Monolayers of the lipopeptide mycosubtilin are studied at the air/water interface. Their structure is investigated using molecular dynamics simulations. All-atom models suggest that the lipopeptide is flexible and aggregates at the interface. To achieve simulation times of several microseconds, a coarse-grained (CG) model based on the MARTINI force field was also used. These CG simulations describe the formation of half-micelles at the interface for surface densities up to 1 lipopeptide per nm(2). In these aggregates, the tyrosine side chain orientation is found to be constrained: on average, its main axis, as defined along the C-OH bond, aligns along the interface normal and points towards the air side. The origin of the optical second harmonic generation (SHG) from mycosubtilin monolayers at the air/water interface is also investigated. The molecular hyperpolarizability of the lipopeptide is obtained from quantum chemistry calculations. The tyrosine side chain contribution to the hyperpolarizability is found to be dominant. The orientation distribution of tyrosine, associated with a dominant hyperpolarizability component along the C-OH bond of the tyrosine, yields a ratio of the susceptibility elements χ((2))(ZZZ)/χ((2))(ZXX) consistent with the experimental measurements recently reported by M. N. Nasir et al. [Phys. Chem. Chem. Phys., 2013, 15, 19919].

Les antibiotiques ituriniques, produits par Bacillus subtilis, representent une famille qui comprend l’iturine A, la mycosubtilline, les bacillomycines D, F et Lc. Ce sont des lipopeptides cycliques possedant des acides gras β-amines relies a un peptide forme par sept acides α-amines avec une sequence chirale invariable dont la configuration type est LDDLLDL. Ils possedent tous en commun les trois premiers acides α-amines incluant le residu tyrosyl. Ils sont connus essentiellement par leur pouvoir antifongique et ils ont egalement des proprietes antibacteriennes et hemolytiques. Ces proprietes proviennent de leur nature amphiphile qui leur permet d’interagir avec differents constituants membranaires. Les sterols, qui se trouvent dans les membranes plasmiques, constituent le partenaire privilegie d’interaction de ces antibiotiques. Aussi, le groupement phenol du residu tyrosine des antibiotiques est suppose avoir un role important pour la fixation des antibiotiques au niveau de la membrane plasmique. La finalite de cette fixation est souvent la lyse cellulaire. Il existe, au sein de la membrane plasmique, des regions particulieres contenant une forte concentration en sterol. Ces microdomaines membranaires different du reste de la membrane plasmique par leur composition ; ils sont egalement enrichis en certains lipides et en certaines proteines. Les microdomaines interviennent dans divers processus cellulaires cles, leur perturbation peut donc engendrer de nombreuses consequences. Du fait de leur composition, ces microdomaines peuvent constituer la cible des activites biologiques des antibiotiques ituriniques. Cette revue a pour but de faire la synthese des travaux realises sur les antibiotiques ituriniques au niveau de leurs activites biologiques en focalisant sur la comprehension de celles-ci a l’echelle moleculaire avec une discussion sur leurs groupements chimiques cles et sur le potentiel des microdomaines membranaires en tant que cible privilegiee pour ces molecules.

Les antibiotiques ituriniques, produits par Bacillus subtilis, representent une famille qui comprend l’iturine A, la mycosubtilline, les bacillomycines D, F et Lc. Ce sont des lipopeptides cycliques possedant des acides gras β-amines relies a un peptide forme par sept acides α-amines avec une sequence chirale invariable dont la configuration type est LDDLLDL. Ils possedent tous en commun les trois premiers acides α-amines incluant le residu tyrosyl. Ils sont connus essentiellement par leur pouvoir antifongique et ils ont egalement des proprietes antibacteriennes et hemolytiques. Ces proprietes proviennent de leur nature amphiphile qui leur permet d’interagir avec differents constituants membranaires. Les sterols, qui se trouvent dans les membranes plasmiques, constituent le partenaire privilegie d’interaction de ces antibiotiques. Aussi, le groupement phenol du residu tyrosine des antibiotiques est suppose avoir un role important pour la fixation des antibiotiques au niveau de la membrane plasmique. La finalite de cette fixation est souvent la lyse cellulaire. Il existe, au sein de la membrane plasmique, des regions particulieres contenant une forte concentration en sterol. Ces microdomaines membranaires different du reste de la membrane plasmique par leur composition ; ils sont egalement enrichis en certains lipides et en certaines proteines. Les microdomaines interviennent dans divers processus cellulaires cles, leur perturbation peut donc engendrer de nombreuses consequences. Du fait de leur composition, ces microdomaines peuvent constituer la cible des activites biologiques des antibiotiques ituriniques. Cette revue a pour but de faire la synthese des travaux realises sur les antibiotiques ituriniques au niveau de leurs activites biologiques en focalisant sur la comprehension de celles-ci a l’echelle moleculaire avec une discussion sur leurs groupements chimiques cles et sur le potentiel des microdomaines membranaires en tant que cible privilegiee pour ces molecules.

Niemann-Pick type C (NPC) disease is a lipid storage disorder characterized by accumulation of lipids in the late endosome/lysosome (LE/LY) compartment. In our previous report we isolated membranes of the LE/LY compartment from NPC L1 skin fibroblasts with a mutation in the NPC1 gene and found that they were characterized by low fluidity which likely contributed to the impaired function of membrane proteins involved in storage and turnover of cholesterol. In this report we isolated lipid microdomains (DRMs) from membranes of various cellular compartments and observed an increased amount of DRMs in the LE/LY compartment of NPC L1 cells in comparison to control cells, with no change in the DRM content in the plasma membrane. In addition, in the NPC cells, the majority of the cholesterol-interacting protein, AnxA6, which participates in the transport and distribution of cholesterol, translocated to DRMs upon a rise in Ca2+ concentration. The mechanism of this translocation was further studied in vitro using Langmuir monolayers. We found that Ca2+ is the main factor which regulates the interaction of AnxA6 with monolayers composed of neutral lipids, such as DPPC and sphingomyelin, and may also determine AnxA6 localization in cholesterol and sphingomyelin enriched microdomains, thus contributing to the etiology of the NPC disease.

The glycoprotein gp41 from the Human Immunodeficiency Virus type 1 (HIV-1) has an amino acid sequence enriched in tryptophan residues, the so-called gp41W peptide (i.e., KWASLWNWFNITNWLWYIK) and plays a crucial role in HIV-1 host cell infection. Using the coupling of Second Harmonic Generation targeting the tryptophan residues with lateral surface tension measurements, we investigate the interaction of gp41W with a neat air/water and a lipid/water interfaces. At the air/water interface, gp41W presents a well-defined orientation and this orientation is strongly modified at the lipid/water interface, depending on the surface pressure. These results show that this strategy is well suited to monitor tryptophan containing α-helices orientation at lipid/water interfaces.

Annexin A6 (AnxA6), a calcium- and membrane-binding protein, is expressed in mammalian cells in two isoforms: AnxA6-1 and AnxA6-2, the latter lacking the 524-VAAEIL-529 sequence at the start of repeat 7. The different intracellular localization of these two isoforms suggests distinct function in membrane dynamics. The aim of this work was to analyze the behavior of AnxA6 isoforms at the air/water interface alone and in the presence of membrane mimicking lipid monolayers. Using Langmuir technique showed that AnxA6-2 was less adsorbed to the neat air-water interface than AnxA6-1 at acidic pH and minor differences in their PM-IRRAS spectra were observed. Both isoforms exhibited similar behavior towards cholesterol monolayer. However, the interactions of AnxA6-2 with cholesterol ester monolayer were most favorable compared to AnxA6-1. Our experimental data are discussed in relation with the different intracellular localization of the two isoforms and with our constructed model of AnxA6-2 with the known crystal structure of AnxA6-1 showing the persistence of the 516-529 alpha-helix in AnxA6-2 despite the absence of the 524-VAAEIL-529 sequence. (C) 2013 Elsevier Inc. All rights reserved.

Iturinic antibiotics are produced by Bacillus subtilis strains and constitute a family including iturin A, mycosubtilin and bacillomycins D, F and Lc. These are cyclic lipopeptides with alpha-amino fatty acids linked up to a peptide constituted by seven alpha-aminoacids with an invariable LDDLLDL chiral sequence. The first three a-aminoacids containing the tyrosyl residue are the same for all members. They are well known for their strong antifungal activities but they also have antibacterial and hemolytic properties. These biological properties are due to their amphiphilic nature, allowing interactions with different membrane components. Sterols found in plasma membranes are the privileged interaction partners of these lipopeptides. Moreover, the tyrosyl residue of the iturinic antibiotics seems to play an important role during their fixation to the plasma membrane, the result of which is often cellular lysis. Within plasma membranes, there are particular regions with a high sterol content. These microdomains have a different composition compared to the rest of the membrane; they are rich in certain lipids and proteins and are involved in many key cellular processes. The perturbation of these microdomains could therefore have an important impact on the cell. Due to their composition, these microdomains may constitute the preferential target of iturin antibiotics. This review aims to summarize the studies relating to the biological activities of iturinic antibiotics. It focuses in particular on the existing knowledge regarding iturin antibiotics at the molecular level and discusses both the key chemical groups of these drugs and the potentiality of microdomains to constitute a target for these molecules.

The second harmonic generation (SHG) response at the air-water interface from the tyrosine-containing natural iturinic cyclo-lipopeptides mycosubtilin, iturin A and bacillomycin D is reported. It is shown that this response is dominated by the single tyrosine residue present in these molecules owing to the large first hyperpolarizability arising from the non-centrosymmetric aromatic ring structure of this amino acid. The SHG response of these iturinic antibiotics is also compared to the response of surfactin, a cyclo-lipopeptide with a similar l,d-amino acid sequence but lacking a tyrosine residue, and PalmATA, a synthetic linear lipopeptide possessing a single tyrosine residue but lacking the amino acid sequence structuring the cycle of the iturinic antibiotics. From the light polarization analysis of the SHG response, it is shown that the tyrosine local environment is critical in defining the SHG response of these peptides at the air-water interface. Our results demonstrate that tyrosine, similar to tryptophan, can be used as an endogenous molecular probe of peptides and proteins for SHG at the air-water interface, paving the way for SHG studies of other tyrosine-containing bioactive molecules.

Mycosubtilin is a strong antimicrobial agent belonging to the iturinic lipopeptide family which contains a single tyrosine residue. Its cell target has been shown to be the cytoplasmic membrane. This tyrosine residue has been previously shown to be essential for the biological activity of mycosubtilin. Since we have previously demonstrated that tyrosine, an aromatic amino acid, can be used as an endogenous probe for the frequency doubling process, the presence of a tyrosine residue in mycosubtilin allowed us to investigate the interactions of mycosubtilin with biomimetic lipid monolayers at the air–water interface by second harmonic generation (SHG). Mycosubtilin was added underneath dipalmitoylphosphatidylcholine or cholesterol monolayers at the air–water interface and significant increases in the surface pressure were observed in both cases. This observation demonstrates that mycosubtilin interacts with these biomimetic membranes. A light polarization resolved analysis of the SHG signals recovered for these two systems was then performed and confirmed that those interactions between the tyrosine residue in mycosubtilin and the membranes could be monitored by SHG. Furthermore, the differences exhibited by the nonlinear optical measurements for different membranes showed that these interactions depend on the nature of the biomimetic membrane present at the air–water interface.

Mycosubtilin, an antimicrobial lipopeptide produced by Bacillus subtilis, is characterized by strong antifungal activities. The molecular mechanisms of its biological activities on the membranes of the sensitive yeasts or fungi have not yet been clearly elucidated. Our purpose was to mimic the mycosubtilin interactions with these membranes using various Langmuir monolayers. Since the major sterol of yeasts or fungi is ergosterol, the interactions of mycosubtilin with monolayers constituted by ergosterol, DPPC/ergosterol or DPPC/sphingomyelin/ergosterol were examined at different initial surface pressures (Πi). Plotting the mycosubtilin-induced surface pressure increases versus Πi allowed to determine that the exclusion pressures of mycosubtilin from these different monolayers is higher than the surface prevailing within the biological membranes. However, this behavior was lost when mycosubtilin was interacting with ergosteryl acetate-containing monolayers. This suggests the involvement of the sterol alcohol group in the mycosubtilin interactions within membranes. Furthermore, the behavior of mycosubtilin with stigmasterol, similar to that observed with ergosterol, differs from that previously observed with cholesterol, suggesting a role of the alkyl side chain of the sterols. The adsorption of mycosubtilin to ergosterol monolayers induced changes in the lipopeptide orientation at the air–water interface as revealed by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). Moreover, imaging the air–water interface by Brewster angle microscopy (BAM) indicates that mycosubtilin induced changes in the organization and morphology of monolayers containing pure ergosterol with the appearance of small condensed dots, suggesting again that the target of mycosubtilin might be the ergosterol present in the membranes of the sensitive yeasts or fungi.

Bacillomycin D is a natural antimicrobial lipopeptide belonging to the iturin family. It is produced by Bacillus subtilis strains. Bacillomycin D is characterized by its strong antifungal and hemolytic properties, due to its interaction with the plasma membrane of sensitive cells. Until now, only few limited analyses were conducted to understand the biological activities of bacillomycin D at the molecular level. Our purpose was to analyze the conformation of bacillomycin D using IR spectroscopy and to model its interactions with cytoplasmic membranes using Langmuir interfacial monolayers. Our findings indicate that bacillomycin D contains turns and allow to model its three-dimensional structure. Bacillomycin D formed a monolayer film at the air–water interface and kept its turn conformation, as shown by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). To identify the membrane lipid target of bacillomycin D, its interactions with pure lipid monolayers were analyzed and an original behavior of the lipopeptide toward cholesterol-containing monolayers was shown. This original behavior was lost when bacillomycin D was interacting with pure cholesteryl acetate monolayers, suggesting the involvement of the alcohol group of cholesterol in the lipopeptide–cholesterol interaction.