Monitoring membrane binding and insertion of peptides by two-color fluorescent label.

Herein, we developed an approach for monitoring membrane binding and insertion of peptides using a fluorescent environment-sensitive label of the 3-hydroxyflavone family. For this purpose, we labeled the N-terminus of three synthetic peptides, melittin, magainin 2 and poly-l-lysine capable to interact with lipid membranes. Binding of these peptides to lipid vesicles induced a strong fluorescence increase, which enabled to quantify the peptide-membrane interaction. Moreover, the dual emission of the label in these peptides correlated well with the depth of its insertion measured by the parallax quenching method. Thus, in melittin and magainin 2, which show deep insertion of their N-terminus, the label presented a dual emission corresponding to a low polar environment, while the environment of the poly-l-lysine N-terminus was rather polar, consistent with its location close to the bilayer surface. Using spectral deconvolution to distinguish the non-hydrated label species from the hydrated ones and two photon fluorescence microscopy to determine the probe orientation in giant vesicles, we found that the non-hydrated species were vertically oriented in the bilayer and constituted the best indicators for evaluating the depth of the peptide N-terminus in membranes. Thus, this label constitutes an interesting new tool for monitoring membrane binding and insertion of peptides.

Neutral fluorescence probe with strong ratiometric response to surface charge of phospholipid membranes.

We report on dramatic differences in fluorescence spectra of 4'-dimethylamino-3-hydroxyflavone (probe F) studied in phospholipid membranes of different charge (phosphatidyl glycerol, phosphatidylcholine (PC), their mixture and the mixture of PC with a cationic lipid). The effect consists in variations of relative intensities at two well-separated band maxima at 520 and 570 nm belonging to normal (N*) and tautomer (T*) excited states of flavone chromophore. Based on these studies we propose a new approach to measure electrostatic potential at the surface layer of phospholipid membranes, which is based on potential-dependent changes of bilayer hydration and involves very sensitive and convenient ratiometric measurements in fluorescence emission.

Neurotrophic activity of 2,4,4-trimethyl-3-(15-hydroxypentadecyl)-2-cyclohexen-1-one in cultured central nervous system neurons.

Endogenous neurotrophic factors are essential for the development and maintenance of the nervous system. This suggests their potential utilization as therapeutic agents for neurodegenerative diseases. However, the clinical use of these proteic factors is still restricted, and brings about undesirable consequences, including adverse side effects, and bioavailability and stability difficulties. Therefore, the development of low-molecular weight, non-proteic synthetic compounds with neurotrophic properties appears as a promising approach. The aim of this study was to explore the biological activity of 2,4,4-trimethyl-3-(15-hydroxypentadecyl)-2-cyclohexen-1-one (tCFA15), a trimethyl cyclohexenonic long-chain fatty alcohol. To this end, neurons from fetal rat cerebral hemispheres were cultured in the presence of increasing doses of tCFA15 ranging from 0.1 to 1000 nM. Quantification of cell numbers after 48-h culture showed that 100 nM tCFA15 induced a significant increase in the number of surviving cells. Measurement of total neurite length in microtubule-associated protein 2-positive cells also revealed a stimulatory effect in a wider range of concentrations. The extent of this neuritogenic action was similar to that induced by dibutyryl-cyclic AMP, a well-known neurite outgrowth stimulator, but used at much higher concentration (1 mM). Analysis of structure-activity relationships with different tCFA15 analogs and derivatives corroborated the neurotrophic activity. Taken together, these findings provide strong evidence that tCFA15 exhibits neurotrophic properties in vitro.

DNA condensation by an oxidizable cationic detergent. Interactions with lipid vesicles.

Cationic amphiphile-mediated delivery of plasmid DNA is the non-viral gene transfer method most often used. In the present work, we considered a new cysteine-detergent, ornithinyl-cysteinyl-tetradecylamide (C(14)-CO), able to convert itself, via oxidative dimerization, into a cationic cystine-lipid. By using fluorescence techniques, we first characterized the structure of complexes of plasmid DNA with C(14)-CO molecules either kept as monomers, or oxidized into dimers. Both forms are able to condense DNA, with the formation of hydrophobic micelle-like domains along the DNA chain. Domains with a larger molecular order were obtained with dimeric C(14)-CO/DNA complexes. In a second step, the interactions of these complexes with lipid vesicles considered as membrane models were investigated. In the presence of vesicles, we observed a decondensation of the DNA involved in complexes obtained with C(14)-CO monomers. With anionic vesicles, the DNA is released into the bulk solution, while with neutral vesicles, it remains bound to the vesicles via electrostatic interactions with inserted C(14)-CO molecules. In sharp contrast, the complexes with C(14)-CO dimers are unaffected by the addition of either neutral or anionic vesicles and show no interaction with them. These results may partly explain the low transfection efficiency of these complexes at the +/-charge ratios used in this study.

Effect of the structure of natural sterols and sphingolipids on the formation of ordered sphingolipid/sterol domains (rafts). Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide.

Ordered lipid domains enriched in sphingolipids and cholesterol (lipid rafts) have been implicated in numerous functions in biological membranes. We recently found that lipid domain/raft formation is dependent on the sterol component having a structure that allows tight packing with lipids having saturated acyl chains (Xu, X., and London, E. (2000) Biochemistry 39, 844-849). In this study, the domain-promoting activities of various natural sterols were compared with that of cholesterol using both fluorescence quenching and detergent insolubility methods. Using model membranes, it was shown that, like cholesterol, both plant and fungal sterols promote the formation of tightly packed, ordered lipid domains by lipids with saturated acyl chains. Surprisingly ergosterol, a fungal sterol, and 7-dehydrocholesterol, a sterol present in elevated levels in Smith-Lemli-Opitz syndrome, were both significantly more strongly domain-promoting than cholesterol. Domain formation was also affected by the structure of the sphingolipid (or that of an equivalent "saturated" phospholipid) component. Sterols had pronounced effects on domain formation by sphingomyelin and dipalmitoylphosphatidylcholine but only a weak influence on the ability of cerebrosides to form domains. Strikingly it was found that a small amount of ceramide (3 mol %) significantly stabilized domain/raft formation. The molecular basis for, and the implications of, the effects of different sterols and sphingolipids (especially ceramide) on the behavior and biological function of rafts are discussed.

Gene transfer by cationic surfactants is essentially limited by the trapping of the surfactant/DNA complexes onto the cell membrane: a fluorescence investigation.

The interaction between complexes of plasmid DNA with cetyltrimethylammonium bromide (CTAB) and L929 fibroblasts was first examined using confocal microscopy. The complexes labeled with the DNA intercalator, YOYO-1, were found to be trapped onto the external face of the plasma membrane; a feature that may constitute a major limiting step in transfection. Moreover, since no cytotoxic effect appeared in these conditions, we further inferred that the CTAB molecules remained bound to the DNA. The interaction of the complexes with the membranes was best modeled with neutral vesicles. From anisotropy thermotropic curves of DPHpPC-labeled vesicles and fluorescence resonance energy transfer measurements between these vesicles and YOYO-labeled complexes, we evidenced that the binding of the complexes to the vesicle surface opened the micelle-like domains and unwound DNA. However, DNA was not released but remained stably bound via electrostatic interactions to the CTAB molecules incorporated in the external liposome leaflet. Consequently, the large diameter of the unwound plasmid DNA is likely the major factor that precludes its internalization into the cells by endocytosis. In contrast, anionic vesicles that mimic the cytoplasmic facing monolayer of the plasma membrane rapidly released DNA from the complex. This may explain the previously reported high transfection efficiency of DNA complexed with liposomes composed of neutral lipids and cationic surfactants, since the latter may destabilize the endosomal membrane and induce the release of DNA in the cytoplasm.

The influence of microtubule integrity on plasma membrane fluidity in L929 cells.

The aim of this work was to examine the possible influence of the integrity of the microtubule network on the plasma membrane fluidity of L929 mouse fibroblasts. The L929 cell line was selected for the ease of culture and the stability of its characteristics. The cells were treated with colchicine, nocodazole and vinblastine, three microtubule-depolymerizing drugs, at various concentrations and for various times. Membrane fluidity was assessed from fluorescence depolarization measurements with the plasma membrane probe TMA-DPH. Each of the drugs induced a significant, dose-dependent decrease in fluorescence anisotropy. The effect levelled off (5-7% decrease) after approximately 90 min of treatment, and could be unambiguously interpreted as resulting from an increase in membrane fluidity. The cumulative action of the drugs did not significantly increase the effect. The effects of colchicine and nocodazole could be reversed by incubation in drug-free medium, but not that of vinblastine. The results are discussed in correlation with the kinetics of the three drugs interaction with tubulin or microtubules. It is concluded that the microtubule integrity contributed to the high plasma membrane lipidic order, but less than other factors, like the lipid composition and the cholesterol content.

Calcium-induced conformational change in fragment 1-86 of factor X.

Time-resolved fluorescence of the single tryptophan residue Trp41 in fragment 1-86 of factor X (FX F1-86) is studied using a time-correlated single photon counting technique with synchrotron radiation as the excitation source. Calcium ions are believed to induce a conformational change in the N-termini of the activated factor X and other vitamin K dependent proteins, which is accompanied by a decrease in fluorescence intensity. The titration with calcium yields a sigmoidal fluorescence titration curve with a transition midpoint concentration of 0.44 mM. The wavelength-dependent tryptophan fluorescence decays of the apo-FX F1-86 (in the absence of calcium) and Ca-FX F1-86 are characterized by conventional multiexponential analysis and fluorescence lifetime distribution analysis. In the absence of calcium there are three significant classes of fluorescence lifetimes (ns) that are nearly wavelength independent: 0.55 +/- 0.08 (component A), 2.6 +/- 0.1 (component B), and 5.3 +/- 0.3 (component C). However, their preexponential amplitudes vary with wavelength. The decay associated emission spectra of the individual components show that components B and C contribute over 85% to the total fluorescence for all examined wavelengths. However, in the presence of calcium, the analysis of the time-resolved fluorescence data of Ca-FX F1-86 yields four wavelength-independent lifetimes (ns) of 0.30 +/- 0.09 (component D), 0.65 +/- 0.10 (component A), 2.7 +/- 0.2 (component B), and 5.4 +/- 0.3 (component C). Calcium addition to the apo-FX F1-86 leads to a decrease in the fluorescence intensities of components B and C while their decay times remain unaffected. In Ca-FX F1-86 an additional component D arises that has a decay time of 0.30 ns and that contributes up to 35% to the total fluorescence intensity. A comparison with a previous investigation of prothrombin fragment 1 demonstrates the extensive structural and functional homology between the N termini of prothrombin and factor X(a).

Interactions of the monomeric and dimeric flavones apigenin and amentoflavone with the plasma membrane of L929 cells; a fluorescence study.

Flavonoids are ubiquitous polyphenolic compounds, found in vascular plants, which are endowed with a large variety of biological effects. Some of these effects have been assumed to result from interactions with the cell plasma membrane. In order to investigate the nature of these interactions a fluorescence study was performed with two flavonoids, currently used in one of the laboratories: apigenin and its homologous dimer amentoflavone. After preliminary assays with DPH in several types of phospholipid liposomes, the effects of these flavonoids on the membrane of mouse L929 fibroblasts were compared, using the non-permeant probe TMA-DPH. Amentoflavone, unlike apigenin, induced a static quenching effect, which denoted an important, but reversible, association of the molecule with the plasma membrane. In addition, amentoflavone treatment induced a dose-dependent increase in TMA-DPH fluorescence anisotropy, which could be interpreted as an increase in membrane lipidic order. For apigenin, the effect was much less important. Moreover, exploiting the capacity of TMA-DPH to label endocytic compartments, it was shown that, after association with the membrane, amentoflavone is not internalized into the cell. Possible correlations of these membrane effects with other biological properties are discussed.

A comparison of the fluorescence properties of TMA-DPH as a probe for plasma membrane and for endocytic membrane.

In earlier studies, the fluorescence probe 1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene (TMA-DPH) was shown to interact with living cells by instantaneous incorporation into the plasma membrane, according to a water (probe not fluorescent)/membrane (probe highly fluorescent) partition equilibrium. This made it interesting both as a fluorescence anisotropy probe for plasma membrane fluidity determinations and as a quantitative tracer for endocytosis and intracellular membrane traffic. In order to ascertain the limiting concentrations for its use in these applications, we performed a systematic study of its fluorescence properties (intensity, lifetime, anisotropy) in the plasma membrane and in endocytic membranes of intact L929 mouse fibroblasts. Some of the experiments were repeated on mouse-bone-marrow-derived macrophages and on phospholipidic LUV to confirm the results. Rather unexpectedly, it was observed that: (i) the incorporation of TMA-DPH into the membranes, monitored by UV absorption measurements, remained proportional to the probe concentration over the wide range explored (5 x 10(-7) M-2.5 x 10(-5) M); (ii) however, concerning fluorescence, quenching effects occurred in the membranes above certain critical concentrations. These effects were shown to result from Förster-type resonance auto-transfer; (iii) strikingly, the critical concentrations were considerably higher in early-endocytic-vesicle membranes than in the bulk plasma membrane. It was established that membrane fluidity was involved and this was confirmed by the parallel study on phospholipidic vesicles. Potential applications of these properties as a novel approach for evaluating membrane fluidity are suggested.

Influence of the length of the spacer on the partitioning properties of amphiphilic fluorescent membrane probes.

Four fluorescent diphenylhexatriene derivatives were considered as membrane probes, namely two ammonium compounds, 3-(diphenylhexatrienyl)propyltrimethylammonium (TMAP-DPH) and 22-(diphenylhexatrienyl)docosyltrimethylammonium (LcTMA-DPH), and two phospholipids, 1-palmitoyl-2-[3-(diphenylhexatrienyl)propanoyl]-sn-glyc ero-3-phosphocholine (DPHpPC) and 1-palmitoyl-2-[21-(diphenylhexatrienyl)henicosanoyl]-sn-phos phocholine (LcDPHpPC). For each pair, the molecules differ by the length of the polymethylenic spacer between the fluorescent moiety and the polar head, so one pair comprises two short chain molecules (C3 spacer) and the other two long chain molecules (C21 or C22 spacer). The partitioning of these probes between gel and liquid crystalline phases of multilamellar vesicles with binary composition (DEPC and DSPC) was measured by a method based on fluorescence anisotropy. The partitioning was shown to depend strongly on the length of the spacer. Short chain probes preferably partition into fluid phases (Kf/s = 1.7 +/- 0.3 for TMAP-DPH; 2.6 +/- 0.11 for DPHpPC), whereas long chain probes show a strong preferential partitioning for gel phases of the vesicles (Kf/s = 0.12 +/- 0.06 for LcTMA-DPH; 0.22 +/- 0.11 for LcDPHpPC). This strong partitioning may be explained by the interdigitation of the long polymethylenic chains across the mid-point of the lipid bilayer (I.E. Mehlhorn et al. (1988) Biochim. Biophys. Acta 939, 151-159), which is enhanced by the better packing provided by a gel phase.

Pore formation by a two-component leukocidin from Staphylococcus aureus within the membrane of human polymorphonuclear leukocytes.

The effects of the Staphylococcus aureus leukocidin (PVL), a two-component non-hemolytic toxin, were investigated on the membrane permeability of human polymorphonuclear leukocytes (PMNs). In the absence of extracellular Ca2+, the fluorescence of ethidium bromide added to the extracellular medium increased after PVL application in a concentration-dependent manner and no variations in the free intracellular [Ca2+] of Fura2-loaded PMNs were detected. In the presence of extracellular Ca2+, the fluorescence of ethidium was not modified but the free intracellular [Ca2+] of PMNs increased after application of PVL in a concentration-dependent manner. The time lag observed before an increase in the ethidium fluorescence was longer than the time lag observed before a Fura2 fluorescence increase. Simultaneous recordings of the two probes fluorescence variations have shown the protective effect of Ca2+ and Zn2+ and the closing of the pore by 50 mM Ca2+ or 2 mM Zn2+. Moreover, the effect of Ca2+ could be reversed by the addition of EGTA. In the presence of 1 mM extracellular Ca2+ or 0.8 mM extracellular Zn2+, the pore induced by PVL had an ionic size allowing Ca2+, Mn2+, Zn2+ and Mg2+ fluxes. The addition of antibodies against either component of PVL inhibits the permeabilization provoked by the toxin even after it was initiated. It is concluded that leukocidin from S. aureus is a pore-forming toxin which, under physiological conditions ([Ca2+] = 1 to 1.5 mM), provokes the formation of an ion-sized pore inducing an increase in the free intracellular Ca2+ which may activate PMN functions.

New diphenylhexatriene derivatives as fluorescent membrane probes: Partitioning properties.

Three new diphenylhexatriene derivatives, two phospholipids and one single-chain amphiphilic molecule, have been synthesized and considered as probes for measuring membrane fluidity by fluorescence anisotropy. The possibility of using these probes to determine specifically fluidity of inner leaflets of cellular plasma membranes was inferred from their partitioning properties between gel and liquid crystalline phases of phospholipid vesicles of binary composition.

Reactions in lipid vesicles. Pyrene excimer formation in restricted geometries. Effect of temperature and concentration.

The decay of pyrene in the presence of excimers in small unilamellar vesicles of 3-sn-phosphatidyl glycerol, dipalmitoyl and 3-sn-phosphatidylglycerol from egg yolk has been analyzed with the use of models appropriate for reactions in restricted geometries. Results are presented with emphasis on probe concentration and temperature. The reaction rate in an organized lipid phase is redefined in a simple manner which allows for a simple treatment of any reaction in such environments. The analysis allows detection of pyrene aggregation in the vesicle lipidic core.

The interactions of the brain-specific calmodulin-binding protein kinase C substrate, neuromodulin (GAP 43), with membrane phospholipids.

The interaction of the brain-specific calmodulin-binding protein kinase C (PKC) substrate, neuromodulin (GAP 43), with membrane phospholipids was studied. Specific binding of neuromodulin to negatively charged phospholipids through electrostatic interactions was demonstrated. Comparison of the binding of neuromodulin to acidic phospholipids with that of neurogranin, a newly characterized calmodulin-binding PKC substrate (Baudier J., Deloulme, J. C., Van Dorsselaer, A., Black, D., and Mathes H. (1991) J. Biol. Chem. 266, 229-237) suggested that the conserved basic amino acid sequence which characterizes the two proteins and which corresponds to the PKC phosphorylation and calmodulin binding domain also serves as phospholipid binding site. In the absence of calmodulin, binding of neuromodulin to phosphatidylserine at low concentration parallels its phosphorylation by PKC, suggesting that formation of a ternary complex between neuromodulin, phosphatidylserine, and PKC is required for optimum neuromodulin phosphorylation. In the presence of calmodulin, the binding of neuromodulin to phosphatidylserine is inhibited, resulting in total inhibition of neuromodulin phosphorylation. Our results suggest that, in vivo, phosphorylation of neuromodulin may not only depend on protein kinase C (PKC)1 activation but also on the accessibility of the neuromodulin phosphorylation domain to activated membrane-bound PKC that could regulated by CaM.

Selectivity of lipid-protein interaction with myelin proteolipids PLP and DM-20. A fluorescence anisotropy study.

The two main myelin proteolipids, PLP (30 kDa) and DM-20 (25 kDa), differ by an internal deletion in DM-20. The deleted fragment, of 35 amino acids (116-150), corresponds to the major hydrophilic domain of PLP. Fluorescence anisotropy experiments using diphenylhexatriene as a fluorescent probe were performed to detect the phase separation induced by these two proteolipids in multilamellar vesicles of binary composition. We found that in vesicles composed of 30% L-alpha-PS and 70% DPPC, the PLP boundary layer contained about 18 motionally restricted phospholipids, almost exclusively L-alpha-PS. On the contrary, the DM-20 boundary layer contained only 14 to 15 phospholipids, with a composition no different from that of the bulk vesicle. In mixtures of DMPG and DPPC, the selectivity of PLP for the acidic phospholipid DMPG was maintained, but was lower than that observed for L-alpha-PS. We assume that this selectivity of PLP stems mainly from electrostatic interactions between the charged residues of the 116-150 fragment, deleted in DM-20, and the acidic phospholipids. These results suggest that fragment 116-150 may play a specific role in the interaction of PLP with the lipid bilayer of the myelin membrane.

Soybean phosphatidylcholine vesicles containing plant sterols: a fluorescence anisotropy study.

The typical plant sterols (sitosterol, stigmasterol and campesterol) were compared with respect to their ability to regulate membrane fluidity of soybean phosphatidylcholine (PC) vesicles. Fluidity changes were monitored by the steady-state fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene as a probe and assigned to a measure of the acyl chain orientational order. Sitosterol and campesterol appear to be the most suitable sterols in ordering the acyl chains of soybean lecithin bilayers, even more efficient than cholesterol, the standard of reference for sterol effects on membranes, suggesting that they play a significant role in the regulation of plant membrane properties. Stigmasterol is shown to be much less active. Cycloartenol, a biosynthetic precursor of plant sterols, increases the acyl chain order with the same efficiency as cholesterol. We also investigated the effects of two unusual sterols, 24-methylpollinastanol and 14 alpha,24-dimethylcholest-8-en-3 beta-ol, which were shown to accumulate in plants treated with fungicides belonging to two important classes, N-substituted morpholines and triazoles, respectively. These two sterols exhibit a behavior very similar to that of stigmasterol. The results are discussed in terms of sterol effects on the molecular packing of soybean PC bilayers.

Sensitive analysis of phospholipid molecular species by high-performance liquid chromatography using fluorescent naproxen derivatives of diacylglycerols.

A sensitive high-performance liquid chromatographic (HPLC) method for the separation and determination of diacylglycerophospholipid and diacylglycerol (DAG) molecular species has been developed. Phospholipids are hydrolysed with phospholipase C and the resulting DAGs are reacted with naproxen chloride in the presence of 4-dimethylaminopyridine. The naproxen-DAGs were purified by thin-layer chromatography on silica gel G plates. Molecular species were separated using reversed-phase HPLC with isocratic elution and determined by measuring the absorbance at 230 nm or fluorescence at 352 nm (excitation at 332 nm). The method was applied to the determination of diacylglycerophosphoethanolamine in rat cerebrum and cerebellum. The molar absorption coefficient of the naproxen derivatives was 53,000 lmol-1 cm-1 at 230 nm, permitting the generation of linear concentration-dependent determinations down to less than 10 pmol. A ten-fold increase in sensitivity was obtained with a fluorescence detection system owing to the fluorescent properties of the proposed adduct.

TMA-DPH a fluorescent probe of membrane dynamics in living cells. How to use it in phagocytosis.

Trimethylammonium-diphenylhexatriene (TMA-DPH), a hydrophobic fluorescent probe, has been shown in earlier studies to possess a variety of particular properties in interaction with intact living cells--specific and rapid incorporation into the plasma membrane and partition equilibrium between the membranes and the buffer. These properties offer promising applications in membrane fluidity studies and in monitoring exocytosis kinetics. Furthermore, these properties offer a method described here for quantitative monitoring of phagocytosis kinetics, by means of simple fluorescence intensity measurements. This method is original in that it evaluates only the particles which have actually been internalized by phagocytosis, and not those adsorbed on the cell surface, and that it gives quantitative information on the amount of plasma membrane involved in the process. It has been tested on mouse bone marrow macrophages.

Effects of random copolymers of lysine on the thermotropic behaviour of dipalmitoylphosphatidylglycerol vesicles. A fluorescence anisotropy study.

The effects of some random copolymers of lysine on the thermotropic behaviour of phospholipid vesicles, mainly dipalmitoylphosphatidylglycerol (DPPG), were studied by monitoring the steady-state fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene (DPH) as a membrane probe. A characteristic effect of the polylysine-tyrosine 4:1 copolymer was observed: DPPG vesicles are strongly stabilized, the gel-to-fluid transition going from 40 degrees C in the absence to 52 degrees C with an excess of copolymer (corresponding to a lipid/amino-acid ratio R = 0.5). For R greater than 0.5, the gel-to-fluid transition shows a three-step profile. This triphasic transition, which appears with a much better resolution than with polylysine, demonstrates the formation of stable distinct domains of reasonable size, coexisting from 41 to 51 degrees C. Such stability, over a temperature range of 10 degrees C, is quite unusual and unexpected for a phase separation of mixed lipid phases. The peculiarity of this copolymer was proved by investigating the interactions of DPPG vesicles with polylysine-tryptophan 4:1, polyornithine-lysine 4:1 and polylysine-tyrosine 1:1. We hypothesize that the observed effects are correlated with the ability of these copolymers to adopt/not adopt an alpha-helical or beta-sheet conformation upon interacting with anionic vesicles.