We have characterized planar lipid films composed of phosphatidylethanolamines (PE) and phosphatidylglycerols (PG) from E. coli bacteria. The nature of the interactions and miscibility of PE and PG components within mixed lipid films was evaluated based on surface pressure measurements and Brewster angle microscopy imaging at the air-water interface. We have found that PE and PG components show tendency to form separated domains at surface pressures relevant for biological membranes. Further, we have directly compared mechanisms of formation of supported lipid bilayers either on mica or Au(111) by spreading of small unilamellar vesicles. The bilayer formation was monitored by in situ atomic force microscopy imaging. The pathways of the vesicles spreading on each substrate are substantially different and the buildup of the bilayer on Au(111) occurs through complex multistep mechanism. The morphology and nanomechanical properties of the resulting PE/PG bilayers were thoroughly compared. We have found that the interactions between lipids and supporting substrate significantly affect molecular organization within the films since the bilayer on Au(111) is uniform in terms of the topography, while the same lipid composition on mica results in formation of distinct gel and liquid disordered domains. Different molecular organization affects also nanomechanical properties of lipid films. The latter were expressed in terms of Young's moduli and bending stiffness.

Floating lipid bilayers composed of phosphatidylglycerols and cardiolipin were deposited on gold electrodes premodified with 1-thio-β-d-glucose monolayer by spreading of small unilamellar vesicles. The resulting lipid membrane was homogeneous, and its thickness was ∼5.0 nm. Electrochemical characterization combined with surface-enhanced infrared absorption spectroscopy revealed that negative polarization of the electrode leads to accumulation of water molecules in the interfacial region between lipid membrane and the thioglucose film. Moreover, the buildup of water layer was demonstrated to affect the nanomechanical properties of the membrane. The latter was manifested by well-pronounced decrease of Young’s modulus of the lipid bilayer correlating with increasing hydration. This effect was ascribed to the decoupling of the membrane from supporting thioglucose film due to the accumulation of interfacial water. As a result, the effective stiffness of the supporting layer is lower and it alters the nanomechanical behavior of lipid membrane. Our results provide strong experimental proof for the correlation between elastic properties of floating lipid membrane and the amount of water accumulated in the submembrane region.

Because of the increasing resistance of pathogens to commonly used antibiotics, there is an urgent need to find alternative antimicrobial compounds with different mechanisms of action. Among them, lipopeptides are recognized as promising candidates. In this work, the Langmuir technique and atomic force microscopy were employed to investigate the interactions of two novel lipopeptides with negatively charged phospholipid membranes, which served as a simplified model of inner membrane of Gram-negative bacteria. Lipid films contained phosphatidylethanolamine and phosphatidylglycerol extracts from E. coli bacteria. Lipopeptides were composed of palmitoyl chain covalently coupled to N-terminus of peptide with Trp-Lys-Leu-Lys amino acid sequence and the conformation of third residue was either d-Leu or l-Leu. It was found that chirality of leucine strongly affects interfacial behavior of these compounds, which was ascribed to the difference in effective size of the peptide portion of the molecules. Although the lipopeptides were the same in terms of amino acid sequence, charge, and identity of lipophilic chain, the experiments revealed that the barrier for their insertion into the lipid membrane is significantly different. Namely, it was lower for lipopeptide containing d-Leu residue. We have also found that insertion of the lipopeptides into the model membranes strongly alters lateral distribution of the membrane components and leads to its substantial fluidization. The dynamics of reorganization was noticeably faster in the presence of lipopeptide with smaller size of peptide moiety, i.e., containing d-Leu. It proves that effective size of the peptide headgroup is an important factor determining lipopeptide activity toward the lipid membranes.