Nanoclusters of Guest Molecules in Lipid Rafts of a Model Membrane Revealed by Pulsed Dipolar EPR Spectroscopy.

Plasma membranes are known to segregate into liquid disordered and ordered nanoscale phases, the latter being called lipid rafts. The structure, lipid composition, and function of lipid rafts have been the subject of numerous studies using a variety of experimental and computational methods. Double electron-electron resonance (DEER, also known as PELDOR) is a member of the pulsed dipole EPR spectroscopy (PDS) family of techniques, allowing the study of nanoscale distances between spin-labeled molecules.

Evidence for capture of spin-labeled ibuprofen drug molecules by lipid rafts in model membranes.

Lipid rafts are lipid-cholesterol nanostructures thought to exist in cell membranes, which are characterized by higher ordering compared to their surroundings. Ibuprofen and other non-steroidal anti-inflammatory drugs (NSAIDs) have a high affinity for phospholipid membranes and can alter their structure and biological properties. Here we use electron paramagnetic resonance (EPR) in its pulsed electron spin echo (ESE) version to study spin-labeled ibuprofen (ibuprofen-SL) in a raft-mimicking bilayer, which consists of an equimolar mixture of the phospholipids dioleoyl-glycero-phosphocholine (DOPC) and dipalmitoyl-glycero-phosphocholine (DPPC), with cholesterol added in various proportions.

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.

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.

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 C 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.

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).

Localization of the ibuprofen molecule in model lipid membranes revealed by spin-label-enhanced NMR relaxation.

Non-steroidal anti-inflammatory drugs (NSAIDs) have antipyretic, anti-inflammatory and analgesic effects, and can be used in the treatment of various diseases. These drugs have also a number of side effects, which may be related to their interaction with lipid membranes. In this study, we use the spin-labeled NSAID ibuprofen (ibuprofen-SL) as a relaxation enhancer to study its interaction with model lipid membranes employing liquid-state H NMR at 500 MHz.

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.

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.

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.

The mechanism of damage recognition by apurinic/apyrimidinic endonuclease Nfo from Escherichia coli.

Apurinic/apyrimidinic (AP) endonuclease Nfo from Escherichia coli recognises AP sites in DNA and catalyses phosphodiester bond cleavage on the 5' side of AP sites and some damaged or undamaged nucleotides. Here, the mechanism of target nucleotide recognition by Nfo was analysed by pulsed electron-electron double resonance (PELDOR, also known as DEER) spectroscopy and pre-steady-state kinetic analysis with Förster resonance energy transfer detection of DNA conformational changes during DNA binding. The efficiency of endonucleolytic cleavage of a target nucleotide in model DNA substrates was ranked as (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran [F-site] > 5,6-dihydro-2'-deoxyuridine > α-anomer of 2'-deoxyadenosine >2'-deoxyuridine > undamaged DNA.

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.

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(Api), able to selectively kill cancer cells, and Tri(Leu), which is completely nontoxic.

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.

Is Cys(MTSL) the Best α-Amino Acid Residue to Electron Spin Labeling of Synthetically Accessible Peptide Molecules with Nitroxides?

Electron paramagnetic resonance spectroscopy, particularly its pulse technique double electron-electron resonance (DEER) (also termed PELDOR), is rapidly becoming an extremely useful tool for the experimental determination of side chain-to-side chain distances between free radicals in molecules fundamental for life, such as polypeptides. Among appropriate probes, the most popular are undoubtedly nitroxide electron spin labels. In this context, suitable biosynthetically derived, helical regions of proteins, along with synthetic peptides with amphiphilic properties and antibacterial activities, are the most extensively investigated compounds.

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--glycero-3-phosphocholine and 1,2-dioleoyl--glycero-3-phosphocholine or of 1-palmitoyl-2-oleoyl--glycero-3-phosphocholine.

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).

Double Electron-Electron Resonance of Spin-Labeled Cholestane in Model Membranes: Evidence for Substructures inside the Lipid Rafts.

Plasma membranes are assumed to be highly compartmentalized, which is believed to be important for the membrane protein functionality. The liquid ordered-disordered phase segregation in the membranes results in nanoscale liquid-ordered assemblies-lipid rafts. Double electron-electron resonance spectroscopy (DEER, also known as PELDOR) is sensitive to spin-spin dipolar interactions between spin labels at the nanoscale range of distances.

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.

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.

Electron spin echo detection of stochastic molecular librations: Non-cooperative motions on solid surface.

In frozen biological media and molecular glasses only restricted motions exist; because of the weakness and disorder of intermolecular bonds these motions may have stochastic nature. Electron spin echo (ESE) spectroscopy of spin-labeled molecules allows detecting their restricted stochastic rotations (stochastic molecular librations). As in molecular disordered media motions may be highly cooperative, it would be desirable to investigate their spectroscopic manifestation also in the systems where cooperative effects would be certainly ruled out.

Normal vibrations of ternary DOPC/DPPC/cholesterol lipid bilayers by low-frequency Raman spectroscopy.

A lipid bilayer containing a ternary mixture of low- and high-melting lipids and cholesterol (Chol) can give rise to domain formation, referred to as lipid rafts. Low-frequency Raman spectroscopy at reduced temperatures allows detection of normal membrane mechanical vibrations. In this work, Raman spectra were obtained in the spectral range between 5 and 90 cm for bilayers prepared from dioleoyl--phosphocholine (DOPC), dipalmitoyl--phosphocholine (DPPC) and Chol.

Lipid chain mobility and packing in DOPC bilayers at cryogenic temperatures.

Low-temperature molecular mobility and packing in biological tissues are important for their survival upon cryopreservation. Electron paramagnetic resonance (EPR) in its pulsed version of electron spin echo (ESE) allows studying stochastic librations of spin-labeled molecules, the type of motion which dominates at low temperatures. These librations are characterized by the parameter <α>τ where <α> is the mean squared angular amplitude and τ is the correlation time for the motion.

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.