In studies of the transfer, distribution and biochemical activity of metal ions it is typically assumed that the phospholipid bilayer acts as an inert barrier. Yet, there is mounting evidence that metal ions can influence the physical properties of membranes. Little is known of the basis of this effect. In this work the location and distribution of common metal ions: Na + , Mg 2+ , and Ca 2+ in phospholipid membranes were studied. Computer simulations of lipid membrane segments in aqueous environment showed that the ions penetrate the membrane headgroup zone and co-localize with the phosphate and the ester moieties. Analysis of the chemical environment of the ions in the simulations suggested that the co-localization is facilitated by coordination to the polar oxygen atoms of the phosphate and ester groups in typical coordination geometries of each ionic species, where the coordination shells are completed by water molecules. In contrast, the counterions do not penetrate the headgroup zone but form a layer over the membrane instead; this layer is also an effective metal exclusion zone. Importantly, the choline groups appear to be distributed almost exactly in the same plane as the phosphate, suggesting that the zwitterion dipole is preferentially horizontally aligned: this suggests that the distribution of the Cl − over the membrane surface is not a direct result of interaction with the choline groups, but rather an effect of the field emanating from the metal ion content of the membrane. Such a well defined ion distribution is expected to have a strong influence on membrane properties, in particular phase transition temperatures via increased in-plane cohesion; this was proven by calorimetry measurements using differential scanning calorimetry of suspended liposomes and quartz crystal microbalance-based measurements on supported single bilayer membranes. These findings shed a new light on the role metal ions play in stabilizing biological membranes.

Cholesterol is believed to induce the formation of membrane domains, “rafts”, which are implicated in a range of natural and pathologic membrane processes. Therefore, it is important to understand the role that cholesterol plays in the formation of these structures. Here, we use label-free spectroscopic imaging to investigate cholesterol fractioning in supported bilayer membranes at nanoscale. Scattering-type scanning near-field optical microscopy (s-SNOM) was used to visualize the formation of cholesterol-induced domains in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes. Our results revealed the coexistence of phase separated domains in DMPC lipids with 10 mol % cholesterol content, whereas a mostly homogeneous bilayer was found at low (5 mol %) and high (15 mol %) cholesterol content. Near-field nano-FTIR spectroscopy was used to identify the cholesterol-rich domains based on their qualitative chemical compositions. It was determined that cholesterol binds to phosphodiester and alkyl glycerol ester moieties, likely via hydrogen bonding of the alcohol to either of the ester oxygens. The results also confirm the existence of an ideal cholesterol-lipid mixture ratio (∼15:85) with a geometrically defined packing. At lower cholesterol content there is phase separation between liquid ordered and almost neat DMPC domains. Thus, the liquid ordered phase exists at an energy minimum at a given lipid–cholesterol ratio.

Caerin1.1 is a 25‐residue antimicrobial peptide secreted by the tree frog Litoria splendida. It is classified as an α‐helical cationic membrane disrupting peptide, with a good activity against Gram positive bacteria. Given its length, it is believed that caerin1.1 acts via transmembrane pore formation. However, the exact mechanism of its action is unknown. Here the interactions of caerin1.1 with biomimetic membranes were studied to establish the molecular mechanism of action. The activity of the peptide against different model membranes was determined by dye leakage experiments. Caerin1.1 showed weak activity against neat dimyristoyl phosphatidylcholine (DMPC) membrane and minimal activity against negatively charged or cholesterol containing membrane mixtures. Circular dichroism spectroscopy revealed that the peptide exhibited only 8‐16% helicity even in the DMPC membrane, yet quartz crystal microbalance fingerprinting suggested that the peptide was able to penetrate the membrane in each case. The results suggest that caerin1.1 is a membrane penetrating peptide but not a membrane disruptor, only causing incidental dye leakage while diffusing across the membrane. Consistently the main mode of action of caerin1.1 is likely exerted through an intracellular target.