Determination of the toxicity of several aromatic carbonylic compounds and their reduced derivatives on Phanerochaete chrysosporium using a Pseudomonas putida test system

We tested four aromatic carbonylic compounds and their corresponding reduced derivatives, possible substrates, and products of a biotransformation for toxicity against the white-rot fungus Phanerochaete chrysosporium. The bacterium Pseudomonas putida, which has been proven to be a good test organism for investigating toxic effects, was used as a primary screen. For both P. chrysosporium and P. putida, all ketones showed a higher toxicity than their corresponding alcohol derivatives. Within one chemical group a direct correlation between the hydrophobicity (logP values) of the compounds and their toxicity could be observed. Furthermore, all tested compounds also caused an isomerization of cis to trans unsaturated fatty acids in P. putida, a mechanism of this bacterium to adapt its membrane to toxic environmental influences. Toxicity of aromatic carbonylic compounds in an established biotransformation system with P. chrysosporium can be estimated by calculating the corresponding logP values of the substrates and potential products. P. putida can be used to test the toxicity of aromatic ketones to the basic diomycete P. chrysosporium.     

A multi-channel continuous toxicity monitoring system using recombinant bioluminescent bacteria for classification of toxicity

A multi-channel system for continuous toxicity monitoring and classification of toxicity was developed based upon a previously developed two-stage minibioreactor system. The multi-channel system consists of a series of a two-stage minibioreactor systems connected by a fiber optic probe to a luminometer. Each channel was used for cultivating different recombinant bacterial strains, such as TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE), and DPD2540 (fabA::luxCDABE), which are induced by protein-, DNA-, and cell membrane damaging-agents, respectively. GC2 (lac::luxCDABE) is a bacterium expressing bioluminescence constitutively, which shows a reduction in its light level as cellular toxicity increases. Artificial wastewater samples were made by combining toxic chemicals, including Mitomycin C (a representative DNA damaging agent), phenol (a representative protein damaging agent), and cerulenin (a representative cell membrane damaging agent), and injecting this sample into each channel in order to simulate the detection of toxicity for mixed chemical samples. Each channel showed a specific bioluminescent response due to the toxic chemicals contained in the sample wastewater, while GC2 showed a general response to cellular toxicity. By using this multi-channel continuous toxicity monitoring system, classification of toxicity in field samples was found to be possible.     

Review on proteomic analyses of benzo[a]pyrene toxicity

Benzo[a]pyrene (BaP), a five-ring polycyclic aromatic hydrocarbon, is a well-recognized environmental pollutant. Coal-processing waste products, petroleum sludge, asphalt, creosote, and tobacco smoke, all contain high levels of BaP. Exposure to BaP elicits many adverse biological effects, including tumor formation, immunosuppression, teratogenicity, and hormonal effects. In addition to the genetic damage caused by BaP exposure, several studies have indicated the disruption of protein-protein signaling pathways. However, contrary to the large number of studies on BaP-induced DNA damage, only few data have been gathered on its effects at the protein level. This review highlights all proteomic studies to date used for assessing the toxicity of BaP and its metabolites in various organ systems. It will also give an overview on the role proteomics may play to elucidate the mechanisms underlying BaP toxicity.     

Comparison of the Acute Toxicity of Corexit 9500 and Household Cleaning Products

Surfactant formulations used in chemical dispersing agents are derived from the same functional components used in numerous household products such as dishwashing soaps and laundry detergents. During the Deep Water Horizon (DWH) oil spill response, a significant volume of chemical dispersant was deployed, causing members of the public and the media to question the role of chemical dispersant (Corexit 9500) usage in mitigating oil spill effects. Consequently, laboratory tests were conducted by regulatory agencies to further evaluate and substantiate the existing aquatic toxicity of Corexit dispersants. To help put dispersant toxicity in context, two independent accredited labs were commissioned to conduct parallel studies that compared the acute toxicity of Corexit 9500 to common household cleaning agents. The results indicate that the acute toxicity of Corexit 9500 to marine aquatic organism is either within the median range or less toxic than the household cleaning agents tested. The median LC50 value for Corexit 9500 exposures to Americamysis bahia was 42.5 mg/L (four products were less toxic and four products were more toxic); whereas, the median LC50 value for Corexit 9500 exposures to Menidia beryllina was 73.1 mg/L (one product was less toxic and seven products were more toxic).     

Target of rapamycin FATC domain as a general membrane anchor: The FKBP‐12 like domain of FKBP38 as a case study

Increased efforts have been undertaken to better understand the formation of signaling complexes at cellular membranes. Since the preparation of proteins containing a transmembrane domain or a prenylation motif is generally challenging an alternative membrane anchoring unit that is easy to attach, water‐soluble and binds to different membrane mimetics would find broad application. The 33‐residue long FATC domain of yeast TOR1 (y1fatc) fulfills these criteria and binds to neutral and negatively charged micelles, bicelles, and liposomes. As a case study, we fused it to the FKBP506‐binding region of the protein FKBP38 (FKBP38‐BD) and used ¹H–¹⁵N NMR spectroscopy to characterize localization of the chimeric protein to micelles, bicelles, and liposomes. Based on these and published data for y1fatc, its use as a C‐terminally attachable membrane anchor for other proteins is compatible with a wide range of buffer conditions (pH circa 6–8.5, NaCl 0 to >150 mM, presence of reducing agents, different salts such as MgCl₂ and CaCl₂). The high water‐solubility of y1fatc enables its use for titration experiments against a membrane‐localized interaction partner of the fused target protein. Results from studies with peptides corresponding to the C‐terminal 17–11 residues of the 33‐residue long domain by 1D ¹H NMR and CD spectroscopy indicate that they still can interact with membrane mimetics. Thus, they may be used as membrane anchors if the full y1fatc sequence is disturbing or if a chemically synthesized y1fatc peptide shall be attached by native chemical ligation, for example, unlabeled peptide to ¹⁵N‐labeled target protein for NMR studies.     

Terrestrial Metals Bioavailability: A Comprehensive Review and Literature-Derived Decision Rule for Ecological Risk Assessment

Interstudy variation among bioavailability studies is a primary deterrent to a universal methodology to assess metals bioavailability to soil-dwelling organisms and is largely the result of specific experimental conditions unique to independent studies. Accordingly, two datasets were established from relevant literature; one includes data from studies related to bioaccumulation (total obs = 520), while the other contains data from studies related to toxicity (total obs = 1264). Experimental factors that affected toxicity and bioaccumulation independent of the effect of soil chemical/physical properties were statistically apportioned from the variation attributed to soil chemical/physical properties for both datasets using a linear mixed model. Residual bioaccumulation data were then used to develop a non-parametric regression tree whereby bootstrap and cross-validation techniques were used to internally validate the resulting decision rule. A similar approach was employed with the toxicity dataset as an independent external validation. A validated decision rule is presented as a quantitative assessment tool that characterizes typical aerobic soils in terms of their potential to sequester common divalent cationic metal contaminants and mitigate their bioavailability to soil-dwelling biota.     

Pharmacotoxicological applications of an equivalent dermis: three measurements of cytotoxicity

The legal procedure for evaluating the toxicity of cosmetic, household, chemical and pharmaceutical products is still the irritancy Draize test on rabbits. Various irritation tests are currently being developed as alternatives toin vivo animal testing. Ourin vitro model system is composed of 24 equivalent dermis (ED) comprising a chitosan-cross-linked collagen-glycosaminoglycan matrix populated by foreskin fibroblasts. In evaluating this system for irritancy testing, three different measures of toxicity were used: MTT (dimethylthiazol diphenyltetrazolium bromide) reduction, and lactate dehydrogenase and interleukin-6 release. The experiments described herein represent a preliminary evaluation to determine the usefulness and predictive value of our 24 ED kit as an alternative method for the prediction of human dermal reaction, versus three chemical products: cadmium chloride, lauryl sulfate, and benzalkonium chloride. Preliminary results suggest that the ED may be a usefulin vitro model for the prediction of cutaneous and ocular toxicity and allow the development of a 24-skin-equivalent kit realized by seeding human normal keratinocytes onto the equivalent dermis.Abbreviations ED equivalent dermis - ECM extracellular matrix - FCM fibroblast culture medium - LDH lactate dehydrogenase - IL-6 interleukin-6 - MTT dimethylthiazol diphenyltetrazolium bromide     

Orientation of cecropin A helices in phospholipid bilayers determined by solid-state NMR spectroscopy

The orientation of the insect antibiotic peptide cecropin A (CecA) in the phospholipid bilayer membrane was determined using (15)N solid-state NMR spectroscopy. Two peptide samples, each specifically labeled with (15)N at Val(11) or Ala(27), were synthesized by solid phase techniques. The peptides were incorporated into phospholipid bilayers, prepared from a mixture of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol, and oriented on glass slides. The (15)N chemical shift solid-state NMR spectra from these uniaxially oriented samples display a single (15)N chemical shift frequency for each labeled residue. Both frequencies are near the upfield end of the (15)N chemical shift powder pattern, as expected for an alpha-helix with its long axis in the plane of the membrane and the NH bonds perpendicular to the direction of the magnetic field. These results support a mechanism of action in which CecA binds to and covers the membrane surface, thereby causing a general destabilization and leakiness of the lipid bilayer membrane. The data are discussed in relation to a proposed mechanism of membrane lysis and bacterial killing via an ion channel activity of CecA.     

Human Normal Bronchial Epithelial Cells: A Novel In Vitro Cell Model for Toxicity Evaluation

Human normal cell-based systems are needed for drug discovery and toxicity evaluation. hTERT or viral genes transduced human cells are currently widely used for these studies, while these cells exhibited abnormal differentiation potential or response to biological and chemical signals. In this study, we established human normal bronchial epithelial cells (HNBEC) using a defined primary epithelial cell culture medium without transduction of exogenous genes. This system may involve decreased IL-1 signaling and enhanced Wnt signaling in cells. Our data demonstrated that HNBEC exhibited a normal diploid karyotype. They formed well-defined spheres in matrigel 3D culture while cancer cells (HeLa) formed disorganized aggregates. HNBEC cells possessed a normal cellular response to DNA damage and did not induce tumor formation in vivo by xenograft assays. Importantly, we assessed the potential of these cells in toxicity evaluation of the common occupational toxicants that may affect human respiratory system. Our results demonstrated that HNBEC cells are more sensitive to exposure of 10~20 nm-sized SiO₂, Cr(VI) and B(a)P compared to 16HBE cells (a SV40-immortalized human bronchial epithelial cells). This study provides a novel in vitro human cells-based model for toxicity evaluation, may also be facilitating studies in basic cell biology, cancer biology and drug discovery.     

Susceptibility of male reproductive system to bisphenol A,an endocrine disruptor: Updates from epidemiological and experimental evidence

Bisphenol A (BPA) is an omnipresent environmental pollutant. Despite being restrictions in-force for its utilization, it is widely being used in the production of polycarbonate plastics and epoxy resins. Direct, low-dose, and long-term exposure to BPA is expected when they are used in the packaging of food products and are used as containers for food consumption. Occupationally, workers are typically exposed to BPA at higher levels and for longer periods during the manufacturing process. BPA is a known endocrine disruptor chemical (EDC), that causes male infertility, which has a negative impact on human life from emotional, physical, and societal standpoints. To minimize the use of BPA in numerous consumer products, efforts and regulations are being made. Despite legislative limits in numerous nations, BPA is still found in consumer products. This paper examines BPA's overall male reproductive toxicity, including its impact on the hypothalamic-pituitary-testicular (HPT) axis, hormonal homeostasis, testicular steroidogenesis, sperm parameters, reproductive organs, and antioxidant defense system. Furthermore, this paper highlighted the role of non-monotonic dose–response (NMDR) in BPA exposure, which will help to improve the overall understanding of the harmful effects of BPA on the male reproductive system.     

What does make an amyloid toxic: Morphology,structure or interaction with membrane?

The toxicity of amyloids is a subject under intense scrutiny. Many studies link this toxicity to the existence of various intermediate structures prior to the fiber formation and/or their specific interaction with membranes. Membranes can also be a catalyst of amyloidogenesis and the composition or the charge of membrane lipids may be of particular importance. Despite intensive research in the field, such intermediates are not yet fully characterized probably because of the lack of adapted methods for their analyses, and the mechanisms of interaction with the membrane are far to be understood. The purpose of this mini-review is to highlight some in vitro characteristics that seem to be convergent to explain the toxicity observed for some amyloids. Based on a comparison between the behavior of a model non-toxic amyloid (the Prion Forming Domain of HET-s) and its toxic mutant (M8), we could establish that short oligomers and/or fibers assembled in antiparallel β-sheets strongly interact with membrane leading to its disruption. Many recent evidences are in favor of the formation of antiparallel toxic oligomers assembled in β-helices able to form pores. We may also propose a new model of amyloid interaction with membranes by a “raft-like” mode of insertion that could explain important destabilization of membranes and thus amyloid toxicity.     

Chemical cytometry for monitoring metabolism of a Ras-mimicking substrate in single cells.

BACKGROUND: Chemical cytometry is an emerging technology that analyzes chemical contents of single cells by means of capillary electrophoresis or capillary chromatography. It has a potential to become an indispensable tool in analyses of heterogeneous cell populations such as those in tumors. Ras oncogenes are found in 30% of human cancers. To become fully functional products, oncogenic Ras proteins require at least three posttranslational modifications: farnesylation, endoproteolysis, and carboxyl-methylation. Therefore, enzymes that catalyze the three reactions, farnesyltransferase (FTase), endoprotease (EPase), and methyltransferase (MTase), are considered highly attractive therapeutic targets. In this work, we used chemical cytometry to study the metabolism of a pentapeptide substrate that can mimic Ras proteins with respect to their posttranslational modifications in solution. METHODS: Mouse mammary gland tumor cells (4T1) and mouse embryo fibroblasts (NIH3T3) were incubated with a fluorescently labeled pentapeptide substrate, 2',7'-difluorofluorescein-5-carboxyl-Gly-Cys-Val-Ilu-Ala. Cells were washed from the substrate and resuspended in phosphate buffered saline. Uptake of the substrate by the cells was monitored by laser scanning confocal microscopy. Single cells were injected into the capillary, lysed, and subjected to capillary electrophoresis. Fluorescent metabolic products were detected by laser-induced fluorescence and compared with products obtained by the conversion of the substrate by FTase, EPase, and MTase in solution. Co-sampling of single cells with the in-vitro products was used for such comparison. RESULTS: Confocal microscopy data showed that the substrate permeated the plasma membrane and clustered in the cytoplasm. Further capillary electrophoresis and chemical cytometry analyses showed that the substrate was converted into three fluorescently labeled products, two of which were secreted in the culture medium and one remained in the cells. The intracellular product was present at approximately 100,000 molecules per cell. The three metabolic products of the substrate were found to be different from the products of its processing by FTase, EPase, and MTase in solution. CONCLUSIONS: This is the first report of chemical cytometry in the context of Ras-signaling studies. The chemical cytometry method used in this work will find applications in the development of suitable peptide substrates for monitoring enzyme activities in single cells.     

Direct evidence that the N-terminal heptad repeat of Sendai virus fusion protein participates in membrane fusion.

Recent studies have demonstrated the importance of heptad repeat regions within envelope proteins of viruses in mediating conformational changes at various stages of viral infection. However, it is not clear if heptad repeats have a direct role in the actual fusion event. Here we have synthesized, fluorescently labeled and functionally and structurally characterized a wild-type 70 residue peptide (SV-117) composed of both the fusion peptide and the N-terminal heptad repeat of Sendai virus fusion protein, two of its mutants, as well as the fusion peptide and heptad repeat separately. One mutation was introduced in the fusion peptide (G119K) and another in the heptad repeat region (I154K). Similar mutations have been shown to drastically reduce the fusogenic ability of the homologous fusion protein of Newcastle disease virus. We found that only SV-117 was active in inducing lipid mixing of egg phosphatidylcholine/phosphatidyiglycerol (PC/PG) large unilamellar vesicles (LUV), and not the mutants nor the mixture of the fusion peptide and the heptad repeat. Functional characterization revealed that SV-117, and to a lesser extent its two mutants, were potent inhibitors of Sendai virus-mediated hemolysis of red blood cells, while the fusion peptide and SV-150 were negligibly active alone or in a mixture. Hemagglutinin assays revealed that none of the peptides disturb the binding of virions to red blood cells. Further studies revealed that SV-117 and its mutants oligomerize similarly in solution and in membrane, and have similar potency in inducing vesicle aggregation. Circular dichroism and FTIR spectroscopy revealed a higher helical content for SV-117 compared to its mutants in 40 % tifluorethanol and in PC/PG multibilayer membranes, respectively, ATR-FTIR studies indicated that SV-117 lies more parallel with the surface of the membrane than its mutants. These observations suggest a direct role for the N-terminal heptad repeat in assisting the fusion peptide in mediating membrane fusion.     

The Negative Effect of Soy Extract on Erythrocyte Membrane Fluidity: An Electron Paramagnetic Resonance Study

A decrease of erythrocyte membrane fluidity can contribute to the pathophysiology of hypertension. Soy products, which are used as alternative therapeutics in some cardiovascular conditions, contain various isoflavones (genistein, daidzein, and their glucosides, genistin and daidzin), which can incorporate cellular membrane and change its fluidity. The aim of this study was to examine the effects of soy extract (which generally corresponds to the soy products of isoflavone composition) on erythrocyte membrane fluidity at graded depths. We used electron paramagnetic resonance spectroscopy and fatty acid spin probes (5-DS and 12-DS), the spectra of which are dependent on membrane fluidity. After being treated with soy extract, erythrocytes showed a significant (P = 0.016) decrease of membrane fluidity near the hydrophilic surface, while there were no significant changes of fluidity in deeper hydrophobic membrane regions. These results suggest that soy products containing high levels of genistein and isoflavone glucosides may not be suitable for use in hypertension because they decrease erythrocyte membrane fluidity.     

Detection of Escherichia coli in potable water using direct impedance technology

Direct impedance measurement utilizing a medium previously described as being specific for Escherichia coli and which contains trimethylamine N -oxide (TMAO) and glucuronic acid was used to detect E. coli in water samples. The system was compared with the Colilert® presence/absence test and the United Kingdom standard membrane nitration technique using membrane lauryl sulphate broth. The impedance method correlated well with both the traditional membrane method (93%) and the Colilert® method (93.95%) for a number of different water types. No interference from Citrobacter spp. (as reported in previous studies) was detected in this study although some Salmonella spp. did give false-positive results. The data presented here suggest that the use of direct impedance may offer an alternative to conventional methods for the detection of E. coli in water.     

Orientation of the Escherichia coli outer membrane protein OmpX in phospholipid bilayer membranes determined by solid-State NMR

The solid-state NMR orientation-dependent frequencies measured for membrane proteins in macroscopically oriented lipid bilayers provide precise orientation restraints for structure determination in membranes. Here we show that this information can also be used to supplement crystallographic structural data to establish the orientation of a membrane protein in the membrane. This is achieved by incorporating a few orientation restraints, measured for the Escherichia coli outer membrane protein OmpX in magnetically oriented lipid bilayers (bicelles), in a simulated annealing calculation with the coordinates of the OmpX crystal structure. The (1)H-(15)N dipolar couplings measured for the seven Phe residues of OmpX in oriented bilayers can be assigned by back-calculation of the NMR spectrum from the crystal structure and are sufficient to establish the three-dimensional orientation of the protein in the membrane, while the (15)N chemical shifts provide a measure of cross-validation for the analysis. In C14 lipid bilayers, OmpX adopts a transmembrane orientation with a 7 degrees tilt of its beta-barrel axis relative to the membrane normal, matching the hydrophobic thickness of the barrel with that of the membrane.     

Psychosine,the cytotoxic sphingolipid that accumulates in globoid cell leukodystrophy,alters membrane architecture

Globoid cell leukodystrophy (GLD) is a neurological disease caused by deficiency of the lysosomal enzyme galactosylceramidase (GALC). In the absence of GALC, the cytotoxic glycosphingolipid, psychosine (psy), accumulates in the nervous system. Psychosine accumulation preferentially affects oligodendrocytes, leading to progressive demyelination and infiltration of activated monocytes/macrophages into the CNS. GLD is characterized by motor defects, cognitive deficits, seizures, and death by 2–5 years of age. It has been hypothesized that psychosine accumulation, primarily within lipid rafts, results in the pathogenic cascade in GLD. However, the mechanism of psychosine toxicity has yet to be elucidated. Therefore, we synthesized the enantiomer of psychosine (ent-psy) to use as a probe to distinguish between protein-psy (stereo-specific enantioselective) or membrane-psy (stereo-insensitive nonenantioselective) interactions. The enantiomer of psychosine has equal or greater toxicity compared with psy, suggesting that psy exerts its toxicity through a nonenantioselective mechanism. Finally, in this study we demonstrate that psy and ent-psy localize to lipid rafts, perturb natural and artificial membrane integrity, and inhibit protein Kinase C translocation to the plasma membrane. Although other mechanisms may play a role in disease, these data strongly suggest that psy exerts its effects primarily through membrane perturbation rather than through specific protein-psy interactions.     

Effect of l-carnitine on liver cell membranes in ethanol-intoxicated rats

Ethanol intoxication is characterized by changes in cell metabolism which alter the structure and function of cell membrane components, including phospholipids and integral membrane proteins. The interaction of food nutrients with ethanol may modulate alcohol toxicity. One such compound is l-carnitine (l-3-hydroxy-4-N,N,N-trimethylaminobutyrate), which is also an antioxidant. Here we investigate l-carnitine as an antioxidant and assess its effect on the composition and electrical charge of liver cell membranes in ethanol-intoxicated rats. Qualitative and quantitative phospholipid composition and the presence of integral membrane proteins were determined by high performance liquid chromatography (HPLC). Electrophoresis was used to determine the surface charge density of the rat liver cell membranes. Ethanol increased phospholipid levels and altered the level of integral proteins as determined by decreased phenylalanine (Phe), cysteine (Cys) and lysine (Lys). Ethanol significantly enhanced changes in the surface charge density of the liver cell membranes. l-Carnitine administration to ethanol-intoxicated rats significantly protects phospholipids and proteins against oxidative modifications. Therefore, the beneficial effect of l-carnitine may be connected to its ability to scavenge free radicals.     

Abeta Peptide Toxicity is Reduced After Treatments Decreasing Phosphatidylethanolamine Content in Differentiated Neuroblastoma Cells

We investigated whether the toxicity of oligomeric amyloid-beta peptide (Abeta1-42) upon differentiated human neuroblastoma SH-SY5Y cells, can be affected by changes of membrane lipid composition. An immunostaining technique, using lipids extracted from the cells and separated by thin layer chromatography, suggested that Abeta preferentially binds to phosphatidylethanolamine (PE), one of the major lipids in the cell extract. For this reason, we utilized treatments with putative inhibitors of phosphatidylethanolamine biosynthesis (choline, phosphocholine, R59949) to decrease its proportion in the cell membrane; choline treatment (2.5 mM, 24 h) showed the best performance, reducing phosphatidylethanolamine content from 5.7 to 3.3 μg phosphorous/mg protein. Either the extent of Abeta binding or its toxicity decreased onto choline-treated cells. These data may open the possibility to develop future strategies aiming to reduce Abeta toxicity in Alzheimer disease.     

Analysis of the amide <Superscript>15</Superscript>N chemical shift tensor of the C<Subscript>α</Subscript> tetrasubstituted constituent of membrane-active peptaibols,the α-aminoisobutyric acid residue,compared to those of di- and tri-substituted proteinogenic amino acid residues

In protein NMR spectroscopy the chemical shift provides important information for the assignment of residues and a first structural evaluation of dihedral angles. Furthermore, angular restraints are obtained from oriented samples by solution and solid-state NMR spectroscopic approaches. Whereas the anisotropy of chemical shifts, quadrupolar couplings and dipolar interactions have been used to determine the structure, dynamics and topology of oriented membrane polypeptides using solid-state NMR spectroscopy similar concepts have been introduced to solution NMR through the measurements of residual dipolar couplings. The analysis of ¹⁵N chemical shift spectra depends on the accuracy of the chemical shift tensors. When investigating alamethicin and other peptaibols, i.e. polypeptides rich in α-aminoisobutyric acid (Aib), the ¹⁵N chemical shift tensor of this C_α-tetrasubstituted amino acid exhibits pronounced differences when compared to glycine, alanine and other proteinogenic residues. Here we present an experimental investigation on the ¹⁵N amide Aib tensor of N-acetyl-Aib-OH and for the Aib residues within peptaibols. Furthermore, a statistical analysis of the tensors published for di- (glycine) and tri-substituted residues has been performed, where for the first time the published data sets are compiled using a common reference. The size of the isotropic chemical shift and main tensor elements follows the order di- < tri- < tetra-substituted amino acids. A ¹⁵N chemical shift-¹H-¹⁵N dipolar coupling correlation NMR spectrum of alamethicin is used to evaluate the consequences of variations in the main tensor elements for the structural analysis of this membrane peptide.