Recently, developments in time-resolved spin-label electron spin resonance (ESR) spectroscopy have contributed considerably to the study of biomembranes. Two different applications of electron spin echo spectroscopy of spin-labelled phospholipids are reviewed here:(1) the use of partially relaxed echo-detected ESR spectra to study the librational lipid-chain motions in the low-temperature phases of phospholipid bilayers;(2) the use of electron spin echo envelope modulation spectroscopy to determine the penetration of water into phospholipid membranes. Results are described for phosphatidylcholine bilayer membranes, with and without equimolar cholesterol, that are obtained with phosphatidylcholine spin probes site-specifically labelled throughout the sn-2 chain.

Membranous Na,K-ATPase from shark salt gland and from pig kidney was spin-labelled on Class I -SH groups in the presence of glycerol, or on Class II -SH groups in the absence of glycerol. The Class I-labelled preparations retain full enzymatic activity, whereas the Class II-labelled preparations are at least partially inactivated. This provides an excellent testbed on which to demonstrate how advanced electron paramagnetic resonance (EPR) can provide novel information on specific residues in unique environments in a complex, membrane-bound transport system. The polarity of the environment, and the librational dynamics and conformational exchange, of the spin-labelled groups were studied with pulsed EPR by using electron spin echo envelope modulation (ESEEM) spectroscopy and spin-echo detected (ED) EPR spectroscopy, respectively. 2H-ESEEM spectra of membranes dispersed in D2O reveal that Class I groups of the shark enzyme are more exposed to water than are those of the pig enzyme or Class II groups of either species, consistent with the more superficial membrane location in the former case. Spin-echo decay curves indicate conformational heterogeneity at low temperatures (< 150 K), but a more homogeneous conformational state at higher temperatures that is characterised by a single phase-memory T2M relaxation time. Conventional EPR lineshapes also demonstrate conformational microheterogeneity at low temperatures: the inhomogeneously broadened lines narrow progressively with increasing temperature reaching an almost pure Lorentzian lineshape at temperatures of ca. 220 K and above. The inhomogeneous broadening at low temperature is well described by a Gaussian distribution of Lorentzian lines. ED-spectra as a function of echo-delay time demonstrate the onset of rapid librational motions of appreciable amplitude, and slower conformational exchange, at temperatures above 220 K. These motions could drive transitions between the different conformational substates, which are frozen in at lower temperatures but contribute to the pathways between the principal enzymatic intermediates at higher temperatures.

Alamethicin F50/5 is a hydrophobic peptide that is devoid of charged residues and which induces voltage-dependent ion channels in lipid membranes. The peptide backbone is likely to be involved in the ion conduction pathway. Electron spin-echo spectroscopy of alamethicin F50/5 analogues in which a selected alpha-aminoisobutyric acid residue (at position n = 1, 8 or 16) is replaced by the TOAC amino-acid spin label was used to study torsional dynamics of the peptide backbone in phosphatidylcholine bilayer membranes. Rapid librational motions of limited angular amplitude were observed at each of the three TOAC sites by recording echo-detected spectra as a function of echo delay time, 2tau. Simulation of the time-resolved spectra, combined with conventional EPR measurements of the librational amplitude, shows that torsional fluctuations of the peptide backbone take place on the subnanosecond to nanosecond timescale, with little temperature dependence. Associated fluctuations in polar fields from the peptide could facilitate ion permeation.

Human serum albumin (HSA) is an abundant plasma protein that transports fatty acids and also binds a wide variety of hydrophobic pharmacores. Echo-detected (ED) EPR spectra and D(2)O-electron spin echo envelope modulation (ESEEM) Fourier-transform spectra of spin-labelled free fatty acids and phospholipids were used jointly to investigate the binding of stearic acid to HSA and the adsorption of the protein on dipalmitoyl phosphatidylcholine (DPPC) membranes. In membranes, torsional librations are detected in the ED-spectra, the intensity of which depends on chain position at low temperature. Water penetration into the membrane is seen in the D(2)O-ESEEM spectra, the intensity of which decreases greatly at the middle of the membrane. Both the chain librational motion and the water penetration are only little affected by adsorption of serum albumin at the DPPC membrane surface. In contrast, both the librational motion and the accessibility of the chains to water are very different in the hydrophobic fatty acid binding sites of HSA from those in membranes. Indeed, the librational motion of bound fatty acids is suppressed at low temperature, and is similar for the different chain positions, at all temperatures. Correspondingly, all segments of the bound chains are accessible to water, to rather similar extents.

Electron spin-echo envelope modulation (ESEEM) spectroscopy of phospholipids spin-labeled systematically down the sn-2 chain was used to detect the penetration of water (D(2)O) into bilayer membranes of dipalmitoyl phosphatidylcholine with and without 50 mol % cholesterol. Three-pulse stimulated echoes allow the resolution of two superimposed (2)H-ESEEM spectral components of different widths, for spin labels located in the upper part of the lipid chains. Quantum chemical calculations (DFT) and ESEEM simulations assign the broad spectral component to one or two D(2)O molecules that are directly hydrogen bonded to the N-O group of the spin label. Classical ESEEM simulations establish that the narrow spectral component arises from nonbonded water (D(2)O) molecules that are free in the hydrocarbon chain region of the bilayer membrane. The amplitudes of the broad (2)H-ESEEM spectral component correlate directly with those of the narrow component for spin labels at different positions down the lipid chain, reflecting the local H-bonding equilibria. The D(2)O-ESEEM amplitudes decrease with position down the chain toward the bilayer center, displaying a sigmoidal dependence on position that is characteristic of transmembrane polarity profiles established by other less direct spin-labeling methods. The midpoint of the sigmoidal profile is shifted toward the membrane center for membranes without cholesterol, relative to those with cholesterol, and the D(2)O-ESEEM amplitude in the outer regions of the chain is greater in the presence of cholesterol than in its absence. For both membrane types, the D(2)O amplitude is almost vanishingly small at the bilayer center. The water-penetration profiles reverse correlate with the lipid-chain packing density, as reflected by (1)H-ESEEM intensities from protons of the membrane matrix. An analysis of the H-bonding equilibria provides essential information on the binding of water molecules to H-bond acceptors within the hydrophobic interior of membranes. For membranes containing cholesterol, approximately 40% of the nitroxides in the region adjacent to the lipid headgroups are H bonded to water, of which ca. 15% are doubly H bonded. Corresponding H-bonded populations in membranes without cholesterol are ca. 20%, of which ca. 6% are doubly bonded.

Spin-echo decays of spin-labelled phospholipids have been recorded to study the chain dynamics in the low-temperature phases of dipalmitoyl phosphatidylcholine membranes with and without 50 mol% cholesterol. The phase-memory relaxation time, T(2M), depends on the position of spin-labelling in the sn-2 chain, and on the presence of cholesterol. A biphasic temperature dependence of T(2M) is obtained over the range 150-270 K. Echo-detected field-swept absorption EPR spectra were recorded as a function of the echo delay time, tau. The echo-detected EPR lineshapes show a strong dependence on tau, revealing anisotropic phase relaxation arising from torsional chain motions. Cholesterol has a large effect on torsional oscillations about the chain long axis. Small-amplitude chain motions in the low-temperature phases may be important for cryopreservation of membranes.

The association of water (D(2)O) with phospholipid membranes was studied by using pulsed-electron spin resonance techniques. We measured the deuterium electron spin echo modulation of spin-labeled phospholipids by D(2)O in membranes of dipalmitoyl phosphatidylcholine with and without 50 mol% of cholesterol. The Fourier transform of the relaxation-corrected two-pulse echo decay curve reveals peaks, at one and two times the deuterium NMR frequency, that arise from the dipolar hyperfine interaction of the deuterium nucleus with the unpaired electron spin of the nitroxide-labeled lipid. For phosphatidylcholine spin-labeled at different positions down the sn-2 chain, the amplitude of the deuterium signal decreases toward the center of the membrane, and is reduced to zero from the C-12 atom position onward. At chain positions C-5 and C-7 closer to the phospholipid headgroups, the amplitude of the deuterium signal is greater in the presence of cholesterol than in its absence. These results are in good agreement with more indirect measurements of the transmembrane polarity profile that are based on the (14)N-hyperfine splittings in the conventional continuous-wave electron spin resonance spectrum.

Membranes grafted with water-soluble polymers resist protein adsorption and adhesion to cellular surfaces. Liposomes with surface-grafted polymers therefore find applications in drug delivery. The physicochemical properties of polymer-grafted lipid membranes are reviewed with mean-field and scaling theories from polymer physics. Topics covered are: mushroom-brush transitions, membrane expansion and elasticity, bilayer-micelle transitions, membrane-membrane interactions and protein-membrane interactions.

The chain-melting transition temperature of dipalmitoyl phosphatidylcholine (DPPC) bilayer membranes containing poly(ethylene glycol)-grafted dipalmitoyl phosphatidylethanolamine (PEG-DPPE) was determined by optical turbidity measurements. The dependence on content, Xp, of PEG-DPPE lipid was studied for different polar headgroup sizes, np, of the polymer lipid, throughout the lamellar phase of the mixtures with DPPC. Mean-field theory for the polymer brush regime predicts that the downward shift in transition temperature should vary with polymer size and content as npXp(5/3)(approximately npXp(11/6) for scaling theory). Any shift induced by the charge on PEG-lipids is independent of polymer size. These predictions are reasonably borne out for the longer polymer lipids (PEG molecular masses 750, 2000 and 5000 Da). Transition temperature shifts in the lamellar phase, before the onset of micellisation, are in the region of -1 to -2 degrees C (+/-0.1-0.2 degrees C) in reasonable accord with theoretical estimates of the lateral pressure exerted by the polymer brush. Shifts of this size are significant to the design of liposomes for controlled release of contents by mild hyperthermia.

The adsorption of human serum albumin (HSA) to dipalmitoyl phosphatidylcholine (DPPC) bilayer membranes containing poly(ethylene glycol)-grafted dipalmitoyl phosphatidylethanolamine (PEG-DPPE) was studied as a function of content and headgroup size of the polymer lipid. In the absence of protein, conversion from the low-density mushroom regime to the high-density brush regime of polymer-lipid content is detected by the change in ESR outer hyperfine splitting, 2A(max), of chain spin-labelled phosphatidylcholine in gel-phase membranes. The values of 2A(max) remain constant in the mushroom regime, but decrease on entering the brush regime. Conversion between the two regimes occurs at mole fractions X(PEG)(m-->b) approximately 0.04, 0.01-0.02 and 0.005-0.01 for PEG-DPPE with mean PEG molecular masses of 350, 2000 and 5000 Da, respectively, as expected theoretically. Adsorption of HSA to DPPC membranes is detected as a decrease of the spin label 2A(max) hyperfine splitting in the gel phase. Saturation is obtained at a protein/lipid ratio of ca. 1:1 w/w. In the presence of polymer-grafted lipids, HSA adsorbs to DPPC membranes only in the mushroom regime, irrespective of polymer length. In the brush regime, the spin-label values of 2A(max) are unchanged in the presence of protein. Even in the mushroom regime, protein adsorption progressively becomes strongly attenuated as a result of the steric stabilization exerted by the polymer lipid. These results are in agreement with theoretical estimates of the lateral pressure exerted by the grafted polymer in the brush and mushroom regimes, respectively.

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 D(2)O that are determined by using (2)H electron spin echo envelope modulation spectroscopy.

FMR measurements on barium ferrite nanoparticles (with an average length of about 13 nm) dispersed within a block copolymer (styrene-butadiene-styrene) are reported. Resonance spectra have been successfully simulated by a convolution of a Dysonian line and a Lorentzian line. The temperature dependence of FMR spectra in the so called in-the-plane and out-of the-plane configurations is reported. The angular dependence of FMR spectra at room temperature is analyzed in detail and simulated within simple thermodynamic model that takes into account the competition between shape and magnetocrystalline anisotropies. FMR data revealed that the local magnetic field acting on uncoupled electronic spin is dominated by the magnetocrystalline contribution, which eventually includes surface effects. The strong connection between FMR spectra and hysteresis loop is demonstrated.

We theoretically investigate intermolecular motions in liquid water in terms of third-order infrared (IR) spectroscopy. We calculate two-dimensional (2D) IR spectra, pump-probe signals, and three-pulse stimulated photon echo signals from the combination of equilibrium and nonequilibrium molecular dynamics simulations. The 2D IR spectra and the three-pulse photon echo peak shift exhibit that the frequency correlation of the librational motion decays with a time scale of 100 fs. The two-color 2D IR spectra and the pump-probe signals reveal that the energy transfer from the librational motion at 700 cm(-1) to the low frequency motion below 300 cm(-1) occurs with a time scale of 60 fs and the subsequent relaxation to the hot ground state takes place on a 500 fs time scale. The time scale of the anisotropy decay of the librational motion is found to be approximately 115 fs. The energy dissipation processes are investigated in detail by using the nonequilibrium molecular dynamics simulation, in which an electric field pulse is applied. We show that the fast energy transfer from the librational motion to the low frequency motion is mainly due to the librational-librational energy transfer. We also show that the fast anisotropy decay mainly arises from the rapid intermolecular energy transfer.

Electron spin echo (ESE) was applied to study transversal spin relaxation of photoexcited triplet state of fullerene C(70) molecules in glassy o-terphenyl and cis-/trans-decalin matrices (glass transition temperatures of 243 and 137 K, respectively). The relaxation rate T(2)(-1) was found to increase sharply above 110 K in o-terphenyl and above 100 K in decalin. It is suggested that this increase arises from interaction of (3)C(70) pseudorotation with fast molecular librations in the matrix. Both these types of motion involve atomic vibrations and are uniaxial in their nature, the known literature data on Raman light scattering and others indicate that molecular librations may be thermally activated in glasses just near 100 K. The increase in T(2)(-1) near 100 K is not observed for photoexcited triplet state of fullerene C(60), for which pseudorotation is not uniaxial. As the fullerene molecule has a size much larger than that for glass solvent molecules, it is likely that molecular librations in the matrix are of collective nature.

This paper describes the design and experimental tests of a novel neutron spin analyzer optimized for wide angle spin echo spectrometers. The new design is based on nonremanent magnetic supermirrors, which are magnetized by vertical magnetic fields created by NdFeB high field permanent magnets. The solution presented here gives stable performance at moderate costs in contrast to designs invoking remanent supermirrors. In the experimental part of this paper we demonstrate that the new design performs well in terms of polarization, transmission, and that high quality neutron spin echo spectra can be measured.

Characterizing the structure and dynamic properties of a single monolayer is a challenge due to the minute amount of material that is probed. Here, EPR spectroscopy is used for investigating the spatial and temporal organization of self-assembled monolayers of 5- and 16-doxyl stearic acid (5 DSA and 16 DSA, respectively) adsorbed on a GaAs substrate. The results are complemented with FTIR and ellipsometery measurements, which provide the evidence for the formation of monolayers. Moreover, a comparison with the FTIR spectrum of a monolayer of stearic acid shows that the monolayers of the spin labeled molecules are less packed due to the hindrance introduced by the labeling group. The EPR spectra provide a new insight on the ordering in the layer and more interestingly, it reveals the time dependence of the organization. For 5DSA, with the spin-label group situated close to the substrate, the EPR spectrum immediately after adsorption is poorly resolved and dominated by the spin-exchange interaction between neighboring molecules. As time increases (up to 1 week) the resolution of the 14N hyperfine coupling increases, revealing a better organized monolayer where the molecules are more homogenously spaced. Moreover, the spectrum of the layer, after reaching equilibrium, shows that there is no motional freedom near the GaAs surface. Orientation dependence measurements on the equilibrated sample show the presence of a preferred orientation of the molecules, although with a wide distribution. The spectrum of the 16DSA monolayer, where the nitroxide spin label is situated at the end of the chain, far from the surface, also showed a poorly resolved spectrum at short times, but unlike 5DSA, it did not exhibit any time dependence. Through EPR line-shape simulations and by comparison with FTIR results, the differences between 5DSA and 16DSA were attributed to difference in coverage caused by the bulky spin label near the surface in the case of 5DSA.

The physical properties of membranes derived from the total lipids extracted from the lens cortex and nucleus of a two-year-old cow were investigated using EPR spin labeling methods. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. Properties of these membranes are very similar to those reported by us for membranes made of the total lipid extract of six-month-old calf lenses (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta 1768 (2007) 1454-1465). However, in membranes made from nuclear lipids, two domains were detected by the EPR discrimination by oxygen transport method using the cholesterol analogue spin label and were assigned to the bulk phospholipid-cholesterol domain (PCD) and the immiscible cholesterol crystalline domain (CCD), respectively. Profiles of the order parameter, hydrophobicity, and the oxygen transport parameter are practically identical in the bulk PCD when measured for either the cortical or nuclear lipid membranes. In both membranes, lipids in the bulk PCD are strongly immobilized at all depths. Hydrophobicity and oxygen transport parameter profiles have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid-ring structure of cholesterol reaches into the membrane. The permeability coefficient for oxygen, estimated at 35 degrees C, across the bulk PCD in both membranes is slightly lower than across the water layer of the same thickness. However, the evaluated upper limit of the permeability coefficient for oxygen across the CCD (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can significantly reduce oxygen transport in the lens nucleus.

We synthesized oligodeoxynucleotide (ODN, 3) containing 2-N-tert-butylaminoxyladenosine (1) and studied EPR spectra of 3 and its duplexes. The h(+)/h(0) values in the EPR spectra of duplexes between 3 and 5-8 well correlated with Tm values. We also synthesized ODN 4 containing 2, which has a cyclic aminoxyl via a linker, to compare with ODN 3. The h(+)/h(0) values in the EPR spectra of duplexes between 4 and 5-8 did not correlate with Tm values. These results indicate that 1 has a potential to monitor of motion of the nucleobase.

Site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy has emerged as an efficient tool to elucidate the structure and conformational dynamics of biomolecules under native-like conditions. This article summarizes the basics as well as recent progress of site-directed spin labeling. Continuous wave EPR spectra analyses and pulse EPR techniques are reviewed with special emphasis on applications to the sensory rhodopsin-transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis and the photosynthetic reaction center from Rhodobacter sphaeroides R26.