Resumen

The eukaryotic cell membrane is a complex mixture of cholesterol, lipids, and proteins. Direct study of the cell membrane must include these multiple variables, which significantly complicates their analysis. Model systems minimize the number of components used in a membrane study. Atomic force microscopy (AFM) can be employed to study the morphology of model supported phospholipid bilayers (SLB). Thermal and diffusive properties of membranes are identified using differential scanning calorimetry (DSC) and fluorescence correlation spectroscopy (FCS). Attenuated total reflectance infrared spectroscopy (ATR-FTIR) can be applied to observe SLB disruption. This suite of techniques has been used to observe interactions of molecules interfering with phospholipid bilayers. Single- and multicomponent SLBs and phospholipid vesicles are employed as models of cell membranes.

The effects of the pore-forming peptide alamethicin are shown for lipid bilayers with varying compositions. A correlation between the presence of sphingomyelin or cholesterol and increased pore formation is identified by the increase of potassium leakage through vesicles and formation of raft structures visible with AFM. ATR-FTIR shows that alamethicin disrupts all types of bilayers studied, suggesting that the observed effects are directly correlated to alamethicin incorporation.

A comparison is made between the interactions of two polychlorinated biphenyl isomers and an SLB. A study of AFM micrographs identifies multiple phase transitions in one isomer, but only one phase transition for the other isomer. Two transitions are consistent with an uninterrupted bilayer, but one transition suggests that an intercalated molecule ties the bilayer leaflets together, or a contaminant molecule disrupts substrate ordering on the bilayer. DSC results show that one isomer is more strongly associated with the bilayer than the other isomer, a difference that is explained based on geometric differences in the isomers. FCS is employed to observe a PCB-lipid complex which highlights the different strengths of interaction between PCB isomers and lipids. A model is proposed where the ortho-substituted PCB isomer inserts into the bilayer and strongly associates with the hydrophobic region. In contrast, the planar PCB isomer does not insert deeply past the hydrophilic region of the bilayer, which allows it to interrupt interactions between the substrate and its closest bilayer leaflet.

We employ atomic force microscopy to show interactions of sphingomyelin or cholesterol with a mixed DSPC/DOTAP bilayer. We report that sphingomyelin, but not cholesterol, supports the formation of hexagonal crystalline domains. These domains are small (ca. 70 nm) and can be observed with AFM as high as 40°C. We use differential scanning calorimetry of DSPC/DOTAP vesicles in the presence of either sphingomyelin or gramicidin A to show the elimination of one thermogram peak. This result suggests that sphingomyelin, like gramicidin A, interacts with both leaflets in the bilayer. We report dichroic ratios measured from each bilayer composition with ATR-FTIR. The methylene dichroic ratios in all bilayers decrease with introduction of both SM and Chol, but the magnitude of decrease is greater in the presence of SM. A decrease in methylene dichroic ratios is related to an increase in lipid chain order. We propose that SM exerts extensive hydrogen-bonding forces on DSPC and sustains structure to high temperatures when incorporated in a 40 mol% ratio. At this same temperature, we propose that Chol does not support the same structure formation because it induces the liquid-ordered phase in DSPC/DOTAP. We note that sphingomyelin promotes multilayer, not bilayer, formation.