalT between the second and third pulses, due to the longitudinal relaxation of nearby nuclei. It is observed for nitroxide radicals in glassy matrices in the temperature range of 130–240 K. Nuclear relaxation is assumed to arise from fluctuation of the proton hyperfine interaction, due to fast rotation of the methyl groups. This contribution to ESEEM and the conventional one that is induced by the simultaneous excitation of allowed and forbidden electron spin transitions were found to be multiplicative. As the latter does not depend on the timeT, both contributions can be easily separated. The rate of nuclear spin relaxation was determined, and correlation time of methyl group rotation was estimated by Redfield theory of spin relaxation. Arrhenius parameters of this motion were estimated on the basis of these data and those at 77 and 90 K, where the previously developed approach was used (L.V. Kulik, I.A. Grigor’ev, E.S. Salnikov, S.A. Dzuba, Yu.D. Tsvetkov, J. Phys. Chem. A 106, 12066–12071, 2003).     

Vibrational spectra of monoisotopic SiH<Subscript>4</Subscript> and GeH<Subscript>4</Subscript> in low-temperature matrices

IR absorption spectra of monoisotopic ²⁸SiH₄ and ⁷⁶GeH₄ are studied in Ar and N₂ matrices at 10 K. It is shown that the absorption spectra of silane and germane are similar in the regions of the stretching ν₃ and bending ν₄ vibrations. Four groups of bands can be separated out in the spectra of each molecule: (1) narrow bands characteristic of the matrix isolation studies, (2) broad bands, (3) diffuse absorption with a large value of the spectral moment M ₂^* the intensity of which increases upon annealing, and (4) bands of dimers the intensity of which increases quadratically with concentration. The spectra of ²⁸SiH₄ and ⁷⁶GeH₄ in nitrogen matrices contain a triplet in the stretching region and a doublet in the bending region, which is explained by the change in the molecular symmetry from T _d to C _3V on passage from the gas phase to solid nitrogen.     

Low-Temperature Molecular Motions in Phospholipid Bilayers in Presence of Glycerol as Studied by Spin-Echo EPR of Spin Labels

Glycerol is used as a cryoprotective agent to protect biological systems under freezing conditions. Electron spin echo (ESE) spectroscopy, a pulsed version of EPR, is capable of studying low-temperature molecular motions of nitroxide spin labels. ESE technique was applied to study molecular motions in phospholipid bilayers prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with added spin-labeled lipids 1-palmitoyl-2-stearoyl-(n-DOXYL)-sn-glycero-3-phosphocholine (n-PCSL, n was optionally 5 or 16). Bilayers were hydrated (solvated) either in pure water or in a 1:1 v/v water–glycerol mixture. In the used ESE approach, there were studied stochastic (or diffusive) orientational vibrations of the molecule as a whole (i.e., stochastic molecular librations). The anisotropic contribution to the echo decay rate, W _anis, was measured, which is proportional, according to theory, to the product of the mean-squared angular amplitude \(\langle \alpha^{ 2} \rangle\) and the correlation time τ _c. W _anis was found to be small below and to sharply increase above 200 K, for the both types of solvents and the both label positions. As compared with hydration by pure water, in presence of glycerol W _anis was larger for the 5th label position while for the 16th one it did not change. Also, for the 5th label position W _anis values were found to be nearly the same as those for a polar spin probe 3,4-dicarboxy-PROXYL which was separately added to the bilayer as a reference and which is assumed to be partitioned only into the solvating shell. These results indicate that motions at the surface of bilayer are governed by the motion of solvating shell while motions in the bilayer interior occur independently. The relation of the obtained data with the dynamical transition phenomenon that is known for biological substances near 200 K from neutron scattering and Mössbauer absorption is discussed.