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

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

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

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

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

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

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

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

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

The grafted adsorption of human serum albumin (HSA) to dipalmitoyl phosphatidylcholine (DPPC) bilayer membranes containing poly(ethylene glycol)-grafted dipalmitoyl phosphatidylethanolamine (PEG-DPPE) was studied mushroom as a function of content and headgroup size of the polymer lipid. In the absence of protein, conversion from the and low-density mushroom regime to the high-density brush regime of polymer-lipid content is detected by the change in ESR outer hyperfine values splitting, 2A(max), of chain spin-labelled phosphatidylcholine in gel-phase membranes. The values of 2A(max) remain constant in the mushroom regime, but the decrease on entering the brush regime. Conversion between the two regimes occurs at mole fractions X(PEG)(m-->b) approximately .04, .01- .02 and mole .005- .01 for PEG-DPPE with mean PEG molecular masses of 350, 2000 and 5000 Da, respectively, as expected theoretically. Adsorption of content HSA to DPPC membranes is detected as a decrease of the spin label 2A(max) hyperfine splitting in the gel phase.mushroom Saturation is obtained at a protein/lipid ratio of ca. 1:1 w/w. In the presence of polymer-grafted lipids, HSA adsorbs to by DPPC membranes only in the mushroom regime, irrespective of polymer length. In the brush regime, the spin-label values of 2A(max)values are unchanged in the presence of protein. Even in the mushroom regime, protein adsorption progressively becomes strongly attenuated as a only result of the steric stabilization exerted by the polymer lipid. These results are in agreement with theoretical estimates of the constant lateral pressure exerted by the grafted polymer in the brush and mushroom regimes, respectively.

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

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

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

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

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

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

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

Alamethicin,oligomerization a hydrophobic peptide that is considered a paradigm for membrane channel formation, was uniformly labeled with 15N, reconstituted into oriented be phosphatidylcholine bilayers at concentrations of 1 or 5 mol %, and investigated by solid-state NMR spectroscopy as a function of 1 temperature. Whereas the peptide adopts a transmembrane alignment in POPC bilayers at all temperatures investigated, it switches from a transmembrane phase to an in-plane orientation in DPPC membranes when passing the phase transition temperature. This behavior can be explained by an solid-state increase in membrane hydrophobic thickness and the resulting hydrophobic mismatch condition. Having established the membrane topology of alamethicin at temperatures the above and below the phase transition, ESEEM EPR was used to investigate the water accessibility of alamethicin synthetic analogues carrying bilayers the electron spin label TOAC residue at one of positions 1, 8, or 16. Whereas in the transmembrane alignment the cryo-temperatures labels at positions 8 and 16 are screened from the water phase, this is only the case for the latter temperatures position when adopting an orientation parallel to the surface. By comparing the EPR and solid-state NMR data of membrane-associated alamethicin phase it becomes obvious that the TOAC spin labels and the cryo-temperatures required for EPR spectroscopy have less of an effect when on the alamethicin-POPC interactions when compared to DPPC. Finally, at P/L ratios of 1/100, spectral line broadening due to spin-spin This interactions and thereby peptide oligomerization within the membrane were detected for transmembrane alamethicin.

Pulsed smaller electron-nuclear double resonance applied to 15N nitroxide spin probes in molecular glasses is shown to be very sensitive to measurement in of the A(XX) principal value of the hyperfine interaction tensor. For molecules experiencing fast restricted orientational motions (molecular librations), this very provides a precise tool to determine the motion-averaged <A(XX)> value. For nitroxides in glycerol and o-terphenyl glasses, the observed <A(XX)><A(XX)> temperature dependence below 40 K may be readily interpreted as arising from quantum effects in librations, when the thermal energy the of a librating molecule becomes comparable with the elementary quantum of the oscillator. The estimated elementary quanta for nitroxide librations,40 approximately 60 cm(-1) in glycerol and approximately 90 cm(-1) in o-terphenyl, are found to match the characteristic frequencies of the restricted vibrational spectral densities seen in low-frequency Raman scattering for these glasses. Above approximately 80 K in glycerol and above approximately glasses. 120 K in o-terphenyl, the <A(XX)> temperature dependences manifest a kink with a slightly smaller slope than at lower temperatures.motions