Vibrational layer eigenmodes of binary phospholipid-cholesterol bilayers at low temperatures.

Raman spectra in the low-frequency spectral range-between 5 and 90cm^{-1}-were studied for multilamellar bilayers prepared with cholesterol (Chol) and phospholipids of three different types: doubly unsaturated lipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), monounsaturated lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and fully saturated lipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The narrow peak seen below 250 K and positioned between 9 and 18cm^{-1}-depending on the system and temperature-was attributed to the vibrational eigenmode of a lipid monolayer. For the DOPC-Chol bilayer, the peak position and the peak width were found to monotonically increase and decrease, respectively, with the Chol concentration. For POPC-Chol and DMPC-Chol bilayers, these parameters revealed nonmonotonic concentration dependences, with an apparent minimum at the intermediate Chol content. The peak intensity was ascribed to interleaflet coupling. As in the literature, a coexistence of liquid-ordered and solid-ordered domains was suggested for the DMPC-Chol and POPC-Chol bilayers; the Chol concentration dependences of Raman peak parameters were discussed in line with this suggestion, under the assumption that the different composition of coexisting domains conserves upon cooling. We demonstrated that the obtained Raman data disagree with the suggested domain coexistence if the domain sizes are substantially larger than the lipid layer thickness.

Normal vibrational modes of phospholipid bilayers observed by low-frequency Raman scattering.

Low-frequency Raman spectra of multilamellar vesicles made either of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) have been studied in a wide temperature range. Below 0^{∘}C two peaks are found at frequencies around 8-9 and 14-17cm^{-1} and attributed to the normal vibrational modes of the phospholipid bilayer, which are determined by the bilayer thickness and stiffness (elastic modulus). The spectral positions of the peaks depend on the temperature and the bilayer composition. It is suggested that the ratio of the intensities of the first and second peaks can serve as a measure of the interleaflet elastic coupling. The addition of cholesterol to the phospholipid bilayer leads to peak shift and broadening, which may be assigned to the composition heterogeneities commonly attributed to the lipid raft formation.

Dynamical Transitions at Low Temperatures in the Nearest Hydration Shell of Phospholipid Bilayers.

For the so-called dynamical transition from harmonic to anharmonic (or diffusive) motions in biological systems, the presence of hydration water is important. To explain the molecular mechanism of this transition, the information on molecular motions in the nearest hydration shell would be helpful. In this work, to study molecular motions in the nearest hydration shell of spin-labeled model biological membranes, a pulsed version of electron paramagnetic resonance, electron spin echo envelope modulation (ESEEM) spectroscopy, is used. For hydration by deuterium water, the 2H ESEEM frequency spectra resemble the solid-state 2H NMR line shape that is widely used for structural and dynamical studies. Two types of model membranes were investigated and compared: bilayers consisting of unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and bilayers consisting of fully saturated lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The lipid chain packing for the POPC bilayer is known to be more defective than that for the DPPC bilayer. For both the POPC and the DPPC bilayers, the 2H ESEEM NMR-like spectra showed a sharp narrowing between 180 and 190 K. From the other side, in both bilayers at 188 K, an inflection was observed for the temperature dependence of molecular motions detected by the spin relaxation of spin labels in the bilayer interior. It was concluded that dynamical transition in the bilayer interior is accompanied by an onset of isotropic water molecular dynamics in the nearest hydration shell of the bilayer with a rate of ∼105 s-1. Also, the 2H ESEEM NMR-like spectra in the POPC bilayer showed slight changes above 100 K that could be ascribed to another dynamical transition resulting in the appearance of restricted orientational motion of water molecules. These data also are interrelated with spin relaxation of spin labels in the POPC bilayer interior and support the hypothesis ascribing the transition at 100 K to excessive lipid chain flexibility.

Electron spin echo envelope modulation of molecular motions of deuterium nuclei.

Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy is a powerful technique for the study of hyperfine interactions between an unpaired electron and nearby nuclei in solids, and is employed in quantitative structural studies. Here, we describe the use of ESEEM to study the slow motion of deuterium nuclei using their nuclear quadrupole resonance (NQR) line shapes. Two ESEEM techniques were employed: the conventional three-pulse ESEEM experiment, π/2 - τ - π/2 - T- π/2 - τ - echo, and the four-pulse ESEEM, π/2 - τ - π/2 - T/2 - π - T/2 - π/2 - τ - echo, with the time variable T scanned in both cases. The nitroxide free radical 4-tert-butyliminomethyl-2,2,5,5-tetramethyl(d12)-3-imidazoline-1-oxyl with four deuterated methyl groups was investigated in a glassy ortho-terphenyl matrix over a wide temperature range. It was shown that four-pulse ESEEM allowed measurement of the nearly pure (2)H NQR line shape. Between 90K and 120K, the ESEEM spectra change drastically. At low temperatures, four-pulse ESEEM spectra show a Pake-like pattern, which evolves into a single line at higher temperatures, which is typical for NQR of rotating methyl CD3 groups. Comparison with literature data on NQR allows estimation of the reorientation rate, k. At ∼100K, where the spectral changes are most pronounced, k was found to be ∼10(5)s(-1). The spectral linewidths for the three-pulse ESEEM were found to decrease similarly with increasing temperature; so the three-pulse technique is also capable to detect motion of this type. The ESEEM approach, along with site-directed spin labeling, may be useful for detection of motional transitions near the spin labels in biological systems, when information on motion is required in a wide temperature range.

Flexibility of phospholipids with saturated and unsaturated chains studied by Raman scattering: the effect of cholesterol on dynamical and phase transitions.

Raman scattering spectra were obtained at 25-320 K for bilayers prepared from saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and mono-unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipids, with and without cholesterol. Raman intensities were measured at modes sensitive to lipid inter-chain interactions and/or intra-chain torsional motion (asymmetric CH2 stretching at 2880 cm(-1)) and to the conformational state of lipids (C-C stretching at 1130 cm(-1)). These intensities decreased with temperature, which could be ascribed to increased lipid flexibility. For cholesterol-free and cholesterol-containing DPPC bilayers, the decrease of Raman intensities observed above ∼200 K could be related to the phenomenon of dynamical transition known for biological systems near these temperatures. For a cholesterol-free POPC bilayer, the decrease of intensity for the asymmetric CH2 stretching mode started at a lower temperature, above 100 K, while the addition of cholesterol shifted this starting temperature to a more normal ∼200 K value. The low-temperature lipid flexibility in the case of POPC was related to the abundance of free-volume holes, which disappeared in presence of cholesterol. Near gel-fluid phase transitions, Raman intensities for cholesterol-free bilayers dropped sharply, while for cholesterol-containing bilayers, they changed smoothly.

Phospholipid bilayer relaxation dynamics as revealed by the pulsed electron-electron double resonance of spin labels.

Electron paramagnetic resonance (EPR) spectroscopy in the form of pulsed electron-electron double resonance (ELDOR) was applied to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid bilayers containing lipids that were spin-labeled at different carbon positions along the lipid acyl chain. Pulsed ELDOR detects motionally induced spin flips of nitrogen nuclei in the nitroxide spin labels, which manifests itself as magnetization transfer (MT) in the nitroxide EPR spectrum. The MT effect was observed over a wide temperature range (100-225 K) on a microsecond time scale. In line with a previous study on molecular glasses [N. P. Isaev and S. A. Dzuba, J. Chem. Phys. 135, 094508 (2011)], the motions that induce MT effect were suggested to have the same nature as those in dielectric secondary (ß) Johari-Goldstein fast relaxation. The results were compared with literature dielectric relaxation data for POPC bilayers, revealing some common features. Molecular motions resulting in MT are faster for deeper spin labels in the membrane interior. The addition of cholesterol to the bilayer suppresses the lipid motions near the steroid nucleus and accelerates the lipid motions beyond the steroid nucleus, in the bilayer interior. This finding was attributed to the lipid acyl chains being more ordered near the steroid nucleus and less ordered in the bilayer interior. The motions are absent in dry lipids, indicating that the motions are determined by intermolecular interactions in the bilayer.

Low-temperature dynamical and structural properties of saturated and monounsaturated phospholipid bilayers revealed by Raman and spin-label EPR spectroscopy.

The Raman scattering and pulsed electron paramagnetic resonance (EPR) of spin-labeled saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and monounsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid bilayers in a wide temperature range were studied. Raman spectra in the frequency range of CH2 and C-C stretching vibrations were obtained between 25 and 320 K. The modes sensitive to phospholipid interchain packing, interaction, and intrachain torsional motions (asymmetric CH2 stretching mode at 2880 cm(-1)) as well as conformational states (C-C stretching mode at 1130 cm(-1)) were analyzed. The Raman intensities of these modes significantly depend on the temperature in the gel phase. In the saturated phospholipid DPPC, changes in the temperature dependence of Raman intensities occur near the same temperature for the CH2 and C-C stretching modes, which is approximately 200-230 K. However, in monounsaturated POPC lipids, the temperature dependence for the C-C stretching mode at 1130 cm(-1) reveals a transition near 170 K, and the temperature dependence for the asymmetric CH2 stretching mode transition was near 120 K. For spin-labeled 5-DOXYL- and 16-DOXYL-stearic acids embedded into lipid bilayers, the anisotropic contribution to the electron spin-echo signal decays was interpreted as a result of nanosecond stochastic librations. The decay rates increased remarkably at temperatures above 200 K for DPPC and POPC, which is consistent with the Raman scattering data. A noticeable increase in the libration-induced relaxation rate was observed in POPC lipids above 120 K, and libration-induced relaxation was nearly temperature-independent in DPPC lipids up to 200 K. In the framework of the suggested interpretation, the bilayer structure of monounsaturated lipids contains defective, free volume-like places that provide freedom for phospholipid acyl-tail motions at low temperatures.

Nitrogen nuclear spin flips in nitroxide spin probes of different sizes in glassy o-terphenyl: possible relation with α- and ß-relaxations.

The pulsed electron-electron double resonance (ELDOR) technique was employed to study nitroxide spin probes of three different sizes dissolved in glassy o-terphenyl. A microwave pulse applied to the central hyperfine structure (hfs) component of the nitroxide electron paramagnetic resonance spectrum was followed by two echo-detecting pulses of different microwave frequency to probe the magnetization transfer (MT) to the low-field hfs component. The MT between hfs components is readily related to flips in the nitrogen nuclear spin, which in turn are induced by molecular motion. The MT on the time scale of tens of microseconds was observed over a wide temperature range, including temperatures near and well below the glass transition. For a bulky nitroxide, it was found that MT rates approach dielectric α (primary) relaxation frequencies reported for o-terphenyl in the literature. For small nitroxides, MT rates were found to match the frequencies of dielectric ß (secondary) Johari-Goldstein relaxation. The most probable motional mechanism inducing the nitrogen nuclear spin flips is large-angle angular jumps, between some orientations of unequal occupation probabilities. The pulsed ELDOR of nitroxide spin probes may provide additional insight into the nature of Johari-Goldstein relaxation in glassy media and may serve as a tool for studying this relaxation in substances consisting of non-rigid molecules (such as branched polymers) and in heterogeneous and non-polar systems (such as a core of biological membranes).

PELDOR analysis of enzyme-induced structural changes in damaged DNA duplexes.

PELDOR (pulsed electron-electron double resonance) spectroscopy was applied to determine spin-spin distances in spin-labeled DNA duplexes (13-mer and 17-mer) containing the damaged sites 8-oxoguanine or uncleavable abasic site analogue tetrahydrofuran. The lesions were located in one strand of the DNA, and two nitroxyl spin labels were attached at the 5'- and 3'-ends of the complementary strand. PELDOR data allow us to obtain distances between the two spin labels in DNAs, which turned out to be around 5 nm for the 13-mer DNA and around 6 nm for 17-mer DNA. Results of PELDOR measurements were supported by molecular dynamics calculations. Study of the interaction of DNA fragments with DNA repair enzyme 8-oxoguanine-DNA glycosylase from E. coli (Fpg protein) showed that this interaction leads to a noticeable decrease of the distance between spin labels, which indicates the enzyme-induced bending of the DNA duplex. This bending may be important for the mechanisms of recognition of damaged sites by DNA repair enzymes.

Conformational changes of lipids in bilayers at the dynamical transition near 200 K seen by Raman scattering.

Dynamical transition in biomolecules (proteins, membranes, etc.) is the phenomenon of increase of mean square atomic displacements, when temperature increases above 180-230 K. This increase is seen in neutron scattering and is ascribed to the increase of anharmonical motion of hydrogen atoms in biomolecules. In this work, Raman scattering study was performed for model synthetic membranes of dipalmitoylphosphatidylcholine in the spectral range of C-C vibrations (1050-1150 cm(-1)). It was demonstrated that dynamical transition is accompanied by appearance of newly achievable conformations of the lipid tails. In particular, the portion of the all-trans conformations decreases above the temperature of the dynamical transition. The data are compared with simple theoretical models.

Electron-nuclear double resonance study of molecular librations of nitroxides in molecular glasses: quantum effects at low temperatures, comparison with low-frequency Raman scattering.

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

On the possible manifestation of harmonic-anharmonic dynamical transition in glassy media in electron paramagnetic resonance of nitroxide spin probes.

Continuous wave (cw) electron paramagnetic resonance (EPR) and echo-detected (ED) EPR were applied to study molecular motions of nitroxide spin probes in glassy glycerol and o-terphenyl. A linear decrease with increasing temperature of the total splitting in the cw EPR line shape was observed at low temperatures in both solvents. Above some temperature points the temperature dependencies become sharper. Within the model of molecular librations, this behavior is in qualitative and quantitative agreement with the numerical data on neutron scattering and Mossbauer absorption for molecular glasses and biomolecules, where temperature dependence of the mean-squared amplitude of the vibrational motion was obtained. In analogy with these data the departure from linear temperature dependence in cw EPR may be ascribed to the transition from harmonic to anharmonic motion (this transition is called dynamical transition). ED EPR spectra were found to change drastically above 195 K in glycerol and above 245 K in o-terphenyl, indicating the appearance of anisotropic transverse spin relaxation. This appearance may also be attributed to the dynamical transition as an estimation shows the anisotropic relaxation rates for harmonic and anharmonic librational motions and because these temperature points correspond well to those known from neutron scattering for these solvents. The low sensitivity of ED EPR to harmonic motion and its high sensitivity to the anharmonic one suggests that ED EPR may serve as a sensitive tool to detect dynamical transition in glasses and biomolecules.

Location and aggregation of the spin-labeled peptide trichogin GA IV in a phospholipid membrane as revealed by pulsed EPR.

The lipopeptaibol trichogin GA IV is a 10 amino acid-long residue and alpha-aminoisobutyric acid-rich antibiotic peptide of fungal origin. TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) spin-labeled analogs of this membrane active peptide were investigated in hydrated bilayers of dipalmitoylphosphatidylcholine by electron spin echo envelope modulation (ESEEM) spectroscopy and pulsed electron-electron double resonance (PELDOR). Since, the ESEEM of the spin label appears to be strongly dependent on the presence of water molecules penetrated into the membrane, this phenomenon was used to study the location of this peptide in the membrane. This was achieved by comparing the ESEEM spectra for peptides labeled at different positions along the amino acid sequence with spectra known for lipids with spin labels at different positions along the hydrocarbon chain. To increase the ESEEM amplitude and to distinguish the hydrogen nuclei of water from lipid protons, membranes were hydrated with deuterated water. The PELDOR spectroscopy technique was chosen to study peptide aggregation and to determine the mutual distance distribution of the spin-labeled peptides in the membrane. The location of the peptide in the membrane and its aggregation state were found to be dependent on the peptide concentration. At a low peptide/lipid molar ratio (less than 1:100) the nonaggregated peptide chain of the trichogin molecules lie parallel to the membrane surface, with TOAC at the 4th residue located near the 9th-11th carbon positions of the sn-2 lipid chain. Increasing this ratio up to 1:20 leads to a change in peptide orientation, with the N-terminus of the peptide buried deeper into membrane. Under these conditions peptide aggregates are formed with a mean aggregate number of about N = 2. The aggregates are further characterized by a broad range of intermolecular distances (1.5-4 nm) between the labels at the N-terminal residues. The major population exhibits a distance of approximately 2.5 nm, which is of the same order as the length of the helical peptide. We suggest that the constituting monomers of the dimer are antiparallel oriented.

The functional sites of chlorophylls in D1 and D2 subunits of photosystem II identified by pulsed EPR.

The functional site of ChlZ, an auxiliary electron donor to P680+, was determined by pulsed ELDOR applied to a radical pair of YD * and Chlz+ in oriented PS II membranes from spinach. The radical-radical distance was determined to be 29.5 A and its direction was 50 degrees from the membrane normal, indicating that a chlorophyll on the D2 protein is responsible for the EPR Chlz+ signal. Spin polarized ESEEM (Electronin Spin Echo Envelop Modulation) of a 3Chl and QA - radical pair induced by a laser flash was observed in reaction center D1D2Cytb559 complex, in which QA was functionally reconstituted with DBMIB and reduced chemically. QA -ESEEM showed a characteristic oscillating time profile due to dipolar coupling with 3Chl. By fitting with the dipolar interaction parameters, the distance between 3Chl and QA - was determined to be 25.9 A, indicating that the accessory chlorophyll on the D1 protein is responsible for the 3Chl signal.

Restricted orientational motion of nitroxides in molecular glasses: direct estimation of the motional time scale basing on the comparative study of primary and stimulated electron spin echo decays.

A comparative study of anisotropic relaxation in two-pulse primary and three-pulse stimulated electron spin echo decays provides a direct way to distinguish fast (correlation time tau(c)<10(-6) s) and slow (tau(c)>10(-6) s) motions. Anisotropic relaxation is detected as a difference of the decay rates for different resonance field positions in anisotropic electron paramagnetic resonance spectra. For fast motion anisotropic relaxation influences the primary echo decay and does not influence the stimulated echo decay. For slow motion it is seen in both two-pulse echo and three-pulse stimulated echo decays. For nitroxide spin probes dissolved in glassy glycerol only fast motion was found below 200 K. Increase of temperature above 200 K results in the appearance of slow motion. Its amplitude increases rapidly with temperature increase. While in glycerol glass slow motion appears above glass transition temperature T(g), in ethanol glass it is observable below T(g). The scenario of motional dynamics in glasses is proposed which involves the broadening of the correlation time distribution with increasing temperature.

Selective excitation in pulsed EPR of a spin-correlated triplet-radical pair.

Experiments are described in which a low-amplitude microwave pulse excites only one out of three allowed transitions of the quinone radical (Q(A)(-)) in a spin-correlated triplet-radical pair 3PQ(A)(-) of the bacterial photosynthetic reaction center. A second high-amplitude pulse produces a FID whose temporal shape is strongly modulated with frequencies determined by electron-electron dipolar interaction in the pair. The FID is detected in both the in-phase and the out-of-phase channels. The out-of-phase FID is a result of switching off the magnetic dipolar interaction between 3P and Q(A)(-) due to decay of 3P during the time interval between the two pulses. Refocusing of FID by an additional non-selective pulse allows a dead-time free measurement of this modulation. The influence of the dead-time problem on the distance determination is discussed.

Electron dipole-dipole ESEEM in field-step ELDOR of nitroxide biradicals.

The use of a rapid stepping of the magnetic field for investigation of electron dipole-dipole ESEEM in pulsed X-band ELDOR is described. The magnetic field jump, synchronized with a microwave pumping pulse, is positioned between the second and the third pulses of the stimulated echo pulse sequence. This echo is measured as a function of the delay between the first and the second pulses. The data are analyzed for a Fourier transform resulting in a Pake resonance pattern. To remove the electron-nuclear contributions to ESEEM, time traces with pumping were divided by those without. This resulted in complete elimination of electron-nuclear contributions, which is seen from the absence of peaks at nuclear frequencies and the similarity of results for protonated and deuterated solvents. For increasing the electron-electron modulation depth, a scanning of the magnetic field during the microwave pumping is proposed. The interspin distances and their distribution are determined for two long-chained (ca. 2 nm) nitroxide biradicals in glassy toluene and in frozen nematic liquid crystal 4-cyano-4'-pentyl-biphenyl. For the latter solvent, the alignment of the axis connecting two nitroxides in biradicals is quantitatively analyzed.

Libration motion of guest spin probe molecules in organic glasses: CW EPR and electron spin echo study.

Echo-detected (ED) EPR spectra of nitroxide spin probes dissolved in glassy materials provide evidence that guest molecules in these media undergo fast librational motion. Theory of spin relaxation of a librating molecule is presented. The mean squared amplitude, , of this motion which can be derived from continuous wave (CW) EPR spectral splitting is found to depend linearly on temperature in the low temperature region. This may be ascribed to thermal harmonic vibrations. The slope of the linear dependence varies from glass to glass and seems to correlate with the strength of the intermolecular bonds and with a degree of the fragility of the glass. Above the glass transition temperature increases sharply. Different applications are discussed: study of molecular properties of glass, intracellular glass formation in plant tissues, structural investigations.

Pulsed EPR spin-probe study of intracellular glasses in seed and pollen.

EPR spectra of 3-carboxy-proxyl (CP) in dry biological tissues exhibited a temperature-dependent change in the principal value A'(zz) of the hyperfine interaction tensor. The A'(zz) value changed sharply at a particular temperature that was dependent on water content. At elevated water contents, the break occurred at lower temperatures and appeared to be associated with the melting of the cytoplasmic glassy state. To investigate the reason for the change in A'(zz), we employed echo-detected EPR (ED EPR) spectroscopy. The shape of the ED EPR spectrum revealed the presence of librational motion of the spin probe, a motion typically present in glassy materials. The similarities in temperature dependency of A'(zz) and librational motion of CP in pea seed axes indicated that the change in A'(zz) arose from librational motion. ED EPR measurements of CP as a function of water content in Typha latifolia pollen showed that librational motion decreased with decreasing water contents until a plateau or minimum was reached. ED EPR spectroscopy is a valuable technique for characterizing the relation between molecular motion and storage kinetics of dry seed and pollen.