Assembly of galvinoxyl doped in polymer-fullerene photovoltaic blends.

Galvinoxyl (Gx) is a stable free radical used as a dopant in active layers of organic solar cells. Here, the nanoscale arrangement of Gx molecules in a composite of the PCDTBT polymer and modified C60 fullerene, PCBM, was studied using a two-pulse electron spin echo (ESE) technique. The results show that the Gx molecules assemble into clusters, which can be described by the model of 8 molecules on the surface of a sphere with a radius of 2.0 nm. Such a structure can arise due to the octahedral packing of 6 PCBM molecules surrounded by 8 Gx molecules. ESE decays also indicate that these clusters repel each other, forming a quasi-regular nanostructure in the matrix. The Gx concentration of 2 wt% at which clusters appear correlates with the literature data on the Gx-induced enhancement of photocurrent, which provides structural insight into the possible molecular mechanism of this enhancement.

Glass-like behavior of intercalated organic solvents in graphite oxide detected by spin-probe EPR.

Membranes based on graphite oxide (GO) are promising materials for the separation of polar liquids and gases. Understanding the properties of solvents immersed in GO is important for the development of various technological applications. Here, the molecular motions of the TEMPO nitroxide spin probe in acetonitrile intercalated into the GO inter-plane space were studied using electron paramagnetic resonance (EPR), including its pulsed version, and electron spin echo (ESE). For a sample containing 75% acetonitrile relative to equilibrium sorption at room temperature, ESE-detected stochastic librations were observed for TEMPO molecules above 135 K. Since these librations are an inherent property of molecular glasses, this fact indicates that intercalated acetonitrile forms a two-dimensional glass state. Above 225 K, an acceleration of stochastic librations was observed, indicating the manifestation of a glass-like dynamical cross-over. Continuous wave (CW) EPR spectra of TEMPO showed the absence of overall tumbling motions in the entire investigated temperature range of up to 340 K, indicating that the intercalated acetonitrile does not behave as a bulk liquid (the melting point of acetonitrile is 229 K). Dynamical librations of TEMPO molecules detected by CW EPR were found to accelerate above 240 K.

Inner Amino Acid Contacts Are Key Factors of Multistage Structural Rearrangements of DNA and Affect Substrate Specificity of Apurinic/Apyrimidinic Endonuclease APE1.

Apurinic/apyrimidinic endonuclease 1 (APE1) is one of the most important enzymes in base excision repair. Studies on this enzyme have been conducted for a long time, but some aspects of its activity remain poorly understood. One such question concerns the mechanism of damaged-nucleotide recognition by the enzyme, and the answer could shed light on substrate specificity control in all enzymes of this class. In the present study, by pulsed electron-electron double resonance (DEER, also known as PELDOR) spectroscopy and pre-steady-state kinetic analysis along with wild-type (WT) APE1 from Danio rerio (zAPE1) or three mutants (carrying substitution N253G, A254G, or E260A), we aimed to elucidate the molecular events in the process of damage recognition. The data revealed that the zAPE1 mutant E260A has much higher activity toward DNA substrates containing 5,6-dihydro-2'-deoxyuridine (DHU), 2'-deoxyuridine (dU), alpha-2'-deoxyadenosine (αA), or 1,N6-ethenoadenosine (εA). Examination of conformational changes in DNA clearly revealed multistep DNA rearrangements during the formation of the catalytic complex. These structural rearrangements of DNA are directly associated with the capacity of damaged DNA for enzyme-induced bending and unwinding, which are required for eversion of the damaged nucleotide from the DNA duplex and for its placement into the active site of the enzyme. Taken together, the results experimentally prove the factors that control substrate specificity of the AP endonuclease zAPE1.

Spin-Labeled Diclofenac: Synthesis and Interaction with Lipid Membranes.

Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) from the group of phenylacetic acid derivatives, which has analgesic, anti-inflammatory and antipyretic properties. The interaction of non-steroidal anti-inflammatory drugs with cell membranes can affect their physicochemical properties, which, in turn, can cause a number of side effects in the use of these drugs. Electron paramagnetic resonance (EPR) spectroscopy could be used to study the interaction of diclofenac with a membrane, if its spin-labeled analogs existed. This paper describes the synthesis of spin-labeled diclofenac (diclofenac-SL), which consists of a simple sequence of transformations such as iodination, esterification, Sonogashira cross-coupling, oxidation and saponification. EPR spectra showed that diclofenac-SL binds to a lipid membrane composed of palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). 2H electron spin echo spectroscopy (ESEEM) was used to determine the position of the diclofenac-SL relative to the membrane surface. It was established that its average depth of immersion corresponds to the 5th position of the carbon atom in the lipid chain.

Structural properties of supercooled deep eutectic solvents: choline chloride-thiourea compared to reline.

Deep eutectic solvents (DESs) are eutectic mixtures of hydrogen bond acceptors and hydrogen bond donors which melt at much lower temperatures than the individual components. DESs are attracting growing interest because of a large variety of possible technological applications. Here, supercooled DESs consisting of choline chloride-urea (1 : 2) (reline) and of choline chloride-thiourea (1 : 2) (ChCl-thiourea), with introduced nitroxide spin probe tempone, were studied by electron paramagnetic resonance (EPR) spectroscopy. Conventional continuous wave (CW) EPR spectra showed the coexistence of solid and liquid microphases, with microviscosity of ∼ 10 P in the latter case. CW EPR spectra taken at different temperatures for ChCl-thiourea showed isosbestic points, which indicates that two phases are separated by sharp boundaries; for reline these points are rather diffuse, which in turn implies diffuse boundaries. Stochastic molecular librations detected by pulsed EPR possess the ability for elucidating nanoscale features of molecular packing; the data obtained showed a drastic difference for the onset of these motions for ChCl-thiourea and for reline, which was interpreted as evidence that the rigidity of molecular packing for ChCl-thiourea is stronger than that for reline. The temperature dependence of stochastic molecular librations for reline was found to be similar to that known for lipid bilayers and globular proteins, which indicates the proximity of the characteristics of molecular packing in these molecular systems.

Synthesis of Spin-Labeled Ibuprofen and Its Interaction with Lipid Membranes.

Ibuprofen is a non-steroidal anti-inflammatory drug possessing analgesic and antipyretic activity. Electron paramagnetic resonance (EPR) spectroscopy could be applied to study its interaction with biological membranes and proteins if its spin-labeled analogs were synthesized. Here, a simple sequence of ibuprofen transformations-nitration, esterification, reduction, Sandmeyer reaction, Sonogashira cross-coupling, oxidation and saponification-was developed to attain this goal. The synthesis resulted in spin-labeled ibuprofen (ibuprofen-SL) in which the spin label TEMPOL is attached to the benzene ring. EPR spectra confirmed interaction of ibuprofen-SL with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers. Using 2H electron spin echo envelope modulation (ESEEM) spectroscopy, ibuprofen-SL was found to be embedded into the hydrophobic bilayer interior.

Probing the E/K Peptide Coiled-Coil Assembly by Double Electron-Electron Resonance and Circular Dichroism.

Double electron-electron resonance (DEER, also known as PELDOR) and circular dichroism (CD) spectroscopies were explored for the purpose of studying the specificity of the conformation of peptides induced by their assembly into a self-recognizing system. The E and K peptides are known to form a coiled-coil heterodimer. Two paramagnetic TOAC α-amino acid residues were incorporated into each of the peptides (denoted as K** and E**), and a three-dimensional structural investigation in the presence or absence of their unlabeled counterparts E and K was performed. The TOAC spin-labels, replacing two Ala residues in each compound, are covalently and quasi-rigidly connected to the peptide backbone. They are known not to disturb the native structure, so that any conformational change can easily be monitored and assigned. DEER spectroscopy enables the measurement of the intramolecular electron spin-spin distance distribution between the two TOAC labels, within a length range of 1.5-8 nm. This method allows the individual conformational changes for the K**, K**/E, E**, and E**/K molecules to be investigated in glassy frozen solutions. Our data reveal that the conformations of the E** and K** peptides are strongly influenced by the presence of their counterparts. The results are discussed with those from CD spectroscopy and with reference to the already reported nuclear magnetic resonance data. We conclude that the combined DEER/TOAC approach allows us to obtain accurate and reliable information about the conformation of the peptides before and after their assembly into coiled-coil heterodimers. Applications of this induced fit method to other two-component, but more complex, systems, like a receptor and antagonists, a receptor and a hormone, and an enzyme and a ligand, are discussed.

Clustering of Stearic Acids in Model Phospholipid Membranes Revealed by Double Electron-Electron Resonance.

Free fatty acids play various important roles in biological membranes. Double electron-electron resonance spectroscopy (DEER, also known as PELDOR) of spin-labeled biomolecules is capable of studying magnetic dipole-dipole (d-d) interactions between spin labels at the nanoscale range of distances. Here, DEER is applied to study intermolecular d-d interactions between doxyl-spin-labeled stearic acids (DSA) in gel-phase phospholipid bilayers composed either of an equimolecular mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine or of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. DEER data obtained for different DSA concentrations showed that DSA molecules at their concentration in the bilayer χ larger than 0.5 mol % are assembled into lateral lipid-mediated clusters, with a characteristic intermolecular distance of 2 nm. Some evidences were obtained indicating that clusters may consist of "subclusters", alternatively appearing in two opposite leaflets. Conventional electron paramagnetic resonance (EPR) spectra for the gel-phase bilayers showed that for χ larger than 2 mol % the molecules in the clusters stick together, forming oligomers. Room-temperature EPR spectra for the liquid-crystalline phase were found to change noticeably for χ larger than 0.5 mol %, which may indicate the clustering in a liquid-crystalline phase similar to that observed by DEER in the gel phase.

Evidence for an Ordering Transition near 120 K in an Intrinsically Disordered Protein, Casein.

Intrinsically disordered proteins (IDPs) are proteins that possess large unstructured regions. Their importance is increasingly recognized in biology but their characterization remains a challenging task. We employed field swept Electron Spin Echoes in pulsed EPR to investigate low-temperature stochastic molecular librations in a spin-labeled IDP, casein (the main protein of milk). For comparison, a spin-labeled globular protein, hen egg white lysozyme, is also investigated. For casein these motions were found to start at 100 K while for lysozyme only above 130 K, which was ascribed to a denser and more ordered molecular packing in lysozyme. However, above 120 K, the motions in casein were found to depend on temperature much slower than those in lysozyme. This abrupt change in casein was assigned to an ordering transition in which peptide residues rearrange making the molecular packing more rigid and/or more cohesive. The found features of molecular motions in these two proteins turned out to be very similar to those known for gel-phase lipid bilayers composed of conformationally ordered and conformationally disordered lipids. This analogy with a simpler molecular system may appear helpful for elucidation properties of molecular packing in IDPs.

Membrane-Sugar Interactions Probed by Low-Frequency Raman Spectroscopy: The Monolayer Adsorption Model.

Small sugars are known to stabilize biological membranes under extreme conditions of freezing and desiccation. The proposed mechanisms of stabilization suggest membrane-sugar interactions to be either attractive or repulsive. To obtain new insight into the problem, we use a recently developed low-frequency Raman scattering approach which allows detecting membrane mechanical vibrations. For model membranes of palmitoyl-oleoyl-glycero-phosphocholine (POPC) hydrated in aqueous sucrose and trehalose solutions, we studied the Raman peak between 12 and 15 cm-1 that is attributed to an eigenmode of the normal mechanical vibrations of a lipid monolayer. For both sugars, similar results were obtained. With an increase in sugar concentration in solution, the frequency position of the peak was found to decrease by ∼13% which was interpreted as a consequence of the membrane thickening due sugar monolayer adsorption on the membrane surface. The concentration dependence of the peak frequency position was satisfactorily described by a Langmuir monolayer adsorption model. It is concluded that, at small sugar concentrations (less than 0.2 M), the membrane-sugar interactions are attractive, while at higher concentrations (more than 0.4 M) the attraction disappears. The data obtained show that one sugar molecule on the surface interacts with approximately 3-4 polar lipid heads.

Substrate specificity of human apurinic/apyrimidinic endonuclease APE1 in the nucleotide incision repair pathway.

Human apurinic/apyrimidinic (AP) endonuclease APE1 catalyses the hydrolysis of phosphodiester bonds on the 5' side of an AP-site (in the base excision repair pathway) and of some damaged nucleotides (in the nucleotide incision repair pathway). The range of substrate specificity includes structurally unrelated damaged nucleotides. Here, to examine the mechanism of broad substrate specificity of APE1, we performed pulsed electron-electron double resonance (PELDOR) spectroscopy and pre-steady-state kinetic analysis with Förster resonance energy transfer (FRET) detection of DNA conformational changes during DNA binding and lesion recognition. Equilibrium PELDOR and kinetic FRET data revealed that DNA binding by APE1 leads to noticeable damage-dependent bending of a DNA duplex. Molecular dynamics simulations showed that the damaged nucleotide is everted from the DNA helix and placed into the enzyme's binding pocket, which is formed by Asn-174, Asn-212, Asn-229, Ala-230, Phe-266 and Trp-280. Nevertheless, no damage-specific contacts were detected between these amino acid residues in the active site of the enzyme and model damaged substrates containing 1,N6-ethenoadenosine, α-adenosine, 5,6-dihydrouridine or F-site. These data suggest that the substrate specificity of APE1 is controlled by the ability of a damaged nucleotide to flip out from the DNA duplex in response to an enzyme-induced DNA distortion.

A Temperature-Driven, Reversible, Helical-Handedness Inversion in Peptaibol Analogues Tuned by the C-Terminal Capping Moiety.

Trichogin is a natural peptide endowed with antimicrobial and antitumor activity. A member of the peptaibol family, trichogin possesses a C-terminal amino alcohol. In the past, this moiety was substituted for a methyl ester for synthetic purposes and it was observed that this apparently slight modification caused significant changes in the peptide bioactivity. With the aim of understanding the reasons behind such observations, a detailed spectroscopic study on a number of trichogin analogues has been performed. Herein, data obtained from synchrotron radiation circular dichroism, NMR spectroscopy, and fluorescence spectroscopy in organic solvents at cryogenic temperatures are compared with those independently acquired by means of EPR spectroscopy at 80 K. It is unambiguously revealed that the presence of a reversible, temperature-driven, screw-sense interconversion from a right- to left-handed helix is determined by the C-terminal capping moiety. Data demonstrate, for the first time, the key role of a C-terminal methyl ester in promoting peptide screw-sense inversion.

Alamethicin self-assembling in lipid membranes: concentration dependence from pulsed EPR of spin labels.

The antimicrobial action of the peptide antibiotic alamethicin (Alm) is commonly related to peptide self-assembling resulting in the formation of voltage-dependent channels in bacterial membranes, which induces ion permeation. To obtain a deeper insight into the mechanism of channel formation, it is useful to know the dependence of self-assembling on peptide concentration. With this aim, we studied Alm F50/5 spin-labeled analogs in a model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane, for peptide-to-lipid (P/L) ratios varying between 1/1500 and 1/100. Pulsed electron-electron double resonance (PELDOR) spectroscopy reveals that even at the lowest concentration investigated, the Alm molecules assemble into dimers. Moreover, under these conditions, electron spin echo envelope modulation (ESEEM) spectroscopy of D2O-hydrated membranes shows an abrupt change from the in-plane to the trans-membrane orientation of the peptide. Therefore, we hypothesize that dimer formation and peptide reorientation are concurrent processes and represent the initial step of peptide self-assembling. By increasing peptide concentration, higher oligomers are formed. A simple kinetic model of equilibrium among monomers, dimers, and pentamers allows for satisfactorily describing the experimental PELDOR data. The inter-label distances in the oligomers obtained from PELDOR experiments become better resolved with increasing P/L ratio, thus suggesting that the supramolecular organization of the higher-order oligomers becomes more defined.

Conformational properties, membrane interaction, and antibacterial activity of the peptaibiotic chalciporin A: Multitechnique spectroscopic and biophysical investigations on the natural compound and labeled analogs.

In this work, an extensive set of spectroscopic and biophysical techniques (including FT-IR absorption, CD, 2D-NMR, fluorescence, and CW/PELDOR EPR) was used to study the conformational preferences, membrane interaction, and bioactivity properties of the naturally occurring synthetic 14-mer peptaibiotic chalciporin A, characterized by a relatively low (≈20%), uncommon proportion of the strongly helicogenic Aib residue. In addition to the unlabeled peptide, we gained in-depth information from the study of two labeled analogs, characterized by one or two residues of the helicogenic, nitroxyl radical-containing TOAC. All three compounds were prepared using the SPPS methodology, which was carefully modified in the course of the syntheses of TOAC-labeled analogs in view of the poorly reactive α-amino function of this very bulky residue and the specific requirements of its free-radical side chain. Despite its potentially high flexibility, our results point to a predominant, partly amphiphilic, α-helical conformation for this peptaibiotic. Therefore, not surprisingly, we found an effective membrane affinity and a remarkable penetration propensity. However, chalciporin A exhibits a selectivity in its antibacterial activity not in agreement with that typical of the other members of this peptide class.

Microscopic rigidity and heterogeneity of ionic liquids probed by stochastic molecular librations of the dissolved nitroxides.

Microscopic molecular organization and heterogeneities in ionic liquids (ILs) are of significant fundamental and applied interest. Although many theoretical studies have been dedicated to this topic, the development of experimental methods for studying such heterogeneities is still in demand. In this work we propose a new approach for the characterization of microscopic rigidity and heterogeneities in ILs using stochastic librations (small angle motions) of the nitroxide radicals as a probe. Stable nitroxides are dissolved in ILs, which are then shock-frozen and investigated using pulse Electron Paramagnetic Resonance (EPR) at variable temperatures. Stochastic molecular librations of nitroxides depend on local rigidity of a medium and manifest themselves in different electron dephasing times across the EPR spectrum. The use of advanced spiro-cyclohexane-substituted nitroxides allows implementation of this approach at temperatures up to ∼150 K and above. Remarkably, we have found that librational motions in ILs arise at much lower temperatures (∼75 K for [Bmim]BF4, [Bmim]PF6, [C10mim]BF4) compared to common organic solvents. This can be rationalized by smaller local rigidity of the medium in ILs, most likely due to localization of nitroxides in low-density heterogeneities formed by non-polar alkyl chains. The structure, size, solubility and other properties of nitroxides are adjustable for particular tasks, therefore the proposed approach can potentially be implemented to probe the rigidity and heterogeneities of any ILs, thus providing vital insights into their molecular-scale self-organization.

Dynamical transition in molecular glasses and proteins observed by spin relaxation of nitroxide spin probes and labels.

In glassy substances and biological media, dynamical transitions are observed in neutron scattering that manifests itself as deviations of the translational mean-squared displacement, 〈x2〉, of hydrogen atoms from harmonic dynamics. In biological media, the deviation occurs at two temperature intervals, at ∼100-150 K and at ∼170-230 K, and it is attributed to the motion of methyl groups in the former case and to the transition from harmonic to anharmonic or diffusive motions in the latter case. In this work, electron spin echo (ESE) spectroscopy-a pulsed version of electron paramagnetic resonance-is applied to study the spin relaxation of nitroxide spin probes and labels introduced in molecular glass former o-terphenyl and in protein lysozyme. The anisotropic contribution to the rate of the two-pulse ESE decay, ΔW, is induced by spin relaxation appearing because of restricted orientational stochastic molecular motion; it is proportional to 〈α2〉τc, where 〈α2〉 is the mean-squared angle of reorientation of the nitroxide molecule around the equilibrium position and τc is the correlation time of reorientation. The ESE time window allows us to study motions with τc < 10-7 s. For glassy o-terphenyl, the 〈α2〉τc temperature dependence shows a transition near 240 K, which is in agreement with the literature data on 〈x2〉. For spin probes of essentially different size, the obtained data were found to be close, which evidences that motion is cooperative, involving a nanocluster of several neighboring molecules. For the dry lysozyme, the 〈α2〉τc values below 260 K were found to linearly depend on the temperature in the same way as it was observed in neutron scattering for 〈x2〉. As spin relaxation is influenced only by stochastic motion, the harmonic motions seen in ESE must be overdamped. In the hydrated lysozyme, ESE data show transitions near 130 K for all nitroxides, near 160 K for the probe located in the hydration layer, and near 180 K for the label in the protein interior. For this system, the two latter transitions are not observed in neutron scattering. The ESE-detected transitions are suggested to be related with water dynamics in the nearest hydration shell: with water glass transition near 130 K and with the onset of overall water molecular reorientations near 180 K; the disagreement with neutron scattering is ascribed to the larger time window for ESE-detected motions.

Communication: Alamethicin can capture lipid-like molecules in the membrane.

Alamethicin (Alm) is a 19-mer antimicrobial peptide produced by fungus Trichoderma viride. Above a threshold concentration, Alm forms pores across the membrane, providing a mechanism of its antimicrobial action. Here we show that at a small concentration which is below the threshold value, Alm participates in formation of nanoscale lipid-mediated clusters of guest lipid-like molecules in the membrane. These results are obtained by electron spin echo (ESE) technique-a pulsed version of electron paramagnetic resonance-on spin-labeled stearic acid in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer with Alm added at 1/200 peptide-to-lipid ratio. ESE decay measurements are interpreted assuming that stearic acid molecules in the membrane are assembling around the Alm molecule. One may suggest that this Alm capturing effect on the guest lipid-like molecules could be important for the peptide antimicrobial action.

Communication: Orientational self-ordering of spin-labeled cholesterol analogs in lipid bilayers in diluted conditions.

Lipid-cholesterol interactions are responsible for different properties of biological membranes including those determining formation in the membrane of spatial inhomogeneities (lipid rafts). To get new information on these interactions, electron spin echo (ESE) spectroscopy, which is a pulsed version of electron paramagnetic resonance (EPR), was applied to study 3ß-doxyl-5α-cholestane (DCh), a spin-labeled analog of cholesterol, in phospholipid bilayer consisted of equimolecular mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine. DCh concentration in the bilayer was between 0.1 mol.% and 4 mol.%. For comparison, a reference system containing a spin-labeled 5-doxyl-stearic acid (5-DSA) instead of DCh was studied as well. The effects of "instantaneous diffusion" in ESE decay and in echo-detected (ED) EPR spectra were explored for both systems. The reference system showed good agreement with the theoretical prediction for the model of spin labels of randomly distributed orientations, but the DCh system demonstrated remarkably smaller effects. The results were explained by assuming that neighboring DCh molecules are oriented in a correlative way. However, this correlation does not imply the formation of clusters of cholesterol molecules, because conventional continuous wave EPR spectra did not show the typical broadening due to aggregation of spin labels and the observed ESE decay was not faster than in the reference system. So the obtained data evidence that cholesterol molecules at low concentrations in biological membranes can interact via large distances of several nanometers which results in their orientational self-ordering.

Spatial Arrangement of the Drug Ibuprofen in a Model Membrane in the Presence of Lipid Rafts.

Many pharmaceutical drugs are known to interact with lipid membranes through nonspecific molecular interactions, which affect their therapeutic effect. Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID) and one of the most commonly prescribed. In the presence of cholesterol, lipid bilayers can separate into nanoscale liquid-disordered and liquid-ordered structures, the latter known as lipid rafts. Here, we study spin-labeled ibuprofen (ibuprofen-SL) in the model membrane consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol in the molar ratio of (0.5-0.5xchol)/(0.5-0.5xchol)/xchol. Electron paramagnetic resonance (EPR) spectroscopy is employed, along with its pulsed version of double electron-electron resonance (DEER, also known as PELDOR). The data obtained indicate lateral lipid-mediated clustering of ibuprofen-SL molecules with a local surface density noticeably larger than that expected for random lateral distribution. In the absence of cholesterol, the data can be interpreted as indicating alternating clustering in two opposing leaflets of the bilayer. In the presence of cholesterol, for xchol ≥ 20 mol %, the results show that ibuprofen-SL molecules have a quasi-regular lateral distribution, with a "superlattice" parameter of ∼3.0 nm. This regularity can be explained by the entrapment of ibuprofen-SL molecules by lipid rafts known to exist in this system with the additional assumption that lipid rafts have a nanoscale substructure.

Self-assembly of spin-labeled antimicrobial peptides magainin 2 and PGLa in lipid bilayers.

The cationic antimicrobial peptides PGLa and magainin 2 (Mag2) are known for their antimicrobial activity and synergistic enhancement in antimicrobial and membrane leakage assays. Further use of peptides in combinatory therapy requires knowledge of the mechanisms of action of both individual peptides and their mixtures. Here, electron paramagnetic resonance (EPR), double electron-electron resonance (DEER, also known as PELDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies were applied to study self-assembly and localization of spin-labeled PGLa and Mag2 in POPE/POPG membranes with a wide range of peptide/lipid ratios (P/L) from ∼1/1500 to 1/50. EPR and DEER data showed that both peptides tend to organize in clusters, which occurs already at the lowest peptide/lipid molar ratio of 1/1500 (0.067 mol%). For individual peptides, these clusters are quite dense with intermolecular distances of the order of ∼2 nm. In the presence of a synergistic peptide partner, these homo-clusters are transformed into lipid-diluted hetero-clusters. These clusters are characterized by a local surface density that is several times higher than expected from a random distribution. ESEEM data indicate a slightly different insertion depth of peptides in hetero-clusters when compared to homo-clusters.