Quantification of Distributions of Local Proton Concentrations in Heterogeneous Soft Matter and Non-Anfinsen Biomacromolecules.

A new method to quantitatively analyze heterogeneous distributions of local proton densities around paramagnetic centers in unstructured and weakly structured biomacromolecules and soft matter is introduced, and its feasibility is demonstrated on aqueous solutions of stochastically spin-labeled polysaccharides. This method is based on the pulse EPR experiment ih-RIDME (intermolecular hyperfine relaxation-induced dipolar modulation enhancement). Global analysis of a series of RIDME traces allows for a mathematically stable transformation of the time-domain data to the distribution of local proton concentrations. Two pulse sequences are proposed and tested, which combine the ih-RIDME block and the double-electron-electron resonance (DEER) experiment. Such experiments can be potentially used to correlate the local proton concentration with the macromolecular chain conformation. We anticipate an application of this approach in studies of intrinsically disordered proteins, biomolecular aggregates, and biomolecular condensates.

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.

Confidence limits in pulse dipolar EPR spectroscopy: estimates for individual measurements.

The distribution of inter-label distances obtained by electron paramagnetic resonance (EPR) pulse dipolar spectroscopies (PDS), such as DEER aka PELDOR, gives a valuable characterization of structure on the nanometer scale. The impact of random experimental noise on such experiments is examined for three independent methods for analysing PDS data: the model-free method with Tikhonov regularization, model-free with Mellin-transformation, and a model-based method. All three methods show negative bias for the mean distance and positive bias for the distribution width. Both biases grow with increasing noise levels. The estimated confidence bands and the uncertainties obtained from a single experimental measurement by the standard bootstrapping or χ2-surface scanning approaches are inconsistent and can exclude the true distance distribution. Yet, both approaches can provide quite valuable support for hypothesis testing in PDS studies.

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.

Pulse EPR spectroscopy and molecular modeling reveal the origins of the local heterogeneity of dietary fibers.

Optimizing human diet by including dietary fibers would be more efficient when the fibers' chain interactions with other molecules are understood in depth. Thereby, it is important to develop methods for characterizing the fiber chain to be able to monitor its structural alterations upon intermolecular interactions. Here, we demonstrate the utility of the electron paramagnetic resonance (EPR) spectroscopy, complemented by simulations in probing the atomistic details of the chain conformations for spin-labeled fibers. Barley ß-glucan, a native polysaccharide with linear chain, was utilized as a test fiber system to demonstrate the technique's capabilities. Pulse dipolar EPR data show good agreement with results of the fiber chain modeling, revealing sinuous chain conformations and providing polymer shape descriptors: the gyration tensor, spin-spin distance distribution function, and information about proton density near the spin probe. Results from EPR measurements point to the fiber aggregation in aqueous solution, which agrees with the results of the dynamic light scattering. We propose that the combination of pulse EPR measurements with modeling can be a perfect experimental tool for in-depth structural investigation of dietary fibers and their interaction under such conditions, and that the presented methodology can be extended to other weakly ordered or disordered macromolecules.

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.

Ferric Pyrophosphate Forms Soluble Iron Coordination Complexes with Zinc Compounds and Solubilizing Agents in Extruded Rice and Predicts Increased Iron Solubility and Bioavailability in Young Women.

BACKGROUND: Co-extrusion of ferric pyrophosphate (FePP) with solubilizers, citric acid/trisodium citrate (CA/TSC), or ethylenediaminetetraacetic acid (EDTA) sharply increases iron absorption. Whether this can protect against the inhibition of iron absorption by phytic acid (PA) is unclear. Sodium pyrophosphate (NaPP) may be a new enhancer of iron absorption from FePP. OBJECTIVES: Our objectives were to 1) investigate the ligand coordination of iron, zinc, and solubilizers in extruded rice and test associations with iron solubility and absorption, 2) assess whether co-extrusion of FePP + CA/TSC rice can protect against inhibition of iron absorption by PA; 3) determine the effect of zinc oxide (ZnO) compared with zinc sulfate (ZnSO4), and 4) quantify iron absorption from FePP + NaPP rice. METHODS: We produced labeled 57FePP rice cofortified with ZnSO4 and EDTA, CA/TSC or NaPP, and FePP + EDTA rice with ZnO. We used electron paramagnetic resonance (EPR) to characterize iron-ligand complexes. We measured in vitro iron solubility and fractional iron absorption (FIA) in young women (n = 21, age: 22 ± 2 y, BMI: 21.3 ± 1.5 kg/m2 geometric mean plasma ferritin, 28.5 µg/L) compared with ferrous sulfate (58FeSO4). FIA was compared by linear mixed-effect model analysis. RESULTS: The addition of zinc and solubilizers created new iron coordination complexes of Fe(III) species with a weak ligand field at a high-spin state that correlated with solubility (r2 = 0.50, P = 0.02) and absorption (r2 = 0.72, P = 0.02). Phytic acid reduced FIA from FePP + CA/TSC rice by 50% (P < 0.001), to the same extent as FeSO4. FIA from FePP + EDTA + ZnO and FePP + EDTA + ZnSO4 rice did not significantly differ. Mean FIAs from FePP + EDTA + ZnSO4, FePP + CA/TSC + ZnSO4, and FePP + NaPP + ZnSO4 rice were 9% to 11% and did not significantly differ from each other or from FeSO4. CONCLUSION: Rice extrusion of FePP with solubilizers resulted in bioavailable iron coordination complexes. In the case of FePP + CA/TSC, PA exerted similar inhibition of FIA as with FeSO4. FePP + NaPP could be a further viable solubilizing agent for rice fortification. This study was registered at clinicaltrials.gov as NCT03703739.

The Cu(ii) - dietary fibre interactions at molecular level unveiled via EPR spectroscopy.

While dietary fibres have a reputation of a healthy food component, the interaction between nutrients and neutral fibers is non-covalent, and its characterization is challenging for most analytical techniques. Here, on the example of barley ß-glucan (BBG) and paramagnetic Cu(ii) ions we demonstrate the performance of different Electron Paramagnetic Resonance (EPR) methods in the fibre studies. EPR techniques were tested on two spin probe systems with different affinity in the interaction with dietary fibres - Cu(OAc)2 salt, which weakly dissociates under physiological conditions and CuSO4 salt, which easily dissociates, so that in the latter case Cu(ii) can be considered as a 'free' ion, only chelated by water molecules. The Cu(ii)-BBG interaction was determined by pulse EPR relaxation measurements, but this interaction appears not strong enough for continuous wave EPR detection. The capability of the fibres for Cu(ii) absorption was successfully analyzed by comparison of the results from the pulse dipolar spectroscopy with numerical simulations. The local distribution of sugar hydrogen atoms around the Cu(ii) ion has been determined by electron spin echo envelope modulation (ESEEM) and electron-nuclei double resonance (ENDOR) techniques.

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. Real-time conformational changes of DNA during interaction with Nfo revealed an increase of distances between duplex ends during the formation of the initial enzyme-substrate complex. The use of rigid-linker spin-labelled DNA duplexes in DEER measurements indicated that double-helix bending and unwinding by the target nucleotide itself is one of the key factors responsible for indiscriminate recognition of a target nucleotide by Nfo. The results for the first time show that AP endonucleases from different structural families utilise a common strategy of damage recognition, which globally may be integrated with the mechanism of searching for specific sites in DNA by other enzymes.

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.

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. A strict requirement for a precise distance measurement has been identified in a minimal dynamic flexibility of the two nitroxide-bearing α-amino acid side chains. To this end, in this study, we have experimentally compared in detail the side-chain mobility properties of the two currently most widely utilized residues, namely, Cys(MTSL) and 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). In particular, two double-labeled, chemically synthesized 20-mer peptide molecules have been adopted as appropriate templates for our investigation on the determination of the model intramolecular separations. These double-Cys(MTSL) and double-TOAC compounds are both analogues of the almost completely rigid backbone peptide ruler which we have envisaged and 3D structurally analyzed as our original, unlabeled compound. Here, we have clearly found that the TOAC side-chain labels are largely more 3D structurally restricted than the MTSL labels. From this result, we conclude that the TOAC residue offers more precise information than the Cys(MTSL) residue on the side chain-to-side chain distance distribution in synthetically accessible peptide molecules.

Non-uniform sampling in pulse dipolar spectroscopy by EPR: the redistribution of noise and the optimization of data acquisition.

Pulse dipolar spectroscopy (PDS) in Electron Paramagnetic Resonance (EPR) is the method of choice for determining the distance distribution function for mono-, bi- or multi- spin-labeled macromolecules and nanostructures. PDS acquisition schemes conventionally use uniform sampling of the dipolar trace, but non-uniform sampling (NUS) schemes can decrease the total measurement time or increase the accuracy of the resulting distance distributions. NUS requires optimization of the data acquisition scheme, as well as changes in data processing algorithms to accommodate the non-uniformly sampled data. We investigate in silico the applicability of the NUS approach in PDS, considering its effect on random, truncation and sampling noise in the experimental data. Each type of noise in the time-domain data propagates differently and non-uniformly into the distance spectrum as errors in the distance distribution. NUS schemes seem to be a valid approach for increasing sensitivity and/or throughput in PDS by decreasing and redistributing noise in the distance spectrum so that it has less impact on the distance spectrum.

Tylopeptin B peptide antibiotic in lipid membranes at low concentrations: Self-assembling, mutual repulsion and localization.

The medium-length peptide Tylopeptin B possesses activity against Gram-positive bacteria. It binds to bacterial membranes altering their mechanical properties and increasing their permeability. This action is commonly related with peptide self-assembling, resulting in the formation of membrane channels. Here, pulsed double electron-electron resonance (DEER) data for spin-labeled Tylopeptin B in palmitoyl-oleoyl-glycero-phosphocholine (POPC) model membrane reveal that peptide self-assembling starts at concentration as low as 0.1 mol%; above 0.2 mol% it attains a saturation-like dependence with a mean number of peptides in the cluster = 3.3. Using the electron spin echo envelope modulation (ESEEM) technique, Tylopeptin B molecules are found to possess a planar orientation in the membrane. In the peptide concentration range between 0.1 and 0.2 mol%, DEER data show that the peptide clusters have tendency of mutual repulsion, with a circle of inaccessibility of radius around 20 nm. It may be proposed that within this radius the peptides destabilize the membrane, providing so the peptide antimicrobial activity. Exploiting spin-labeled stearic acids as a model for free fatty acids (FFA), we found that at concentrations of 0.1-0.2 mol% the peptide promotes formation of lipid-mediated FFA clusters; further increase in peptide concentration results in dissipation of these clusters.

Improving B1 field homogeneity in dielectric tube resonators for EPR spectroscopy via controlled shaping of the dielectric insert.

We propose an approach for improving the homogeneity of microwave magnetic field amplitude in a dielectric tube resonator for electron paramagnetic resonance spectroscopy at X-band. The improvement is achieved by "shaping" (controllable variation of the outer diameter of a dielectric insert along its axial direction). Various shaping scenarios based on the principle of discrete solenoids and electromagnetic calculations have been considered. The dielectric insert having the most promising shape was manufactured from a bismuth germanate single crystal. The shaped insert increases the area at B1 > 0.9 B1max from 5.06 to 7.36 mm. Higher sensitivity and lower likelihood of quantitative errors have been achieved in pulse EPR experiments for "long" samples (whose length was comparable to that of the dielectric insert) in a shaped dielectric insert.

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.

Peptide antibiotic trichogin in model membranes: Self-association and capture of fatty acids.

The antimicrobial action of peptides in bacterial membranes is commonly related to their mode of self-assembling which results in pore formation. To optimize peptide antibiotic use for therapeutic purposes, a study on the concentration dependence of self-assembling process is thus desirable. In this work, we investigate this dependence for peptaibol trichogin GA IV (Tric) in the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model membrane in the range of peptide concentrations between 0.5 and 3.3 mol%. Pulsed double electron-electron resonance (PELDOR) applied on spin-labeled peptide analogs highlights the onset of peptide dimerization above a critical peptide concentration value, namely ~ 2 mol%. Electron spin echo (ESE) envelope modulation (ESEEM) for D2O-hydrated bilayers shows that dimerization is accompanied by peptide re-orientation towards a trans-membrane disposition. For spin-labeled stearic acids (5-DSA) in POPC bilayers, the study of ESE decays and ESEEM in the presence of a deuterated peptide analog indicates that above the critical peptide concentration the 5-DSA molecules are attracted by peptide molecules, forming nanoclusters. As the 5-DSA molecules represent a model for the behavior of fatty acids participating in bacterial membrane homeostasis, such capturing action by Tric may represent an additional mechanism of its antibiotic activity.

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.

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.

A molecular view on the role of cholesterol upon membrane insertion, aggregation, and water accessibility of the antibiotic lipopeptide trichogin GA IV as revealed by EPR.

Trichogin GA IV is a membrane-active lipopeptide, the antibiotic activity of which was proposed to be based on its capability to induce leakage due to formation of pores into the bacterial cell membrane. However, less attention has been paid to its biological selectivity, i.e., discrimination between bacterial versus cholesterol containing (mammalian) membranes. This is the reason which motivated us to study the role of cholesterol on penetration of the peptide into the membrane and formation of water channels. The ESEEM technique was used to measure the modulation amplitudes for TOAC spin-labeled trichogin GA IV peptide analogues in hydrated membranes of phosphatidylcholine (PC) lipid in the presence of 50 mol % cholesterol-d7. From the interaction between the nitroxide spin-label and the nearby located deeply membrane inserted deuterons, the N-terminus was found to be positioned at the core of the membrane. Separately, ESEEM measurements for the FTOAC-8 labeled peptide, but in D2O hydrated cholesterol/PC membranes, provide strong evidence for the polar C-terminus situated near the membrane surface. The apparently too high modulation amplitude measured for the buried FTOAC-1 label is likely attributed to the presence of peptide associated water. In cholesterol depleted membrane, however, the long axes of the helical molecules are found oriented parallel to the membrane surface even at high peptide concentration. Continuous wave EPR spectroscopy indicates that, for cholesterol containing membrane, peptide insertion is accompanied by self-aggregation of parallelly aligned transmembrane peptide molecules, while for cholesterol lacking membranes they are monomolecularly distributed. Thus, cholesterol tends to stabilize the transmembrane peptide aggregate.