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Oxylipins are lipid-derived molecules that are ubiquitous in eukaryotes and whose functions in plant physiology have been widely reported. They appear to play a major role in plant immunity by orchestrating reactive oxygen species (ROS) and hormone-dependent signalling pathways. The present work focuses on the specific case of fatty acid hydroperoxides (HPOs). Although some studies report their potential use as exogenous biocontrol agents for plant protection, evaluation of their efficiency in planta is lacking and no information is available about their mechanism of action. In this work, the potential of 13(S)-hydroperoxyoctadeca-(9Z,11E)-dienoic acid (13-HPOD) and 13(S)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid (13-HPOT), as plant defence elicitors and the underlying mechanism of action are investigated. Arabidopsis thaliana leaf resistance to Botrytis cinerea was observed after root application with HPOs. They also activate early immunity-related defence responses, like ROS. As previous studies have demonstrated their ability to interact with plant plasma membranes (PPM), we have further investigated the effects of HPOs on biomimetic PPM structure using complementary biophysics tools. Results show that HPO insertion into PPM impacts its global structure without solubilizing it. Relationship between biological assays and biophysical analysis suggests that lipid amphiphilic elicitors that directly act on membrane lipids might trigger early plant defence events

Linoleic and linolenic acid hydroperoxides (HPOs) constitute key intermediate oxylipins playing an important role as signaling molecules during plant defense processes in response to biotic or abiotic stress. They have also been demonstrated in vitro as antimicrobial agents against plant fungi and bacteria. To reach the phytopathogens in vivo, the HPOs biosynthesized in the plant cells must cross the plant plasma membrane (PPM) where they can also interact with plasma membrane lipids and have an effect on their organization. In the present study, we have investigated the interaction properties of HPOs with PPM at a molecular level using biophysical tools combining in vitro and in silico approaches and using plant biomimetic lipid systems. Our results have shown that HPOs are able to interact with PPM lipids and perturb their lateral organization. Glucosylceramide (GluCer) is a privileged partner, sitosterol lessens their binding and the presence of both GluCer and sitosterol further reduces their interaction. Hydrophobic effect and polar interactions are involved in the binding. The chemical structure of HPOs influences their affinity for PPM lipids. The presence of three double bonds in the HPO molecule gives rise to a higher affinity comparatively to two double bonds, which can be explained by their differential interaction with the lipid polar headgroups.

Fatty acid hydroperoxides (HPOs) are amphiphilic molecules naturally produced by plants in stressed conditions and involved in plant immunity as signalling molecules. Although some studies report their potential use as exogenous biocontrol agents for plant protection, evaluation of their efficiency in planta is lacking and no information is available about their mechanism of action. In this work, the potential of two HPO forms, 13-HPOD and 13-HPOT, as plant defence elicitors and the underlying mechanism of action are investigated. Both HPOs trigger Arabidopsis innate immunity. They increase plant resistance to the pathogenic fungi Botrytis cinerea and activate early immunity-related defence responses, like ROS production. As our previous study has suggested that HPOs are able to interact with the plant plasma membrane (PPM) lipid fraction, we have further investigated the effects of HPOs on biomimetic PPM structure using complementary biophysics tools. Results show that HPO insertion into PPM impacts its global structure without solubilizing it. 13-HPOT, with an additional double bond compared to 13-HPOD, exerts a higher effect by fluidifying and reducing the thickness of the bilayer. Correlation between biological assays and biophysical analysis suggests that lipid amphiphilic elicitors that directly act on membrane lipids might trigger early plant defence events.

Cellular membranes are composed of a wide diversity of lipid species in varying proportions and these compositions are representative of the organism, cellular type and organelle to which they belong. Because models of these molecular systems simulated by MD steadily gain in size and complexity, they are increasingly representative of specific compositions and behaviors of biological membranes. Due to the number of lipid species involved, of force fields and topologies and because of the complexity of membrane objects that have been simulated, LIMONADA has been developed as an open database allowing to handle the various aspects of lipid membrane simulation. LIMONADA presents published membrane patches with their simulation files and the cellular membrane it models. Their compositions are then detailed based on the lipid identification from LIPID MAPS database plus the lipid topologies and the force field used. LIMONADA is freely accessible on the web at https://limonada.univ-reims.fr/.

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph–mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.

Hydrophobic organic soil contaminants such as polycyclic aromatic hydrocarbons (PAH) are poorly mobile in the aqueous phase and tend to sorb to the soil matrix, resulting in low bioavailability. Some filamentous fungi are efficient in degrading this kind of pollutants. However, the mechanism of mobilization of hydrophobic compounds by non-motile microorganisms such as filamentous fungi needs investigations to improve pollutant bioavailability and bioremediation efficiency. Usual homogeneous media for microbial growth in the lab are poorly suited to model the soil, which is a compartmentalized and heterogeneous habitat. A microfluidic device was designed to implement a compartmentalization of the fungal inoculum and the source of the pollutant benzo[a]pyrene (BaP) as a deposit of solid crystals in order to gain a further insight into the mechanisms involved in the access to the contaminant and its uptake in soils. Thus in this device, two chambers are connected by an array of parallel microchannels that are wide enough to allow individual hyphae to grow through them. Macro-cultures of Talaromyces helicus in direct contact with BaP have shown its uptake and intracellular storage in lipid bodies despite the low propensity of BaP to cross aqueous phases as shown by simulation. Observations of T. helicus in the microfluidic device through laser scanning confocal microscopy indicate preferential uptake of BaP at a close range and through contact with the cell wall. However faint staining of some hyphae before contact with the deposit also suggests an extracellular transport phenomenon. Macro-culture filtrates analyses have shown that T. helicus releases extracellular non-lipidic surface-active compounds able to lower the surface tension of culture filtrates to 49.4 mN/m. Thus, these results highlight the significance of active mechanisms to reach hydrophobic contaminants before their uptake by filamentous fungi in compartmentalized micro-environments and the potential to improve them through biostimulation approaches for soil mycoremediation.

International audience; Glycolipids constitute a class of molecules with various biological activities. Among them, sugar-based bolaamphiphiles characterized by their biocompatibility, biodegradability and lower toxicity, became interesting for the development of efficient and low cost lipid-based drug delivery systems. Their activity seems to be closely related to their interactions with the lipid components of the plasma membrane of target cells. Despite many works devoted to the chemical synthesis and characterization of sugar-based bolaamphiphiles, their interactions with plasma membrane have not been completely elucidated. In this work, two sugar-based bolaamphiphiles differing only at the level of their sugar residues were chemically synthetized. Their interactions with membranes have been investigated using model membranes containing or not sterol and with in silico approaches. Our findings indicate that the nature of sugar residues has no significant influence for their membrane interacting properties, while the presence of sterol attenuates the interactions of both bolaamphiphiles with the membrane systems. The understanding of this distinct behavior of bolaamphiphiles towards sterol-containing membrane systems could be useful for their applications as drug delivery systems.

With the emergence of multi-drug resistant bacteria and hospital-acquired infections, there is an urgent need to develop new antibiotics. Here, we report the synthesis, physico-chemical characterizations, and antimicrobial activity assays of four Azo compounds that differ in their alkyl chain length. The molecular mechanism of their antibacterial activity was investigated by complementary in vitro and in silico biophysical studies. The compounds with alkyl chain lengths of four or six carbons showed a low MIC50 against Escherichia coli and Bacillus subtilis. Our investigations into the mechanism of their action revealed that phosphatidylethanolamine in the bacterial plasma membrane plays an important role in their antibacterial activity.

Natural phytotoxic compounds could become an alternative to traditional herbicides in the framework of sustainable agriculture. Nonanoic acid, sarmentine and sorgoleone are such molecules extracted from plants and able to inhibit the growth of various plant species. However, their mode of action is not fully understood and despite clues indicating that they could affect the plant plasma membrane, molecular details of such phenomenon are lacking. In this paper, we investigate the interactions between those natural herbicides and artificial bilayers mimicking the plant plasma membrane. First, their ability to affect lipid order and fluidity is evaluated by means of fluorescence measurements. It appears that sorgoleone has a clear ordering effect on lipid bilayers, while nonanoic acid and sarmentine induce no or little change to these parameters. Then, a thermodynamic characterization of interactions of each compound with lipid vesicles is obtained with isothermal titration calorimetry, and their respective affinity for bilayers is found to be ranked as follows: sorgoleone > sarmentine > nonanoic acid. Finally, molecular dynamics simulations give molecular details about the location of each compound within a lipid bilayer and confirm the rigidifying effect of sorgoleone. Data also suggest that mismatch in alkyl chain length between nonanoic acid or sarmentine and lipid hydrophobic tails could be responsible for bilayer destabilization. Results are discussed regarding their implications for the phytotoxicity of these compounds.

International audience; The lipid composition of plasma membrane (PM) and the corresponding detergent-insoluble membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl inositol phosphorylceramides (GIPCs). Using tobacco (Nicotiana tabacum) 'Bright Yellow 2' cell suspension and leaves, evidence is provided that GIPCs represent up to 40 mol % of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35 +/- 7 nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed.

In eukaryotes, membrane contact sites (MCS) allow direct communication between organelles. Plants have evolved unique MCS, the plasmodesmata intercellular pores, which combine endoplasmic reticulum (ER) - plasma membrane (PM) contacts with regulation of cell-to-cell signalling. The molecular mechanism and function of membrane tethering within plasmodesmata remains unknown.Here we show that the Multiple C2 domains and Transmembrane region Protein (MCTP) family, key regulators of cell-to-cell signalling in plants, act as ER - PM tethers specifically at plasmodesmata. We report that MCTPs are core plasmodesmata proteins that insert into the ER via their transmembrane region whilst their C2 domains dock to the PM through interaction with anionic phospholipids. A mctp3/4 loss-of-function mutant induces plant developmental defects while MCTP4 expression in a yeast Δtether mutant partially restores ER-PM tethering. Our data suggest that MCTPs are unique membrane tethers controlling both ER-PM contacts and cell-cell signalling.

An extra sugar protects Many microbial pathogens produce proteins that are toxic to the cells that they are targeting. Broad-leaved plants are susceptible to NLP (necrosis and ethylene-inducing peptide 1–like protein) toxins. Lenarčič et al. identified the receptors for NLP toxins to be GIPC (glycosylinositol phosphorylceramide) sphingolipids (see the Perspective by Van den Ackerveken). Their findings reveal why these toxins only attack broad-leaved plants (so-called eudicots): If the sphingolipid carries just two hexoses, as is the case for eudicots, the toxin binds and causes cell lysis. But in monocots with sphingolipids that have three hexoses, the toxin is ineffective. Science , this issue p. 1431 ; see also p. 1383

Plasma membranes are complex entities common to all living cells. The basic principle of their organization appears very simple, but they are actually of high complexity and represent very dynamic structures. The interactions between bioactive molecules and lipids are important for numerous processes, from drug bioavailability to viral fusion. The cell membrane is a carefully balanced environment and any change inflicted upon its structure by a bioactive molecule must be considered in conjunction with the overall effect that this may have on the function and integrity of the membrane. Conceptually, understanding the molecular mechanisms by which bioactive molecules interact with cell membranes is of fundamental importance.Lipid specificity is a key factor for the detailed understanding of the penetration and/or activity of lipid-interacting molecules and of mechanisms of some diseases. Further investigation in that way should improve drug discovery and development of membrane-active molecules in many domains such as health, plant protection or microbiology.In this review, we will present complementary biophysical approaches that can give information about lipid specificity at a molecular point of view. Examples of application will be given for different molecule types, from biomolecules to pharmacological drugs. A special emphasis is given to cyclic lipopeptides since they are interesting molecules in the scope of this review by combining a peptidic moiety and a lipidic tail and by exerting their activity via specific interactions with the plasma membrane.

The olfactory sense is the dominant sensory perception for many animals. When Richard Axel and Linda B. Buck received the Nobel Prize in 2004 for discovering the G protein-coupled receptors’ role in olfactory cells, they highlighted the importance of olfaction to the scientific community. Several theories have tried to explain how cells are able to distinguish such a wide variety of odorant molecules in a complex context in which enantiomers can result in completely different perceptions and structurally different molecules. Moreover, sex, age, cultural origin, and individual differences contribute to odor perception variations that complicate the picture. In this article, recent advances in olfaction theory are presented, and future trends in human olfaction such as structure-based odor prediction and artificial sniffing are discussed at the frontiers of chemistry, physiology, neurobiology, and machine learning.

We extended the use of Peplook, an in silico procedure for the prediction of three-dimensional (3D) models of linear peptides to the prediction of 3D models of cyclic peptides and thanks to the ab initio calculation procedure, to the calculation of peptides with non-proteinogenic amino acids. Indeed, such peptides cannot be predicted by homology or threading. We compare the calculated models with NMR and X-ray models and for the cyclic peptides, with models predicted by other in silico procedures (Pep-Fold and I-Tasser). For cyclic peptides, on a set of 38 peptides, average root mean square deviation of backbone atoms (BB-RMSD) was 3.8 and 4.1 A for Peplook and Pep-Fold, respectively. The best results are obtained with I-Tasser (2.5 A) although evaluations were biased by the fact that the resolved Protein Data Bank models could be used as template by the server. Peplook and Pep-Fold give similar results, better for short (up to 20 residues) than for longer peptides. For peptides with non-proteinogenic residues, performances of Peplook are sound with an average BB-RMSD of 3.6 A for ‘non-natural peptides’ and 3.4 A for peptides combining non-proteinogenic residues and cyclic structure. These results open interesting possibilities for the design of peptidic drugs. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

Class I fusion glycoproteins of viruses are involved in the fusion between viral envelope and cell membrane. A region located in the N-terminal domain of these glycoproteins, called the fusion peptide, is essential for fusion. Fusion peptides are able to induce by themselves in vitro membrane fusion. In this paper, we review the properties of those peptides related to their fusogenicity, in particular the correlation existing between their ability to insert obliquely in membranes and fusogenicity. This relation notably allows predicting successfully the minimal region of some fusion peptides sufficient to induce significant in vitro fusion. The notion of obliquity and fusogenicity is discussed in terms of the existing proposed mechanisms for viral fusion.

Interfacial properties of surfactants are dependent on the conformation adopted by the hydrophilic headgroup or/and the hydrophobic tail at the boundary limit of two immiscible phases. Here, we demonstrate the impacts of the carbonyl group (-CO-) location of the ester bond of sugar-based surfactants by comparing some properties of two closely related esters, octyl glucuronate and glucose octanoate, at the air-water interface. The carbonyl group location influences the rate and extent of interfacial adsorption and the rheology properties of sugar esters at the air-water interface, which were evaluated by dynamic surface tension and complex surface viscoelastic measurements. Octyl glucuronate adsorbs the fastest at the air-water interface whereas glucose octanoate reduces the dynamic surface tension at the lowest value and exhibits the highest film viscoelasticity. Differences are attributed to molecular conformation constraints inducing relevant changes to the surface coverage kinetic capacity and the interaction strengths of the octyl sugar ester adsorbed films at the air-water interface. All of the results are supported by the minimum cross-sectional area values per molecule determined by both experimental and computational approaches.

Serine protease inhibitors (serpins) are a structurally related but functionally diverse family of ubiquitous proteins. We previously described Ixodes ricinus immunosuppressor (Iris) as a serpin from the saliva of the tick I. ricinus displaying high affinity for human leukocyte elastase. Iris also displays pleotropic effects because it interferes with both the immune response and hemostasis of the host. It thus inhibits lymphocyte proliferation and the secretion of interferon-gamma or tumor necrosis factor-alpha by peripheral blood mononuclear cells, and also platelet adhesion, coagulation and fibrinolysis. Its ability to interfere with coagulation and fibrinolysis, but not platelet adhesion, depends on the integrity of its antiproteolytic reactive center loop domain. Here, we dissect the mechanisms underlying the interaction of recombinant Iris with peripheral blood mononuclear cells. We show that Iris binds to monocytes/macrophages and inhibits their ability to secrete tumor necrosis factor-alpha. Recombinant Iris also has a protective role in endotoxemic shock. The anti-inflammatory ability of Iris does not depend on its antiprotease activity. Moreover, we pinpoint the exosites involved in this activity.

The interactions between a drug and lipids may be critical for the pharmacological activity. We previously showed that the ability of a fluoroquinolone antibiotic, ciprofloxacin, to induce disorder and modify the orientation of the acyl chains is related to its propensity to be expelled from a monolayer upon compression [1]. Here, we compared the binding of ciprofloxacin on DPPC and DPPG liposomes (or mixtures of phospholipids [DOPC:DPPC], and [DOPC:DPPG]) using quasi-elastic light scattering and steady-state fluorescence anisotropy. We also investigated ciprofloxacin effects on the transition temperature (T(m)) of lipids and on the mobility of phosphate head groups using Attenuated Total Reflection Fourier Transform Infrared-Red Spectroscopy (ATR-FTIR) and (31)P Nuclear Magnetic Resonance (NMR) respectively. In the presence of ciprofloxacin we observed a dose-dependent increase of the size of the DPPG liposomes whereas no effect was evidenced for DPPC liposomes. The binding constants K(app) were in the order of 10(5) M(-1) and the affinity appeared dependent on the negative charge of liposomes: DPPG>DOPC:DPPG (1:1; M:M)>DPPC>DOPC:DPPC (1:1; M:M). As compared to the control samples, the chemical shift anisotropy (Deltasigma) values determined by (31)P NMR showed an increase of 5 and 9 ppm for DPPC:CIP (1:1; M:M) and DPPG:CIP (1:1; M:M) respectively. ATR-FTIR experiments showed that ciprofloxacin had no effect on the T(m) of DPPC but increased the order of the acyl chains both below and above this temperature. In contrast, with DPPG, ciprofloxacin induced a marked broadening effect on the transition with a decrease of the acyl chain order below its T(m) and an increase above this temperature. Altogether with the results from the conformational analysis, these data demonstrated that the interactions of ciprofloxacin with lipids depend markedly on the nature of their phosphate head groups and that ciprofloxacin interacts preferentially with anionic lipid compounds, like phosphatidylglycerol, present at a high content in these membranes.

Physical properties of membranes, such as fluidity, charge or curvature influence their function. Proteins and peptides can modulate those properties and conversely, the lipids can affect the activity and/or the structure of the former. Tilted peptides are short hydrophobic protein fragments characterized by an asymmetric distribution of their hydrophobic residues when helical. They were detected in viral fusion proteins and in proteins involved in different biological processes that need membrane destabilization. Those peptides and non lamellar lipids such as PE or PA appear to cooperate in the lipid destabilization process by enhancing the formation of negatively-curved domains. Such highly bent lipidic structures could favour the formation of the viral fusion pore intermediates or that of toroidal pores. Structural flexibility appears as another crucial property for the interaction of peptides with membranes. Computational analysis on another kind of lipid-interacting peptides, i.e. cell penetrating peptides (CPP) suggests that peptides being conformationally polymorphic should be more prone to traverse the bilayer. Future investigations on the structural intrinsic properties of tilted peptides and the influence of CPP on the bilayer organization using the techniques described in this chapter should help to further understand the molecular determinants of the peptide/lipid inter-relationships.

Probing drug/lipid interactions at the molecular level represents an important challenge in pharmaceutical research and membrane biophysics. Previous studies showed differences in accumulation and intracellular activity between two fluoroquinolones, ciprofloxacin and moxifloxacin, that may actually result from their differential susceptibility to efflux by the ciprofloxacin transporter. In view of the critical role of lipids for the drug cellular uptake and differences observed for the two closely related fluoroquinolones, we investigated the interactions of these two antibiotics with lipids, using an array of complementary techniques. Moxifloxacin induced, to a greater extent than ciprofloxacin, an erosion of the DPPC domains in the DOPC fluid phase (atomic force microscopy) and a shift of the surface pressure-area isotherms of DOPC/DPPC/fluoroquinolone monolayer toward lower area per molecule (Langmuir studies). These effects are related to a lower propensity of moxifloxacin to be released from lipid to aqueous phase (determined by phase transfer studies and conformational analysis) and a marked decrease of all-trans conformation of acyl-lipid chains of DPPC (determined by ATR-FTIR) without increase of lipid disorder and change in the tilt between the normal and the germanium surface (also determined by ATR-FTIR). All together, differences of ciprofloxacin as compared to moxifloxacin in their interactions with lipids could explain differences in their cellular accumulation and susceptibility to efflux transporters.

We had previously predicted successfully the minimal fusion peptides (FPs) of the human immunodeficiency virus 1 (HIV-1) gp41 and the bovine leukemia virus (BLV) gp30 using an original approach based on the obliquity/fusogenicity relationship of tilted peptides. In this paper, we have used the same method to predict the shortest FP capable of inducing optimal fusion in vitro of the simian immunodeficiency virus (SRT) mac isolate and of other SIVs and human immunodeficiency virus (HIV-2) isolates. In each case, the 11-residue-long peptide was predicted as the minimal FP. For the SfV mac isolate, liposome lipid-mixing and leakage assays confirmed that this peptide is the shortest peptide inducing optimal fusion in vitro, being therefore the minimal FP. These results are another piece of evidence that the tilted properties of FPs are important for the fusion process and that our method can be used to predict the minimal FPs of other viruses. Copyright (c) 2007 European Peptide Society and John Wiley & Sons, Ltd.

The elastic properties of membrane bilayers are key parameters that control its deformation and can be affected by pharmacological agents. Our previous atomic force microscopy studies revealed that the macrolide antibiotic, azithromycin, leads to erosion of DPPC domains in a fluid DOPC matrix [A. Berquand, M. P. Mingeot-Leclercq, Y. F. Dufrene, Real-time imaging of drug-membrane interactions by atomic force microscopy, Biochim. Biophys. Acta 1664 (2004) 198-205.]. Since this observation could be due to an effect on DOPC cohesion, we investigated the effect of azithromycin on elastic properties of DOPC giant unilamellar vesicles (GUVs). Microcinematographic and morphometric analyses revealed that azithromycin addition enhanced lipid membranes fluctuations, leading to eventual disruption of the largest GUVs. These effects were related to change of elastic moduli of DOPC, quantified by the micropipette aspiration technique. Azithromycin decreased both the bending modulus (k(c), from 23.1+/-3.5 to 10.6+/-4.5 k(B)T) and the apparent area compressibility modulus (K(app), from 176+/-35 to 113+/-25 mN/m). These data suggested that insertion of azithromycin into the DOPC bilayer reduced the requirement level of both the energy for thermal fluctuations and the stress to stretch the bilayer. Computer modeling of azithromycin interaction with DOPC bilayer, based on minimal energy, independently predicted that azithromycin (i) inserts at the interface of phospholipid bilayers, (ii) decreases the energy of interaction between DOPC molecules, and (iii) increases the mean surface occupied by each phospholipid molecule. We conclude that azithromycin inserts into the DOPC lipid bilayer, so as to decrease its cohesion and to facilitate the merging of DPPC into the DOPC fluid matrix, as observed by atomic force microscopy. These investigations, based on three complementary approaches, provide the first biophysical evidence for the ability of an amphiphilic antibiotic to alter lipid elastic moduli. This may be an important determinant for drug: lipid interactions and cellular pharmacology.

Angiogenesis is a crucial step in many pathologies, including tumor growth and metastasis. Here, we show that tilted peptides exert antiangiogenic activity. Tilted (or oblique-oriented) peptides are short peptides known to destabilize membranes and lipid cores and characterized by an asymmetric distribution of hydrophobic residues along the axis when helical. We have previously shown that 16-kDa fragments of the human prolactin/growth hormone (PRL/GH) family members are potent angiogenesis inhibitors. Here, we demonstrate that all these fragments possess a 14-aa sequence having the characteristics of a tilted peptide. The tilted peptides of human prolactin and human growth hormone induce endothelial cell apoptosis, inhibit endothelial cell proliferation, and inhibit capillary formation both in vitro and in vivo . These antiangiogenic effects are abolished when the peptides' hydrophobicity gradient is altered by mutation. We further demonstrate that the well known tilted peptides of simian immunodeficiency virus gp32 and Alzheimer's β-amyloid peptide are also angiogenesis inhibitors. Taken together, these results point to a potential new role for tilted peptides in regulating angiogenesis.

Serpins (serine protease inhibitors) are a large family of structurally related proteins found in a wide variety of organisms, including hematophagous arthropods. Protein analyses revealed that Iris, previously described as an immunomodulator secreted in the tick saliva, is related to the leukocyte elastase inhibitor and possesses serpin motifs, including the reactive center loop (RCL), which is involved in the interaction between serpins and serine proteases. Only serine proteases were inhibited by purified recombinant Iris (rIris), whereas mutants L339A and A332P were found devoid of any protease inhibitory activity. The highest Ka was observed with human leukocyte-elastase, suggesting that elastase-like proteases are the natural targets of Iris. In addition, mutation M340R completely changed both Iris substrate specificity and affinity. This likely identified Met-340 as amino acid P1 in the RCL. The effects of rIris and its mutants were also tested on primary hemostasis, blood clotting, and fibrinolysis. rIris increased platelet adhesion, the contact phase-activated pathway of coagulation, and fibrinolysis times in a dose-dependent manner, whereas rIris mutant L339A affected only platelet adhesion. Taken together, these results indicate that Iris disrupts coagulation and fibrinolysis via the anti-proteolytic RCL domain. One or more other domains could be responsible for primary hemostasis inhibition. To our knowledge, this is the first ectoparasite serpin that interferes with both hemostasis and the immune response.

Elucidation of the molecular mechanism leading to biomembrane fusion is a challenging issue in current biomedical research in view of its involvement in controlling cellular functions and in mediating various important diseases. According to the generally admitted stalk mechanism described for membrane fusion, negatively curved lipids may play a central role during the early steps of the process. In this study, we used atomic force microscopy (AFM) to address the crucial question of whether negatively curved lipids influence the interaction of the simian immunodeficiency virus (SIV) fusion peptide with model membranes. To this end, dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers containing 0.5 mol % dioleoylphosphatidic acid (DOPA) were incubated with the SIV peptide and imaged in real time using AFM. After a short incubation time, we observed a 1.9 nm reduction in the thickness of the DPPC domains, reflecting either interdigitation or fluidization of lipids. After longer incubation times, these depressed DPPC domains evolved into elevated domains, composed of nanorod structures protruding several nanometers above the bilayer surface and attributed to cylindrical reverse micelles. Such DOPC/DPPC/DOPA bilayer modifications were never observed with nontilted peptides. Accordingly, this is the first time that AFM reveals the formation of cylindrical reverse micelles in lipid bilayers promoted by fusogenic peptides.

In this study, we describe an in silico method to design peptides that can be made of non-natural amino acids and elicit specific membrane-interacting properties. The originality of the method holds in the capacities developed to design peptides from any non-natural amino acids as easily as from natural ones, and to test the structure stability by an angular dynamics rather than the currently-used molecular dynamics. The goal of this study was to design a non-natural tilted peptide. Tilted peptides are short protein fragments able to destabilize lipid membranes and characterized by an asymmetric distribution of hydrophobic residues along their helix structure axis. The method is based on the random generation of peptides and their selection on three main criteria: mean hydrophobicity and the presence of at least one polar residue; tilted insertion at the level of the acyl chains of lipids of a membrane; and conformational stability in that hydrophobic phase. From 10,000,000 randomly-generated peptides, four met all the criteria. One was synthesized and tested for its lipid-destabilizing properties. Biophysical assays showed that the “de novo” peptide made of non-natural amino acids is helical either in solution or into lipids as tested by Fourier transform infrared spectroscopy and is able to induce liposome fusion. These results are in agreement with the calculations and validate the theoretical approach.

Understanding the molecular bases of biomembrane fusion events is a challenging issue in current biomedical research in view of its involvement in controlling cellular functions and in mediating various important diseases. In this study, we used atomic force microscopy (AFM) to address the crucial question as to whether negatively curved lipids influence the ability of a viral fusion peptide to perturb the organization of supported lipid bilayers. To this end, an original approach was developed that makes use of an AFM tip functionalized with phospholipase D (PLD) enzymes to generate in situ small amounts of negatively curved phosphatidic acid (PA) in mixed dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers. Real-time AFM imaging revealed that this nanomodification dramatically enhanced subsequent interaction with the simian immunodeficiency virus (SIV) fusion peptide. At short incubation time, the SIV peptide induced a 1.9 nm thickness reduction of the DPPC domains, reflecting either interdigitation or fluidification of the lipids. At longer incubation time, these depressed domains transformed into elevated striated domains, protruding one to several nanometers above the bilayer surface. Two complementary experiments, i.e. addition of the peptide onto DOPC/DPPC/DOPA bilayers or onto DOPC/DPPC bilayers pretreated with a PLD solution, confirmed that both PA and SIV peptides are required to induce depressed and striated domains. Accordingly, this is the first time that a high-resolution imaging technique is used to demonstrate that negatively curved lipids affect the membrane activity of fusion peptides. We believe the nanoscale approach presented here, i.e. use of enzyme-functionalized AFM tips to modify lipid bilayers, will find exciting new applications in nanobiotechnology for the design of biomimetic surfaces.

Thiamine triphosphate (ThTP) is found in most living organisms and it may act as a phosphate donor for protein phosphorylation. We have recently cloned the cDNA coding for a highly specific mammalian 25 kDa thiamine triphosphatase (ThTPase; EC 3.6.1.28). As the enzyme has a high catalytic efficiency and no sequence homology with known phosphohydrolases, it was worth investigating its structure and catalytic properties. For this purpose, we expressed the untagged recombinant human ThTPase (hThTPase) in E. coli, produced the protein on a large scale and purified it to homogeneity. Its kinetic properties were similar to those of the genuine human enzyme, indicating that the recombinant hThTPase is completely functional. Mg2+ ions were required for activity and Ca2+ inhibited the enzyme by competition with Mg2+. With ATP as substrate, the catalytic efficiency was 10(-4)-fold lower than with ThTP, confirming the nearly absolute specificity of the 25 kDa ThTPase for ThTP. The activity was maximum at pH 8.5 and very low at pH 6.0. Zn2+ ions were inhibitory at micromolar concentrations at pH 8.0 but activated at pH 6.0. Kinetic analysis suggests an activator site for Mg2+ and a separate regulatory site for Zn2+. The effects of group-specific reagents such as Woodward's reagent K and diethylpyrocarbonate suggest that at least one carboxyl group in the active site is essential for catalysis, while a positively charged amino group may be involved in substrate binding. The secondary structure of the enzyme, as determined by Fourier-transform infrared spectroscopy, was predominantly beta-sheet and alpha-helix.

Being able to participate in the reverse cholesterol transport (RCT), high density lipoproteins (HDL) are known to be anti-atherogenic. In order to understand such a process, it is thus essential to have a detailed knowledge of the structure and molecular organisation of HDL. Reconstituted nascent high density lipoproteins (r-HDL), consisting of synthetic phospholipids together with different apolipoproteins (apo A-I, A-IV and E), were thus analysed by scanning tunnelling microscopy (STM). Both shape and dimensions of the discoidal HDL particles measured by this technique were found in good agreement with the data available from the literature. The accuracy of the STM pictures presented in this paper enables for the first time the visualisation of the molecular organisation of such macromolecules. The arrangement of the protein as antiparallel helical segments, is consistent with the general mode of organisation of apolipoprotein/phospholipid discoidal particles previously reported.

The lipid-destabilizing properties of the N-terminal domain of the GP2 of Ebola virus were investigated. Our results suggest that the domain of Ebola virus needed for fusion is shorter than that previously reported. The fusogenic properties of this domain are related to its oblique orientation at the lipid/water interface owing to an asymmetric distribution of the hydrophobic residues when helical.

The hepatitis C virus (HCV) glycoproteins E1 and E2 should be anchored in the viral membrane by their C-terminal domains. During synthesis, they are translocated to the endoplasmic reticulum (ER) lumen where they remain. The 31 C-terminal residues of the E1 protein and the 29 C-terminal residues of the E2 protein are implicated in the ER retention. Moreover, the E1 and E2 C termini are implicated in E1-E2 heterodimerization. We studied the E1 and E2 C-terminal sequences of 25 HCV strains in silico using molecular modeling techniques. We conclude that both C-terminal domains should adopt a similar and peculiar configuration: one amphipathic α-helix followed by a pair of transmembrane β-strands. Several three-dimensional (3-D) models were generated. After energy minimization, their ability to interact with membranes was studied using the molecular hydrophobicity potentials calculation and the IMPALA procedure. The latter simulates interactions with a membrane by a Monte Carlo minimization of energy. These methods suggest that the β-hairpins could anchor the glycoproteins in the ER membrane at least transiently. Anchoring could be stabilized by the adsorption of the nearby amphipathic α-helices at the membrane surface. The 3-D models correlate with experimental results which indicate that the E1-E2 transmembrane domains are involved in the heterodimerization and have ER retention properties.

Plasma membranes are complex entities common to all living cells. The basic principle of their organization appears very simple, but they are actually of high complexity and represent very dynamic structures. The interactions between bioactive molecules and lipids are important for numerous processes, from drug bioavailability to viral fusion. The cell membrane is a carefully balanced environment and any change inflicted upon its structure by a bioactive molecule must be considered in conjunction with the overall effect that this may have on the function and integrity of the membrane. Conceptually, understanding the molecular mechanisms by which bioactive molecules interact with cell membranes is of fundamental importance. Lipid specificity is a key factor for the detailed understanding of the penetration and/or activity of lipid-interacting molecules and of mechanisms of some diseases. Further investigation in that way should improve drug discovery and development of membrane-active molecules in many domains such as health, plant protection or microbiology. In this review, we will present complementary biophysical approaches that can give information about lipid specificity at a molecular point of view. Examples of application will be given for different molecule types, from biomolecules to pharmacological drugs. A special emphasis is given to cyclic lipopeptides since they are interesting molecules in the scope of this review by combining a peptidic moiety and a lipidic tail and by exerting their activity via specific interactions with the plasma membrane.

Saponins and triterpenic acids have been shown to be able to interact with lipid membranes and domains enriched with cholesterol (rafts). How saponins are able to modulate lipid phase separation in membranes and the role of the sugar chains for this activity is unknown. We demonstrate in a binary membrane model composed of DMPC/Chol (3:1 mol/mol) that the saponin α-hederin and its aglycone presenting no sugar chain, the triterpenic acid hederagenin, are able to induce the formation of lipid domains. We show on multilamellar vesicles (MLV), giant unilamellar vesicles (GUV), and supported planar bilayers (SPB) that the presence of sugar units on the sapogenin accelerates domain formation and increases the proportion of sterols within these domains. The domain shape is also influenced by the presence of sugars because α-hederin and hederagenin induce the formation of tubular and spherical domains, respectively. These highly curved structures should result from the induction of membrane curvature by both compounds. In addition to the formation of domains, α-hederin and hederagenin permeabilize GUV. The formation of membrane holes by α-hederin comes along with the accumulation of lipids into nonbilayer structures in SPB. This process might be responsible for the permeabilizing activity of both compounds. In LUV, permeabilization by α-hederin was sterol-dependent. The biological implications of our results and the mechanisms involved are discussed in relation to the activity of saponins and triterpenic acids on membrane rafts, cancer cells, and hemolysis.

The human VPAC(1) receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide belongs to the class II family of G-protein-coupled receptors with seven transmembrane segments. Like for all class II receptors, the extracellular N-terminal domain of the human VPAC(1) receptor plays a predominant role in peptide ligand recognition. To determine the three-dimensional structure of this N-terminal domain (residues 1-144), the Protein Data Bank (PDB) was screened for a homologous protein. A subdomain of yeast lipase B was found to have 27% sequence identity and 50% sequence homology with the N-terminal domain (8) of the VPAC(1) receptor together with a good alignment of the hydrophobic clusters. A model of the N-terminal domain of VPAC(1) receptor was thus constructed by homology. It indicated the presence of a putative signal sequence in the N-terminal extremity. Moreover, residues (Glu(36), Trp(67), Asp(68), Trp(73), and Gly(109)) which were shown to be crucial for VIP binding are gathered around a groove that is essentially negatively charged. New putatively important residues for VIP binding were suggested from the model analysis. Site-directed mutagenesis and stable transfection of mutants in CHO cells indicated that Pro(74), Pro(87), Phe(90), and Trp(110) are indeed important for VIP binding and activation of adenylyl cyclase activation. Combination of molecular modeling and directed mutagenesis provided the first partial three-dimensional structure of a VIP-binding domain, constituted of an electronegative groove with an outspanning tryptophan shell at one end, in the N-terminal extracellular region of the human VPAC(1) receptor.

Tilted peptides are short sequence fragments (10–20 residues long) that possess an asymmetric hydrophobicity gradient along their sequence when they are helical. Due to this gradient, they adopt a tilted orientation towards a single lipid/water interface and destabilize the lipids. We have detected those peptides in many different proteins with various functions. While being all tilted-oriented at a single lipid/water interface, no consensus sequence can be evidenced. In order to better understand the relationships between their lipid-destabilizing activity and their properties, we used IMPALA to classify the tilted peptides. This method allows the study of interactions between a peptide and a modeled lipid bilayer using simple restraint functions designed to mimic some of the membrane properties. We predict that tilted peptides have access to a wide conformational space in membranes, in contrast to transmembrane and amphipathic helices. In agreement with previous studies, we suggest that those metastable configurations could lead to the perturbation of the acyl chains organization and could be a general mechanism for lipid destabilization. Our results further suggest that tilted peptides fall into two classes: those from proteins acting on membrane behave differently than destabilizing fragments from interfacial proteins. While the former have equal access to the two layers of the membrane, the latter are confined within a single lipid layer. This could be in relation with the organization of lipid substrate on which the peptides physiologically act. Proteins 2001;44:435–447. © 2001 Wiley-Liss, Inc.

The widespread neuropeptide vasoactive intestinal peptide (VIP) has two receptors VPAC(1) and VPAC(2). Solid-phase syntheses of VIP analogs in which each amino acid has been changed to alanine (Ala scan) or glycine was achieved and each analog was tested for: (i) three-dimensional structure by ab initio molecular modeling; (ii) ability to inhibit (125)I-VIP binding (K(i)) and to stimulate adenylyl cyclase activity (EC(50)) in membranes from cell clones stably expressing human recombinant VPAC(1) or VPAC(2) receptor. The data show that substituting residues at 14 positions out of 28 in VIP resulted in a >10-fold increase of K(i) or EC(50) at the VPAC(1) receptor. Modeling of the three-dimensional structure of native VIP (central alpha-helice from Val(5) to Asn(24) with random coiled N and C terminus) and analogs shows that substitutions of His(1), Val(5), Arg(14), Lys(15), Lys(21), Leu(23), and Ile(26) decreased biological activity without altering the predicted structure, supporting that those residues directly interact with VPAC(1) receptor. The interaction of the analogs with human VPAC(2) receptor is similar to that observed with VPAC(1) receptor, with three remarkable exceptions: substitution of Thr(11) and Asn(28) by alanine increased K(i) for binding to VPAC(2) receptor; substitution of Tyr(22) by alanine increased EC(50) for stimulating adenylyl cyclase activity through interaction with the VPAC(2) receptor. By combining 3 mutations at positions 11, 22, and 28, we developed the [Ala(11,22,28)]VIP analog which constitutes the first highly selective (>1,000-fold) human VPAC(1) receptor agonist derived from VIP ever described.

Nisin is a 34-residue lantibiotic widely used as food preservative. Its mode of action on the bacterial cytoplasmic membrane is unclear. It should form ion channels but a molecular description of the interaction between nisin and phospholipids is lacking. The interactions between nisin and a membrane and the influence of phospholipids are here analysed by molecular modelling. The NMR structures of nisin in a micellar environment were previously determined (Van den Hooven et al., Eur. J. Biochem. 235 (1996) 382–393) Those structures were used to start with. They were refined by running a Monte Carlo procedure at a model lipid/water interface. It was shown that nisin is adsorbing onto the interface, with its N-terminal moiety more deeply inserted in lipids than the C-end, indicating distinct hydrophobic properties of the N- and C-domains. Therefore, we suggest that the N-terminal part is implied in the insertion of nisin in lipids, while the C-terminal moiety could be involved in the initial interaction with the membrane surface. Modelling the interaction of nisin with different neutral or anionic phospholipids shows that it disturbs the lipid organisation. The disturbance is maximal with phosphatidylglycerol. In this system, nisin curves the surface of phosphatidylglycerol layer round suggesting it could induce micelle formation. This could be a preliminary step to pore formation. It suggests that phosphatidylglycerol could have a direct action on nisin insertion and on ion channel formation. Appearance of a curvature also agrees with the ‘wedge model’ proposed in the literature for the nisin pore formation.

Growing evidence indicates the involvement of apolipoprotein E (apoE) in the development of late-onset and sporadic forms of Alzheimer's disease, although its exact role remains unclear. We previously demonstrated that beta-amyloid peptide (Abeta) displays membrane-destabilizing properties and that only apoE2 and E3 isoforms inhibit these properties. In this study, we clearly demonstrate that the carboxy-terminal lipid-binding domain of apoE (e.g., residues 200-299) is responsible for the Abeta-binding activity of apoE and that this interaction involves pairs of apoE amphipathic alpha-helices. We further demonstrate that Abeta is able to inhibit the association of the C-terminal domain of apoE with lipids due to the formation of Abeta/apoE complexes resistant to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the contrary, the amino-terminal receptor-binding domain of apoE (e.g., residues 129-169) is not able to form stable complexes with Abeta. These data extend our understanding of human apoE-dependent binding of Abeta by involving the C-terminal domain of apoE in the efficient formation of apoE/Abeta complex.

C-terminal fragments of the Alzheimer amyloid peptide (amino acids 29–40 and 29–42) have physico-chemical properties related to those of the fusion peptides of viral proteins and they are able to induce the fusion of liposomes in vitro. We proposed that these properties could mediate a direct interaction of the amyloid peptide with cell membranes and account for part of the cytotoxicity of the amyloid peptide. In view of the epidemiologic and biochemical linkages between the pathology of Alzheimer's disease and apolipoprotein E (apoE) polymorphism, we examined the potential interaction between the three common apoE isoforms and the C-terminal fragments of the amyloid peptide. We show that, at low concentration, only apoE2 and apoE3 are potent inhibitors of the amyloid peptide fusogenic and aggregational properties, whereas the apoE4 isoform has no effect. We further show that the protective effect of apoE is mediated by the formation of stable apoE/amyloid peptide complexes, as determined by trypto-phan emission fluorescence measurements and by gel electrophoresis. The interaction specificity between apoE2 and apoE3 and the amyloid fragments is demonstrated here, since other apolipoproteins (e.g. apolipoprotein A-I and A-II), with similar amphipathic structures, do not interact with the amyloid C-terminal fragments. Finally, we show that, reciprocally, the amyloid peptide can interact directly with the apoE2 and apoE3 isoforms to decrease or perturb their normal association with lipids. These data suggest that the 29-40 and 29–42 domains of the amyloid peptide could be critical for the amyloid-apoE interaction, and that apoE2 and apoE3 isoforms, but not apoE4, could play a protective role against the formation of amyloid aggregates and/or against their interaction with cellular membranes.

Molecular Dynamics is a method of choice for membrane simulations and the rising of coarse-grained forcefields has opened the way to longer simulations with reduced calculations times. Here, we present an elastic network, SAHBNET (Surface Accessibility Hydrogen-Bonds elastic NETwork), that will maintain the structure of soluble or membrane proteins based on the hydrogen bonds present in the atomistic structure and the proximity between buried residues. This network is applied on the coarse-grained beads defined by the MARTINI model, and was designed to be more physics-based than a simple elastic network. The SAHBNET model is evaluated against atomistic simulations, and compared with ELNEDYN models. The SAHBNET is then used to simulate two membrane proteins inserted in complex lipid bilayers. These bilayers are formed by self-assembly and the use of a modified version of the GROMACS tool genbox (which is accessible through the gcgs.gembloux.ulg.ac.be website). The results show that SAHBNET keeps the structure close to the atomistic one and is successfully used for the simulation of membrane proteins.

A series of natural peptides and mutants, derived from the Alzheimer β-amyloid peptide, was synthesized, and the potential of these peptides to induce fusion of unilamellar lipid vesicles was investigated. These peptide domains were identified by computer modeling and correspond to respectively the C-terminal (e.g. residues 29-40 and 29-42) and a central domain (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) of the β-amyloid peptide. The C-terminal peptides are predicted to insert in an oblique way into a lipid membrane through their N-terminal end, while the mutants are either parallel or perpendicular to the lipid bilayer. Peptide-induced vesicle fusion was demonstrated by several techniques, including lipid-mixing and core-mixing assays using pyrene-labeled vesicles. The effect of peptide elongation toward the N-terminal end of the entire β-amyloid peptide was also investigated. Peptides corresponding to residues 22-42 and 12-42 were tested using the same techniques. Both the 29-40 and 29-42 β-amyloid peptides were able to induce fusion of unilamellar lipid vesicles and calcein leakage, and the amyloid 29-42 peptide was the most potent fusogenic peptide. Neither the two mutants or the 13-28 β-amyloid peptide had any fusogenic activity. Circular dichroism measurements showed an increase of the α-helical content of the two C-terminal peptides at increasing concentrations of trifluoroethanol, which was accompanied by an increase of the fusogenic potential of the peptides. Our data suggest that the α-helical content and the angle of insertion of the peptide into a lipid bilayer are critical for the fusogenic activity of the C-terminal domain of the amyloid peptide. The differences observed between the fusogenic capacity of the amyloid 29-40 and 29-42 peptides might result from differences in the degree of penetration of the peptides into the membrane and the resulting membrane destabilization. The longer peptides, residues 22-42 and 12-42, had decreased, but significant, fusogenic properties associated with perturbation of the membrane permeability. These data suggest that the fusogenic properties of the C-terminal domain of the β-amyloid peptide might contribute to the cytotoxicity of the peptide by destabilizing the cell membrane.

There are two models of topology for the membrane domains of the erythrocyte/brain facilitative glucose transporter, GLUT1. The first is composed of 12 membrane–spanning α–helices, the second of 16 membrane-spanning β-strands. We have used Jahnig′s and Eisenberg′s methods to identify possible transmembrane segments (10 spanning α-helices and 4 β-strands). The topology proposed is more consistent with available experimental data from FTIR, CD and mapping experiment than the previous models . We suggest that GLUT1 might form two channels, one of which is responsible for glucose transport. This agrees with the theoretical and experimental arguments. Finally, an analysis of the mutation periodicity and of the mean hydrophobicity for the GLUT family is provided in order to evaluate the packing of the protein in the membrane.

Fengycin is a natural lipopeptide with antifungal and eliciting properties and able to inhibit the activity of phospholipase A2. A combination of CD, FT-IR, NMR and fluorescence spectroscopic techniques was applied to elucidate its conformation in a membrane-mimicking environment and to investigate the effect of calcium ions on it. We mainly observed that fengycin adopts a turn conformation. Our results showed that calcium ions are bound by the two charged glutamates. The calcium binding has an influence on the fengycin conformation and more particularly, on the environment of the tyrosine residues. The modulation of the fengycin conformation by the environmental conditions may influence its biological properties.

The sequences of the plasma apolipoproteins have a high degree of internal homology as they contain several 22-mer internal repeats. These amphipathic helical repeats are considered as the structural and functional units of this class of proteins. We proposed that the 22-mer repeats of the plasma apolipoproteins consist of 17-mer helical segments separated by extended beta-strands comprising five amino acid residues with a proline in the center of this segment. These beta-strand segments help reverse the orientation of the consecutive helices of apoA-I, A-IV, and E in a discoidal apolipoprotein-phospholipid complex. In order to support this hypothesis, we synthesized apoA-I fragments consisting of, respectively, one putative helix (residues 166-183), one helix plus a beta-strand (residues 161-183), and a pair of helices separated by a beta-strand (residues 145-183). The structural and lipid-binding properties of these peptides were investigated by turbidity, fluorescence, binding studies with unilamellar phospholipid vesicles, electron microscopy, and circular dichroism measurements. Our data show that one single putative helical segment or one helical segment plus one extended beta-strand do not form stable complexes with phospholipids. The addition of a second adjacent helix has no influence on the lipid affinity of the apoA-I 145-183 peptide compared to the shorter segments but substantially improves the stability of the complexes. The helical content of the peptide increases upon lipid association as observed with apoA-I. The complexes generated with the apoA-I 145-183 peptide appear as discoidal particles by negative staining electron microscopy, with heterogeneous sizes ranging between 250 and 450 A. The relative orientation of the peptide and the phospholipid is the same as in a DMPC/apoA-I complex as the helices are oriented parallel to the acyl chains of the phospholipid. However, the stability of these complexes is significantly lower than that of the corresponding DMPC/apoA-I complexes. The transition temperature, fluidity, and cooperativity of the phospholipid bilayer are only weakly affected by the association with the apoA-I 145-183 peptide. These data suggest that a pair of helical peptides linked through a beta-strand associates more tightly with lipids and can form discoidal lipid-peptide complexes, than a single helix. A comparison with the properties of native apoA-I suggests, however, that the cooperativity between pairs of helices in native apoA-I further contributes to strengthen the lipid-protein association.