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

Conducting a three-pulse DEER experiment without dead time: A review

Double electron-electron resonance spectroscopy (DEER, also known as PELDOR) is used to study spin-spin dipolar interactions between spin labels, at the nanoscale range of distances. The DEER effect is obtained as a signal generated by echo-forming microwave (mw) pulses with an additional mw pump pulse applied at a different frequency. It is important to carry out measurements without artefacts induced by overlap of the pulses in the time scale. Such an experiment without the dead-time effect is achieved using the 4-pulse (4p) DEER method. The analysis of the literature performed here shows however that the 3-pulse (3p) DEER can also be free of the dead time problem, for which there are two possibilities. The first occurs using a specially designed bimodal resonator, for which the two frequencies are completely decoupled. The second possibility, which can be implemented for any commercial spectrometer, involves the signal correction based on an additional “blank” measurement with the pump pulse applied outside the EPR resonance. A detailed comparison of the 3p and 4p DEER data obtained previously by Milov et al. [Appl. Magn. Reson. 41 (2011) 59–67] shows that 3p and 4p approaches give similar results. The advantages of the 3p DEER techniques are discussed

Probing Small-Angle Molecular Motions with EPR Spectroscopy: Dynamical Transition and Molecular Packing in Disordered Solids

Disordered molecular solids present a rather broad class of substances of different origin—amorphous polymers, materials for photonics and optoelectronics, amorphous pharmaceutics, simple molecular glass formers, and others. Frozen biological media in many respects also may be referred to this class. Theoretical description of dynamics and structure of disordered solids still does not exist, and only some phenomenological models can be developed to explain results of particular experiments. Among different experimental approaches, electron paramagnetic resonance (EPR) applied to spin probes and labels also can deliver useful information. EPR allows probing small-angle orientational molecular motions (molecular librations), which intrinsically are inherent to all molecular solids. EPR is employed in its conventional continuous wave (CW) and pulsed—electron spin echo (ESE)—versions. CW EPR spectra are sensitive to dynamical librations of molecules while ESE probes stochastic molecular librations. In this review, different manifestations of small-angle motions in EPR of spin probes and labels are discussed. It is shown that CW-EPR-detected dynamical librations provide information on dynamical transition in these media, similar to that explored with neutron scattering, and ESE-detected stochastic librations allow elucidating some features of nanoscale molecular packing. The possible EPR applications are analyzed for gel-phase lipid bilayers, for biological membranes interacting with proteins, peptides and cryoprotectants, for supercooled ionic liquids (ILs) and supercooled deep eutectic solvents (DESs), for globular proteins and intrinsically disordered proteins (IDPs), and for some other molecular solids

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

Ibuprofen in a Lipid Bilayer: Nanoscale Spatial Arrangement

Ibuprofen is a non-steroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic effects. Understanding the molecular mechanisms of drug interaction with cell membranes is important to improving drug delivery, uptake by cells, possible side effects, etc. Double electron-electron resonance spectroscopy (DEER, also known as PELDOR) provides information on the nanoscale spatial arrangement of spin-labeled molecules. Here, DEER was applied to study (mono-)spin-labeled ibuprofen (ibuprofen-SL) in a bilayer of palmitoyl-oleoyl-sn-glycerophosphocholine (POPC). The results obtained show that the ibuprofen-SL molecules are located within a plane in each bilayer leaflet. At their low molar concentration in the bilayer χ, the found surface concentration of ibuprofen-SL is two times higher than χ, which can be explained by alternative assembling in the two leaflets of the bilayer. When χ > 2 mol%, these assemblies merge. The findings shed new light on the nanoscale spatial arrangement of ibuprofen in biological membranes

Low-Temperature Dynamical Transition in Lipid Bilayers Detected by Spin-Label ESE Spectroscopy

Data on neutron scattering in biological systems show low-temperature dynamical transition between 170 and 230 K manifesting itself as a drastic increase of the atomic mean-squared displacement, aOE (c) x (2)>, detected for hydrogen atoms in the nano- to picosecond time scale. For spin-labeled systems, electron spin echo (ESE) spectroscopy-a pulsed version of electron paramagnetic resonance-is also capable of detection of dynamical transition. A two-pulse ESE decay in frozen matrixes is induced by spin relaxation arising from stochastic molecular librations, and allows to obtain the aOE (c)alpha (2)>tau (c) parameter, where aOE (c)alpha (2)> is a mean-squared angular amplitude of the motion and tau (c) is the correlation time lying in the sub- and nanosecond time ranges. In this work, the ESE technique was applied to spin-labeled amphiphilic molecules of three different kinds embedded in bilayers of fully saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and mono-unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids. Two-pulse ESE data revealed the appearance of stochastic librations above 130 K, with the parameter aOE (c)alpha (2)>tau (c) obeying the Arrhenius type of temperature dependence and increasing remarkably above 170-180 K. A comparison with a dry sample suggests that onset of motions is not related with lipid internal motions. Three-pulse ESE experiments (resulting in stimulated echos) in DPPC bilayers showed the appearance of slow molecular rotations above 170-180 K. For D2O-hydrated bilayers, ESE envelope modulation experiments indicate that isotropic water molecular motions in the nearest hydration shell of the bilayer appear with a rate of similar to 10(5) s(-1) in the narrow temperature range between 175 and 179 K. The similarity of the experimental data found for three different spin-labeled compounds suggests a cooperative character for the ESE-detected molecular motions. The data were interpreted within a model suggesting that dynamical transition is related with overcoming barriers, of 10-20 kJ/mol height, existing in the system for the molecular reorientations

Low-Temperature Molecular Motions in Lipid Bilayers in the Presence of Sugars: Insights into Cryoprotective Mechanisms

Sugars and sugar alcohols can stabilize biological systems under extreme conditions of desiccation and freezing. Phospholipid bilayers solvated by aqueous solutions of sucrose, trehalose, and sorbitol at concentrations of 0.2 and 1 M and containing incorporated spin-labeled stearic acids were studied by electron spin echo (ESE) spectroscopy, a pulsed version of electron paramagnetic resonance (EPR). The phospholipids were 1,2-dipalmitoyl-<i>sn</i>-glycero-3-phosphocholine (DPPC), and the stearic acids were labeled with nitroxide 4,4-dimethyl-oxazolidine-1-oxyl (DOXYL) attached rigidly at either the 5th or 16th carbon positions. The ratio of the echo time traces for the two field positions in the EPR spectrum possessing the largest and smallest anisotropies gave the anisotropic contribution to the echo decay, which obeys exponential time dependence with good accuracy. At low temperatures, the anisotropic contribution is induced by stochastic (or diffusive) orientational vibrations of the molecule as a whole (i.e., stochastic molecular librations), with the exponential decay rate <i>W</i> _anis proportional to <α²>τ_c, where <α²> is the mean angular amplitude of the motion and τ_c is the correlation time. In all cases, it was found that <i>W</i> _anis begins to increase sharply above 170–200 K, which was ascribed to the dynamical transition known for biological systems at these temperatures. For hydration by the sucrose and trehalose solutions, <i>W</i> _anis was found to increase noticeably also above ∼120 K, which was explained by bilayer expansion due to direct bonding of sugar molecules to the bilayer surface. The <i>W</i> _anis temperature dependencies were found to be close to those obtained for the simple systems of the nitroxide spin probe TEMPONE in aqueous sorbitol and sugar 1 M solutions. This correlation suggests a possible mechanism of cryoprotective action of sorbitol and sugars due to the similarity of low-temperature motions in the membrane and in the cryoprotectant-containing surrounding liquid

Peptide-membrane binding is not enough to explain bioactivity: A case study

Membrane-active peptides are a promising class of antimicrobial and anticancer therapeutics. For this reason, their molecular mechanisms of action are currently actively investigated. By exploiting Electron Paramagnetic Resonance, we study the membrane interaction of two spin-labeled analogs of the antimicrobial and cytotoxic peptide trichogin GA IV (Tri), with opposite bioactivity: Tri(Api8), able to selectively kill cancer cells, and Tri (Leu4), which is completely nontoxic. In our attempt to determine the molecular basis of their different biological activity, we investigate peptide impact on the lateral organization of lipid membranes, peptide localization and oligomerization, in the zwitter-ionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model membrane We show that, despite their divergent bioactivity, both peptide analogs (i) are membrane-bound, (ii) display a weak tendency to oligomerization, and (iii) do not induce significant lipid rearrangement. Conversely, literature data show that the parent peptide trichogin, which is cytotoxic without any selectivity, is strongly prone to dimerization and affects the reorganization of POPC membranes. Its dimers are involved in the rotation around the peptide helix, as observed at cryogenic temperatures in the millisecond timescale. Since this latter behavior is not observed for the inactive Tri(Leu4), we propose that for short-length peptides as trichogin oligomerization and molecular motions are crucial for bioactivity, and membrane binding alone is not enough to predict or explain it. We envisage that small changes in the peptide sequence that affect only their ability to oligomerize, or their molecular motions inside the membrane, can tune the peptide activity on membranes of different compositions