The N-terminal domain of chromogranin A (CgA1-40) interacts with monolayers of membrane lipids of fungal and mammalian compositions.
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Alternaria↗
Biomedical subjects
R Maget-Dana
Publications and source records attributed to R Maget-Dana.
Structural and biological characterization of chromofungin, the antifungal chromogranin A (47-66)-derived peptide.
The antifungal peptide named chromofungin is the most active vasostatin-I-derived peptide, corresponding to the sequence 47-66 of chromogranin A. (1)H-NMR analysis revealed that it adopts a helical structure. The mechanism implicated in the interaction of chromofungin with fungi and yeast cells was studied by penetration of monolayers and confocal laser microscopy. Chromofungin is able to interact with the cell wall, to cross the plasma membrane, to accumulate in the microorganism, and to inhibit calcineurin activity.
Alternaria↗
Structural and biological characterization of chromofungin, the antifungal chromogranin A-(47-66)-derived peptide.
Vasostatin-I, the natural fragment of chromogranin A-(1-76), is a neuropeptide able to kill a large variety of fungi and yeast cells in the micromolar range. We have examined the antifungal properties of synthetic vasostatin-I-related peptides. The most active shortest peptide, named chromofungin, corresponds to the sequence Arg(47)-Leu(66). Extensive (1)H NMR analysis revealed that it adopts a helical structure. The biophysical mechanism implicated in the interaction of chromofungin with fungi and yeast cells was studied, showing the penetration of this peptide with different lipid monolayers. In order to examine thoroughly the antifungal activity of chromofungin, confocal laser microscopy was used to demonstrate the ability of the rhodamine-labeled peptide to interact with the fungal cell wall, to cross the plasma membrane, and to accumulate in Aspergillus fumigatus, Alternaria brassicola, and Candida albicans. Our present data reveal that chromofungin inhibits calcineurin activity, extending a previous observation that the N-terminal region of chromogranin A interacts with calmodulin in the presence of calcium. Therefore, the destabilization of fungal wall and plasma membrane, together with the possible intracellular inhibition of calmodulin-dependent enzymes, is likely to represent the mechanism by which vasostatin-I and chromofungin exert antifungal activity.
Amino Acid Sequence↗
Comparative interaction of alpha-helical and beta-sheet amphiphilic isopeptides with phospholipid monolayers.
The two sequential amphiphilic peptide isomers, (Leu-Lys-Lys-Leu)4 and (Leu-Lys)8, were chosen as models for alpha-helical and beta-sheet peptides, respectively. In order to evaluate the contribution of the secondary structure of a peptide to its penetration into cellular membranes, interactions of these isopeptides with L-alpha-dimyristoyl phosphatidylcholine (DMPC) monolayers were studied. Both isopeptides penetrate into DMPC monolayers up to an exclusion pressure of approximately 27 mN/m, but a discontinuity is observed in the penetration profile of the alpha-helical (LKKL)4. The main parameters extracted from the compression isotherms of the mixed peptide/DMPC monolayers-namely, transition pressure, mean area, elasticity modulus, and energy of mixing-were analyzed. These analyses indicate that the alpha-helical isomer interacts strongly with DMPC by forming a 1:32 (LKKL)4-DMPC complex. When engaged in this complex, (LKKL)(4) behaves as an hydrophobic helix and has a tendency to become vertically oriented in the course of the compression process. The beta-sheet (LK)8 interacts more weakly with DMPC and no complex can be detected.
Dimyristoylphosphatidylcholine↗
Behaviour of bovine phosphatidylethanolamine-binding protein with model membranes. Evidence of affinity for negatively charged membranes.
The ability of phosphatidylethanolamine-binding protein (PEBP) to bind membranes was tested by using small and large unilamellar vesicles and monolayers composed of l-alpha-1,2-dimyristoylphosphatidylcholine, l-alpha-1,2-dimyristoylphosphatidylglycerol and l-alpha-1,2-dimyristoylphosphatidylethanolamine. PEBP only bound to model membranes containing l-alpha-1,2-dimyristoylphosphatidylglycerol; the interaction was primarily due to electrostatic forces between the basic protein and the acidic phospholipids. Further experiments indicated that the interaction was not dependent on the length and unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane. PEBP affinity for negatively charged membranes is puzzling considering the previous identification of the protein as a phosphatidylethanolamine-binding protein, and suggests that the association of PEBP with phospholipid membranes is driven by a mechanism other than its binding to solubilized phosphatidylethanolamine. An explanation was suggested by its three-dimensional structure: a small cavity at the protein surface has been reported to be the binding site of the polar head of phosphatidylethanolamine, while the N-terminal and C-terminal parts of PEBP, exposed at the protein surface, appear to be involved in the interaction with membranes. To test this hypothesis, we synthesized the two PEBP terminal regions and tested them with model membranes in parallel with the whole protein. Both peptides displayed the same behaviour as whole PEBP, indicating that they could participate in the binding of PEBP to membranes. Our results strongly suggest that PEBP directly interacts with negatively charged membrane microdomains in living cells.
Amino Acid Sequence↗
Lichenysin: a more efficient cation chelator than surfactin.
The lipopeptide lichenysin (cyclo-[L-Gln1-->D-Leu2-->L-Leu3-->L-Val4--> L-Asp5-->D-Leu6-->L-Ile7-beta-OH fatty acid]) produced by Bacillus licheniformis structurally resembles surfactin from Bacillus subtilis. The main difference is the presence of a glutaminyl residue in position 1 of the peptide sequence in place of glutamic acid in surfactin. This local variation causes significant changes in the properties of the molecule compared to surfactin. Lichenysin has a higher surfactant power, the critical micellar concentration (c.m.c.) being strongly reduced from 220 to 22 microM and a much higher hemolytic activity because 100% hemolysis was observed with only 15 microM instead of 200 microM. Lichenysin is also a better chelating agent because its association constants with Ca2+ and Mg2+ are increased by a factor of 4 and 16, respectively. This effect is assigned to an increase in the accessibility of the carboxyl group to cations owing to a change in the side chain topology induced by the Glu/Gln exchange. Additionally, the propensity of the lipopeptide for extensive hydrophobic interactions, as illustrated by its low c.m.c., contributes to further stabilization of the cation and an increase in the partitioning of lichenysin into the erythrocyte membrane. Our data support the formation of a lichensyin-Ca2+ complex in a molar ratio of 2:1 instead of 1:1 with surfactin, suggesting an intermolecular salt bridge between two lichenysin molecules. Therefore, when Ca2+ ions are present in the solution, micellization occurs via a dimer assembly, with a possible long-range effect on the spatial arrangement of the micelles or other supramolecular structures. Finally, among all the surfactin peptidic variants so far known, lichenysin is the one for which the three tested activities are the most substantially improved.
Bacterial Proteins↗
The monolayer technique: a potent tool for studying the interfacial properties of antimicrobial and membrane-lytic peptides and their interactions with lipid membranes.
Erudites of the antiquity already knew the calming effect of oil films on the sea waves. But one had to wait until 1774 to read the first scientific report on oil films from B. Franklin and again 1878 to learn the thermodynamic analysis on adsorption developed by J. Gibbs. Then, in 1891, Agnes Pockels described a technique to manipulate oil films by using barriers. Finally, in 1917, I. Langmuir introduced the experimental and theoretical modern concepts on insoluble monolayers. Since that time, and because it has been found to provide invaluable information at the molecular scale, the monolayer technique has been more and more extensively used, and, during the past decade, an explosive increase in the number of publications has occurred. Over the same period, considerable and ever-increasing interest in the antimicrobial peptides of various plants, bacteria, insects, amphibians and mammals has grown. Because many of these antimicrobial peptides act at the cell membrane level, the monolayer technique is entirely suitable for studying their physicochemical and biological properties. This review describes monolayer experiments performed with some of these antimicrobial peptides, especially gramicidin A, melittin, cardiotoxins and defensin A. After giving a few basic notions of surface chemistry, the surface-active properties of these peptides and their behavior when they are arranged in monomolecular films are reported and discussed in relation to their tridimensional structure and their amphipathic character. The penetration of these antimicrobial peptides into phospholipid monolayer model membranes, as well as their interactions with lipids in mixed films, are also emphasized.
Amino Acid Sequence↗
Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein.
The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature Tm = 54 degrees C, an enthalpy change DeltaH = 119 kcal.mol-1 and a free energy change DeltaG(H2O, 25 degrees C) = 5 kcal.mol-1. The urea-induced unfolding profiles of PEBP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol-1 at 25 degrees C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment.
Air↗
Structural investigations of basic amphipathic model peptides in the presence of lipid vesicles studied by circular dichroism, fluorescence, monolayer and modeling.
A cationic amphiphilic peptide made of 10 leucine and 10 lysine residues, and four of its fluorescent derivatives in which leucines were substituted by Trp residues at different locations on the primary sequence have been synthesized. The interactions of these five peptides with neutral anionic or cationic vesicles were investigated using circular dichroism, steady state and time-resolved fluorescence with a combination of Trp quenching by brominated lipid probes, monolayers, modeling with minimization and simulated annealing procedures. We show that all the five peptides interact with neutral and anionic DMPC, DMPG, DOPC or egg yolk PC vesicles. The binding takes place whatever the peptide conformation in solution is. In the case of DMPC bilayers the binding free energy DeltaG is estimated at -8 kcal mole-1 and the number of phospholipid molecules involved is about 20-25 per peptide molecule. Peptides are bound as single-stranded alpha helices orientated parallel to the bilayer surface. In the anchoring of phospholipid head groups around the peptides, the lipid molecules are not smeared out in a plane parallel to the membrane surface but are organized around the hydrophilic face of the alpha helices like 'wheat grains around an ear' and protrude outside the bilayer towards the solvent. We suggest that such a lipid arrangement generates transient structural defects responsible for the membrane permeability enhancement. When an electrical potential is applied, the axis of the peptide helices remains parallel to the membrane surface and does not reorient to give rise to a bundle of helix monomers that forms transmembrane channels via a 'barrel stave' mechanism. The penetration depth of alpha helices in relation to the position of phosphorus atoms in the unperturbed lipid leaflet is estimated at 3.2 A.
Amino Acid Sequence↗
Penetration of the insect defensin A into phospholipid monolayers and formation of defensin A-lipid complexes.
Defensin A is an inducible cationic protein secreted in the hemolymph of fleshfly Phormia terranovae larvae in response to bacterial or septic injuries. Defensin A is known to permeabilize the bacteria cell membranes by forming voltage-dependent channels. The penetration of this small protein into lipid monolayers was studied as a function of the polar head and acyl chain length of phospholipids. The extent of penetration by defensin A is higher in monolayers made of anionic phospholipids than in monolayers made of zwitterionic phospholipids (phosphatidylcholines), because of electrostatic interactions. From the analysis of the compression isotherm parameters of mixed defensin A/phospholipid monolayers, it appears that defensin A interacts with phospholipid by forming 1:4 complexes. These complexes are not miscible in the lipid phase and induce microheterogeneity in the lipid membrane. These clusters might be related to the ion-channel structures responsible for the biological activity of defensin A.
Animals↗
Synthesis and interfacial behavior of poly(Lys-Tyr-Tyr-Lys).
Poly(Lys-Tyr-Tyr-Lys) was synthesized by polycondensation of the tetrapeptide unit using paranitrophenyl esters. The conformation of poly(Lys-Tyr-Tyr-Lys) is very dependent on its environment. CD spectra in bulk are difficult to interpret owing to the contribution of Tyr residues, but from ir spectra it seems that poly(Lys-Tyr-Tyr-Lys) adopts preferentially an unordered conformation in water. Addition of salts induces a partial transition to a beta structure. The behavior is different at interfaces. When poly(Lys-Tyr-Tyr-Lys) is spread as a film on a water subphase, the shape of the compression isotherm curves is compatible with a stacking of two beta-sheets. On a KCl subphase, the polymer film is more expanded and more compressible, and the isotherm curve resembles that of a polymer in a random conformation. The analysis by CD and ir spectroscopy of transferred monolayers using the Langmuir-Blodgett technique allowed us to confirm and make these data more precise: on a water subphase the spectra are those of an antiparallel beta structure. At the interface of a saline solution the spectra are compatible with a mixture of random coil (largely) and a small content of beta structures.
Amino Acid Sequence↗
The secondary structure of the insect defensin A depends on its environment. A circular dichroism study.
Defensin A is an inducible antibacterial protein isolated from the larvae of Phormia terranovae. The conformation of defensin A has been previously determined by two-dimensional 1H-NMR for concentrations in the range of 4-8 mM in water (Bonmatin JM et al (1992) J Biomol NMR 2, 235-256). CD spectroscopic data of defensin A at lower concentrations (10(-5) to 10(-3) M) are reported herein. The ellipticity in the 200-240 nm wavelength range for various solvents varies as follows: acetonitrile < water < methanol < HFIP. The magnitude of theta 222 is strongly dependent on defensin concentration in a buffer solution, suggesting an aggregation process. The helical content of defensin A is maximum at a pH value range (7.5-8) for which the optimum antibacterial activity was observed (Cociancich S et al (1993) J Biol Chem 268, 19239-19245).
Acetonitriles↗
Interactions of surfactin with membrane models.
Surfactin, an acidic cyclic lipopeptide produced by strains of Bacillus subtilis, is a powerful biosurfactant possessing biological activities. Interactions of ionized surfactin (two negative charges) with lecithin vesicles have been monitored by changes in its CD spectra. These changes are more important in the presence of Ca2+ ions. We have studied the penetration of ionized surfactin into lipid monolayers. Using dimyristoyl phospholipids, the surfactin penetration is more important in DMPC than in DMPE monolayers and is greatly reduced in DMPA monolayers because of electrostatic repulsion. The surfactin penetration is lowered when the acyl chain length of the phospholipids increases. The exclusion pressure varies from 40 mN m-1 for DMPC to 30 mN m-1 for DPPC and 18 mN m-1 for egg lecithin. The presence of Ca2+ ions, which neutralize the charges of both surfactin and lipids in the subphase, leads to an important change of the penetration process that is enhanced in the case of acidic, but also of long chain (higher than C14) zwitterionic phospholipids (DPPC and lecithin). From compression isotherms of mixed surfactin/phospholipid monolayers, it appears that surfactin is completely miscible with phospholipids. The present study shows that surfactin penetrates spontaneously into lipid membranes by means of hydrophobic interactions. The insertion in the lipid membrane is accompanied by a conformation change of the peptide cycle.
1,2-Dipalmitoylphosphatidylcholine↗
Iturins, a special class of pore-forming lipopeptides: biological and physicochemical properties.
Iturins are a family of lipopeptides extracted from the culture media of various strains of Bacillus subtilis. These amphiphilic compounds are characterized by a peptide ring of seven amino acid residues including an invariable D-Tyr2, with the constant chiral sequence LDDLLDL closed by a C14-C17 aliphatic beta-amino acid. They exhibit strong antifungal activities against a wide variety of pathogenic yeasts and fungi but their antibacterial activities are restricted to some bacteria such as Micrococcus luteus. The biological activity of the iturin lipopeptides is modulated by the primary structure of the peptide cycle as illustrated by the methylation of the D-Tyr2 residue which dramatically decreases the activity or by the inversion of the two adjacent Ser6-Asn7 residues which makes mycosubtilin more active than iturin A. The antifungal activity is related to the interaction of the iturin lipopeptides with the cytoplasmic membrane of target cells, the K+ permeability of which is greatly increased. The ability of iturin compounds to increase the membrane cell permeability is due to the formation of ion-conducting pores, the characteristics of which depend both on the lipid composition of the membrane and on the structure of the peptide cycle. From monolayer experiments it has been suggested that these ionic pores are the consequence of the presence of aggregates (lipopeptide aggregates or lipopeptide/phospholipid complex aggregates) in the phospholipid membrane. It has also been shown that, when active, iturins interact strongly with sterols, forming lipopeptide/cholesterol complexes. Therefore, the biologically efficient structure might be a ternary structure: iturin/phospholipid/sterol.
Amino Acid Sequence↗
Surfactin/iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturin A.
Iturin A and surfactin are two lipopeptides extracted from a same strain of Bacillus subtilis. Iturin A possesses antibiotic and antifungal activities and surfactin is a strong surfactant. The presence of surfactin, at a concentration at which, alone, it is inactive, increases to a very large extent the haemolysis percent induced by iturin A. This synergistic effect seems to be in relation with interactions between iturin A and surfactin. Iturin A adsorbs to and penetrates into surfactin monolayers. Iturin A and surfactin are miscible and interact specifically in mixed monolayers.
Adsorption↗
Interactions of bioactive lipopeptides, iturin A and surfactin from Bacillus subtilis.
The antifungal activity of iturin A and its interaction with erythrocyte membranes were enhanced in the presence of surfactin. The modification of the properties of iturin A was explained by the formation of mixed iturin A-surfactin micelles. Such mixed micelles were easily generated when both lipopeptides were in aqueous solutions in the absence of mineral salts but the formation of these micelles did not occur when the solutions contained a high molarity of mineral cations.
Amino Acid Sequence↗
Iturin lipopeptides: interactions of mycosubtilin with lipids in planar membranes and mixed monolayers.
The interactions between the antifungal lipopeptide mycosubtilin and lipids are studied. Mycosubtilin increases the ion permeability of planar lipid membranes by forming ion conducting pores. The lifetime of these pores is greatly increased when the membrane contains cholesterol. In mixed monolayers the interaction between mycosubtilin and DMPC leads to the formation of a mycosubtilin/DMPC 1:2 complex non miscible in the excess DMPC monolayer but miscible in the mycosubtilin monolayer. Mycosubtilin and cholesterol interact strongly in monolayers in all proportions and form a mycosubtilin-cholesterol (1:2) complex. These results are analyzed with reference to the overall view of the activity of iturins and the importance of the lipopeptide conformation is outlined.
Amino Acid Sequence↗
The lipopeptide antibiotic A21978C has a specific interaction with DMPC only in the presence of calcium ions.
The A21978C group are lipopeptide antibiotics which kill Gram-positive bacteria only in the presence of calcium ions. The calcium requirement of the antibacterial activity of A21978C correlates well with an in vitro calcium-dependent insertion into phospholipid vesicles. In this paper the interaction of A21978C with phosphatidylcholine is investigated in mixed monomolecular films. The spontaneity of the antibiotic-lipid mixing was determined by calculating the free energy change. On a Ca2+ containing subphase there is a specific interaction between the components at all antibiotic-lipid ratios. This is not true on K+ subphases, where specific interactions never occur. On Mg2+ subphases specific interactions occur only in monolayers containing very little lipid. By analysing the fluorescence of the kynurenine residue we have followed the effects of two factors on the penetration of the antibiotic into lipid bilayer vesicles. Firstly, the phospholipid gel to liquid crystalline phase transition which in the absence of calcium leads to an exclusion of the antibiotic from the bilayer. This trend is completely reversed in the presence of Ca2+. Secondly, the role of this lipopeptide's lipid tail was clarified by use of a series of versions of increasing fatty acyl chain length. The results indicate that the interaction promoted by calcium is not simply a hydrophobic attraction between fatty acyl chains but is more likely to be a specific interaction between polar headgroups.
Anti-Bacterial Agents↗