Location and orientation relative to the micelle surface for glucagon in mixed micelles with dodecylphosphocholine: EPR and NMR studies

The spin labels, 5-doxylstearate, 12-doxylstearate, 16-doxylstearate and 1-oxyl-2,2,6,6-tetramethyl-4-dodecylphosphopiperidine, have been incorporated into dodecylphosphocholine micelles and mixed dodecylphosphocholine glucagon micelles. The EPR spectral parameters for the different spin labels and the 1H- and 13C-NMR relaxation rates for nuclei of the detergent molecules indicated that inclusion of up to one spin label molecule per micelle had little influence on the spatial organization of the micelles. Furthermore, the location and environment of the spin labels in the dodecylphosphocholine micelles were not noticeably affected by the addition of glucagon and the 1H-NMR spectra observed for glucagon in mixed spin label/deuterated dodecylphosphocholine/glucagon micelles showed that the different spin labels had essentially no effect on the conformation of glucagon. Approximate spatial locations within the micelle for the nitroxide moieties of the different spin labels were determined from the NMR relaxation rates observed for different nuclei of dodecylphosphocholine. On this basis, the line broadening of individually assigned glucagon 1H-NMR lines by the different spin labels was used to determine the approximate orientation of the polypeptide chain with respect to the micelle surface. Overall, the data indicate that the glucagon backbone runs roughly parallel to the micelle surface, with the depth of immersion adjusted so that polar and apolar side chains can be oriented towards the surface or interior of the micelle, respectively.     

High resolution nuclear magnetic resonance studies of the conformation and orientation of melittin bound to a lipid-water interface.

Previously, the size and stoichiometry of mixed micelles of perdeuterated dodecylphosphocholine and melittin were characterized and the 1H NMR spin systems of most amino acid residues of micelle-bound melittin identified. One- and two-dimensional 1H-1H Overhauser experiments have now been used to obtain qualitative information on intramolecular proton-proton distances. These data show that the N-terminal and the C-terminal segments of melittin form two spatially distinct, compact domains; using lipid spin labels these could be located near the micelle surface. For the C-terminal domain a detailed conformation was determined by using the distance contraints from the Overhauser studies as input for a distance geometry algorithm.     

Melittin bound to dodecylphosphocholine micelles 1H-NMR assignments and global conformational features

Assignments have been obtained for most of the ¹H-NMR lines of melittin bound to fully deuterated dodecylphosphocholine micelles by combined use of two-dimensional spin echo correlated spectroscopy and one-dimensional NMR methods. Nuclear Overhauser enhancement measurements showed that the mobility of the entire polypeptide chain is reduced by binding of melittin to the detergent micelle and that the amino-terminal and carboxy-terminal halves of the primary structure constitute separate, compact domains within the conformation of micelle-bound melittin. p²H titration experiments showed that the presence of positive charges on the four amino groups of melittin had little influence on the conformation of the micelle-bound polypeptide. Titration of tetrameric melittin with detergent provided evidence that melittin assumes similar conformations as a self-aggregated tetramer and as a monomer bound to micelles.     

Physicochemical characterization of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes.

The stoichiometry of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes and the location of the protein in the micelles have been investigated by electron paramagnetic resonance, ultracentrifugation, small-angle X-ray scattering, 31P, 13C, and 1H nuclear magnetic resonance spectroscopy, and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes are formed by association of one protein molecule with approximately 133 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into detergent/protein aggregates. Electron paramagnetic resonance spectral parameters and 13C and 1H nuclear magnetic resonance relaxation times showed that the addition of myelin basic protein does not affect the environment and location of the labels or the organization of the micelles. Previous results suggesting that the protein lies primarily near the surface of the micelles have been confirmed by comparing 13C spectra of the detergents with and without protein with spectra of detergent/protein aggregates containing the spin labels. Electron micrographs of the complexes taken by using the freeze-fracture technique revealed the presence of particles with an estimated radius about three times the radius of the micelles measured by small-angle X-ray scattering. The structural integrity of the complexes appears to be based on intramolecular protein interactions as well as protein-detergent interactions.     

Physicochemical characterization of glucagon-containing lipid micelles

Fluorescence studies showed that glucagon binds to a variety of micellar lipids. By means of ultracentrifugation and quasi-elastic light-scattering, it was found that stoichiometrically well defined complexes were formed between glucagon and perdeuterated dodecylphosphocholine micelles consisting of one glucagon molecule and approx. 40 detergent molecules. Well resolved ¹H-NMR spectra were obtained for glucagon in the deuterated micelles. Studies of nuclear Overhauser effects between individually assigned protons in different regions of the amino acid sequence indicated that micelle-bound glucagon adopts a well defined, predominantly extended conformation. Evidence obtained from circular dichroism indicates that the conformation of glucagon bound to various micellar lipids is largely independent of the type of lipid and, furthermore, appears to be very similar to that of glucagon bound to lipid bilayers.     

Interactions of myelin basic protein with palmitoyllysophosphatidylcholine: characterization of the complexes and conformations of the protein

The stoichiometry of palmitoyllysophosphatidylcholine/myelin basic protein (PLPC/MBP) complexes, the location of the protein in the lysolipid micelles, and the conformational changes occurring in the basic protein and peptides derived from it upon interaction with lysolecithin micelles were investigated by circular dichroic spectropolarimetry, ultracentrifugation, electron paramagnetic resonance (EPR) and ³¹P, ¹³C, and ¹H nuclear magnetic resonance spectroscopy (NMR), and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes were formed by the association of one protein molecule with approximately 141 lysolipid molecules. Small-angle X-ray scattering data indicated that the PLPC/MBP complexes form particles with a radius of gyration of 3.8 nm. EPR spectral parameters of the spin labels 5–, and 16-doxylstearate incorporated into lysolecithin/basic protein aggregates, and ¹³C- and ¹H-NMR relaxation times of PLPC indicated that the addition of the protein did not affect the environment and location of the labels and the organization of the lysolipid micelles. The data suggested that MBP lies primarily near the surface of the micelles, with segments penetrating beyond the interfacial region into the hydrophobic interior, but without any part of the protein being protected against rapid exchange of its amide groups with the aqueous environment. The basic protein acquired about 20% -helix when bound to lysolipid micelles. Circular dichroic spectra of sequential peptides derived by cleavage of the protein revealed the formation of -helical regions in the association with lysolecithin. Specific residues in myelin basic protein that participated in binding to the micelles were identified from magnetic resonance data on changes in the chemical shifts and intensities of assigned resonances, and line broadening of peaks by fatty acid spin-labels incorporated into the micelles. Correspondence to: G. L. Mendz     

Despite a Conserved Cystine Knot Motif, Different Cyclotides Have Different Membrane Binding Modes

Cyclotides are cyclic proteins produced by plants for defense against pests. Because of their remarkable stability and diverse bioactivities, they have a range of potential therapeutic applications. The bioactivities of cyclotides are believed to be mediated through membrane interactions. To determine the structural basis for the biological activity of the two major subfamilies of cyclotides, we determined the conformation and orientation of kalata B2 (kB2), a Möbius cyclotide, and cycloviolacin O2 (cO2), a bracelet cyclotide, bound to dodecylphosphocholine micelles, using NMR spectroscopy in the presence and absence of 5- and 16-doxylstearate relaxation probes. Analysis of binding curves using the Langmuir isotherm indicated that cO2 and kB2 have association constants of 7.0 × 10³ M⁻¹ and 6.0 × 10³ M⁻¹, respectively, consistent with the notion that they are bound near the surface, rather than buried deeply within the micelle. This suggestion is supported by the selective broadening of micelle-bound cyclotide NMR signals upon addition of paramagnetic Mn ions. The cyclotides from the different subfamilies exhibited clearly different binding orientations at the micelle surface. Structural analysis of cO2 confirmed that the main element of the secondary structure is a β-hairpin centered in loop 5. A small helical turn is present in loop 3. Analysis of the surface profile of cO2 shows that a hydrophobic patch stretches over loops 2 and 3, in contrast to the hydrophobic patch of kB2, which predominantly involves loops 2 and 5. The different location of the hydrophobic patches in the two cyclotides explains their different binding orientations and provides an insight into the biological activities of cyclotides.     

Conformation and mode of membrane interaction in cyclotides. Spatial structure of kalata B1 bound to a dodecylphosphocholine micelle

Cyclotides are a family of bioactive plant peptides that are characterized by a circular protein backbone and three conserved tightly packed disulfide bonds. The antimicrobial and hemolytic properties of cyclotides, along with the relative hydrophobicity of the peptides, point to the biological membrane as a target for cyclotides. To assess the membrane-induced conformation and orientation of cyclotides, the interaction of the M?bius cyclotide, kalata B1, from the African perennial plant Oldenlandia affinis, with dodecylphosphocholine micelles was studied using NMR spectroscopy. Under conditions where the cyclotide formed a well-defined complex with micelles, the spatial structure of kalata B1 was calculated from NOE and J couplings data, and the model for the peptide-micelle complex was built using 5- and 16-doxylstearate relaxation probes. The binding of divalent cations to the peptide-micelle complex was quantified by Mn2+ titration. The results show that the peptide binds to the micelle surface, with relatively high affinity, via two hydrophobic loops (loop 5, Trp19-Val21; and loop6, Leu27-Val29). The charged residues (Glu3 and Arg24), along with the cation-binding site (near Glu3) are segregated on the other side of the molecule and in contact with polar head groups of detergent. The spatial structure of kalata B1 is only slightly changed during incorporation into micelles and represents a distorted triple-stranded beta-sheet cross-linked by a cystine knot. Detailed structural analysis and comparison with other knottins revealed structural conservation of the two-disulfide motif in cyclic and acyclic peptides. The results thus obtained provide the first model for interaction of cyclotides with membranes and permit consideration of the cyclotides as membrane-active cationic antimicrobial peptides.     

Characterization of dodecylphosphocholine/myelin basic protein complexes

The stoichiometry of myelin basic protein (MBP)/dodecylphosphocholine (DPC) complexes and the location of protein segments in the micelle have been investigated by electron paramagnetic resonance (EPR), ultracentrifugation, photon correlation light scattering, 31P, 13C, and 1H nuclear magnetic resonance (NMR), and electron microscopy. Ultracentrifugation measurements indicate that MBP forms stoichiometrically well-defined complexes consisting of 1 protein molecule and approximately 140 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into DPC/MBP aggregates. EPR spectral parameters and 13C and 1H NMR relaxation times indicate that the addition of MBP does not affect the environment and location of the labels or the organization of the micelles except for a slight increase in size. Previous results indicating that the protein lies primarily near the surface of the micelle have been confirmed by comparing 13C NMR spectra of the detergent with and without protein with spectra of protein/detergent aggregates containing spin-labels. Electron micrographs of the complexes taken by using the freeze-fracture technique confirm the estimated size obtained by light-scattering measurements. Overall, these results indicate that mixtures of MBP and DPC can form highly porous particles with well-defined protein and lipid stoichiometry. The structural integrity of these particles appears to be based on protein-lipid interactions. In addition, electron micrographs of aqueous DPC/MBP suspensions show the formation of a small amount of material consisting of large arrays of detergent micelles, suggesting that MBP is capable of inducing large changes in the overall organization of the detergent.     

Impact of membrane partitioning on the spatial structure of an S-type cobra cytotoxin

Cobra cytotoxins (CTs) belong to the three-fingered protein family. They are classified into S- and P-types, the latter exhibiting higher membrane-perturbing capacity. In this work, we investigated the interaction of CTs with phospholipid bilayers, using coarse-grained (CG) and full-atom (FA) molecular dynamics (MD). The object of this work is a CT of an S-type, cytotoxin I (CT1) from N.oxiana venom. Its spatial structure in aqueous solution and in the micelles of dodecylphosphocholine (DPC) were determined by ¹H-NMR spectroscopy. Then, via CG- and FA MD-computations, we evaluated partitioning of CT1 molecule into palmitoyloleoylphosphatidylcholine (POPC) membrane, using the toxin spatial models, obtained either in aqueous solution, or detergent micelle. The latter model exhibits minimal structural changes upon partitioning into the membrane, while the former deviates from the starting conformation, loosing the tightly bound water molecule in the loop-2. These data show that the structural changes elicited by CT1 molecule upon incorporation into DPC micelle take place likely in the lipid membrane, although the mode of the interaction of this toxin with DPC micelle (with the tips of the all three loops) is different from its mode in POPC membrane (primarily with the tip of the loop-1 and both the tips of the loop-1 and loop-2).     

Dodecylphosphocholine micelles as a membrane-like environment: new results from NMR relaxation and paramagnetic relaxation enhancement analysis

To further examine to what extent a dodecylphosphocholine (DPC) micelle mimics a phosphatidylcholine bilayer environment, we performed ¹³C, ²H, and ³¹P NMR relaxation measurements. Our data show that the dynamic behavior of DPC phosphocholine groups at low temperature (12 °C) corresponds to that of a phosphatidylcholine interface at high temperature (51 °C). In the presence of helical peptides, a PMP1 fragment, or an annexin fragment, the DPC local dynamics are not affected whereas the DPC aggregation number is increased to match an appropriate area/volume ratio for accommodating the bound peptides. We also show that quantitative measurements of paramagnetic relaxation enhancements induced by small amounts of spin-labeled phospholipids on peptide proton signals provide a meaningful insight on the location of both PMP1 and annexin fragments in DPC micelles. The paramagnetic contributions to the relaxation were extracted from intra-residue cross-peaks of NOESY spectra for both peptides. The location of each peptide in the micelles was found consistent with the corresponding relaxation data. As illustrated by the study of the PMP1 fragment, paramagnetic relaxation data also allow us to supply the missing medium-range NOEs and therefore to complete a standard conformational analysis of peptides in micelles. Received: 16 April 1998 / Revised version: 19 June 1998 / Accepted: 30 July 1998     

Spatial structure of zervamicin IIB bound to DPC micelles: implications for voltage-gating.

Zervamicin IIB is a 16-amino acid peptaibol that forms voltage-dependent ion channels with multilevel conductance states in planar lipid bilayers and vesicular systems. The spatial structure of zervamicin IIB bound to dodecylphosphocholine micelles was studied by nuclear magnetic resonance spectroscopy. The set of 20 structures obtained has a bent helical conformation with a mean backbone root mean square deviation value of approximately 0.2 A and resembles the structure in isotropic solvents (Balashova et al., 2000. NMR structure of the channel-former zervamicin IIB in isotropic solvents. FEBS Lett 466:333-336). The N-terminus represents an alpha-helix, whereas the C-terminal part has a mixed 3(10)/alpha(R) hydrogen-bond pattern. In the anisotropic micelle environment, the bending angle on Hyp10 (23 degrees) is smaller than that (47 degrees) in isotropic solvents. In the NOESY (Nuclear Overhauser Effect Spectroscopy) spectra, the characteristic attenuation of the peptide signals by 5- and 16-doxylstearate relaxation probes indicates a peripheral mode of the peptaibol binding to the micelle with the N-terminus immersed slightly deeper into micelle interior. Analysis of the surface hydrophobicity reveals that the zervamicin IIB helix is amphiphilic and well suited to formation of a tetrameric transmembrane bundle, according to the barrel-stave mechanism. The results are discussed in a context of voltage-driven peptaibol insertion into membrane.     

Membrane surface-associated helices promote lipid interactions and cellular uptake of human calcitonin-derived cell penetrating peptides

hCT(9-32) is a human calcitonin (hCT)-derived cell-penetrating peptide that has been shown to translocate the plasma membrane of mammalian cells. It has been suggested as a cellular carrier for drugs, green fluorescent protein, and plasmid DNA. Because of its temperature-dependent cellular translocation resulting in punctuated cytoplasmatic distribution, its uptake is likely to follow an endocytic pathway. To gain insight into the molecular orientation of hCT(9-32) when interacting with lipid models, and to learn more about its mode of action, various biophysical techniques from liposome partitioning to high-resolution NMR spectroscopy were utilized. Moreover, to establish the role of individual residues for the topology of its association with the lipid membrane, two mutants of hCT(9-32), i.e., W30-hCT(9-32) and A23-hCT(9-32), were also investigated. Although unstructured in aqueous solution, hCT(9-32) adopted two short helical stretches when bound to dodecylphosphocholine micelles, extending from Thr10 to Asn17 and from Gln24 to Val29. A23-hCT(9-32), in which the helix-breaking Pro23 was replaced by Ala, displayed a continuous alpha-helix extending from residue 12 to 26. Probing with the spin label 5-doxylstearate revealed that association with dodecylphosphocholine micelles was such that the helix engaged in parallel orientation to the micelle surface. Moreover, the Gly to Trp exchange in W30-hCT(9-32) resulted in a more stable anchoring of the C-terminal segment close to the interface, as reflected by a twofold increase in the partition coefficient in liposomes. Interestingly, tighter binding to model membranes was associated with an increase in the in vitro uptake in human cervix epithelial adenocarcinoma cell line cells. Liposome leakage studies excluded pore formation, and the punctuated fluorescence pattern of internalized peptide indicated vesicular localization and, in conclusion, strongly suggested an endocytic pathway of translocation.     

On the surface properties of oleate micelles and oleic acid/oleate vesicles studied by spin labeling

Dilute aqueous systems composed of sodium oleate micelles and sodium oleate/oleic acid vesicles were investigated as a function of pH by electron spin resonance spectroscopy with TEMPO-stearate TEMPO-stearamide as well as with a positively charged water soluble spin label, TEMPO-choline. The dynamics of the three TEMPO-spin labels were found to be sensitive to changes in the interfacial region of the aggregates as a function of pH. The results obtained are consistent with the formation of a hydrogen bond network (RCOO⁻ ↔ HOOCR) at the surface of the sodium oleate/oleic acid system in the course of the transformation of micelles into the closed bilayers (vesicles). Vesicles formation below pH 10 was determined independently with a spin labeled glucose derivative.     

The interactions of the HIV gp41 fusion peptides with zwitterionic membrane mimics determined by NMR spectroscopy

The wild-type (wt) N-terminal 23-residue fusion peptide (FP) of the human immunodeficiency virus (HIV) fusion protein gp41 and its V2E mutant have been studied by nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles as membrane mimics. A number of NMR techniques have been used. Pulsed field-gradient diffusion measurements in DPC and in 4:1 DPC/sodium dodecylsulfate mixed micelles showed that there is no major difference between the partition coefficients of the fusogenic wt peptide and the V2E mutant in these micelles, indicating that there is no correlation between the activity of the fusion peptides and their membrane affinities. The nuclear Overhauser enhancement (NOE) patterns and the chemical shift index for these two peptides indicated that both FP are in an α helical conformation between the Ile⁴ to Leu¹² or to Ala¹⁵ region. Simulated annealing showed that the helical region extends from Ile⁴ to Met¹⁹. The two FPs share similar conformational characteristics, indicating that the conformation of the FP is not an important factor determining its activity. The spin-label studies, utilizing spin labels 5- and 16-doxystearic acids in the DPC micelles, provided clear indication that the wt FP inserts its N-terminus into the micelles while the V2E mutant does not insert into the micelles. The conclusion from the spin-label results is corroborated by deuterium amide proton exchange experiments. The correlation between the oblique insertion of the FP and its fusogenic activity is in excellent agreement with results from our molecular dynamics simulation and from other previous studies.     

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra: Glucagon bound to perdeuterated dodecylphosphocholine micelles

The assignment of the ¹H nuclear magnetic resonance spectrum of glucagon bound to perdeuterated dodecylphosphocholine micelles with the use of two-dimensional ¹H nuclear magnetic resonance techniques at 360 MHz is described. Sequential resonance assignments were obtained for all backbone and C^β protons except the N-terminal amino group and the amide proton of Ser2. The assignments of the non-labile amino acid side-chain protons are complete except for the γ-methylene protons of Gln20 and Gln24. These assignments provide a basis for the determination of the three-dimensional structure of lipid-bound glucagon.     

Micelle-bound structures and dynamics of the hinge deleted analog of melittin and its diastereomer: Implications in cell selective lysis by d-amino acid containing antimicrobial peptides

Melittin, the major component of the honey bee venom, is a 26-residue hemolytic and membrane active peptide. Structures of melittin determined either in lipid environments by NMR or by use of X-ray demonstrated two helical regions at the N- and C-termini connected by a hinge or a bend at the middle. Here, we show that deletion of the hinge residues along with two C-terminal terminal Gln residues (Q25 and Q26), yielding a peptide analog of 19-residue or Mel-H, did not affect antibacterial activity but resulted in a somewhat reduced hemolytic activity. A diastereomer of Mel-H or Mel-^dH containing d-amino acids [^dV5, ^dV8, ^dL11 and ^dK16] showed further reduction in hemolytic activity without lowering antibacterial activity. We have carried out NMR structures, dynamics (H-D exchange and proton relaxation), membrane localization by spin labeled lipids, pulse-field-gradient (PFG) NMR and isothermal titration calorimetry (ITC) in dodecylphosphocholine (DPC) micelles, as a mimic to eukaryotic membrane, to gain insights into cell selectivity of these melittin analogs. PFG-NMR showed Mel-H and Mel-^dH both were similarly partitioned into DPC micelles. ITC demonstrated that Mel-H and Mel-^dH interact with DPC with similar affinity. The micelle-bound structure of Mel-H delineated a straight helical conformation, whereas Mel-^dH showed multiple β-turns at the N-terminus and a short helix at the C-terminus. The backbone amide-proton exchange with solvent D₂O demonstrated a large difference in dynamics between Mel-H and Mel-^dH, whereby almost all backbone protons of Mel-^dH showed a much faster rate of exchange as compared to Mel-H. Proton T₁ relaxation had suggested a mobile backbone of Mel-^dH peptide in DPC micelles. Resonance perturbation by paramagnetic lipids indicated that Mel-H inserted deeper into DPC micelles, whereas Mel-^dH is largely located at the surface of the micelle. Taken together, results presented in this study demonstrated that the poor hemolytic activity of the d-amino acid containing analogs of antimicrobial peptides may be correlated with their flexible dynamics at the membrane surface.     

Phase memory relaxation times of spin labels in human carbonic anhydrase II: pulsed EPR to determine spin label location.

Phase memory relaxation times (T(M) or T(2)) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels (N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters T(M) and x. T(M) values of seven positions are between 1.6 micros for the most buried residue (L79C) and 4.7 micros for a residue at the protein surface (W245C). In deuteriated buffer, longer T(M) are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose T(M) as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism.     

Structural and positional studies of the antimicrobial peptide brevinin‐1BYa in membrane‐mimetic environments

Brevinin‐1BYa (FLPILASLAAKFGPKLFCLVTKKC), first isolated from skin secretions of the foothill yellow‐legged frog Rana boylii, shows broad‐spectrum activity, being particularly effective against opportunistic yeast pathogens. The structure of brevinin‐1BYa was investigated in various solution and membrane‐mimicking environments by proton nuclear magnetic resonance (¹H‐NMR) spectroscopy and molecular modelling. The peptide does not possess a secondary structure in aqueous solution. In a 33% 2,2,2‐trifluoroethanol (TFE‐d₃)‐H₂O solvent mixture, as well as in membrane‐mimicking sodium dodecyl sulfate and dodecylphosphocholine micelles, the peptide's structure is characterised by a flexible helix‐hinge‐helix motif, with the hinge located at the Gly¹³/Pro¹⁴ residues, and the two α‐helices extending from Pro³ to Phe¹² and from Pro¹⁴ to Thr²¹. Positional studies involving the peptide in sodium dodecyl sulfate and dodecylphosphocholine micelles using 5‐doxyl‐labelled stearic acid and manganese chloride paramagnetic probes show that the peptide's helical segments lie parallel to the micellar surface, with the residues on the hydrophobic face of the amphipathic helices facing towards the micelle core and the hydrophilic residues pointing outwards, suggesting that the peptide exerts its biological activity by a non–pore‐forming mechanism.