Using monomolecular films to characterize lipid lateral interactions

Abstract

Membrane lipids are structurally diverse in ways that far exceed the role envisioned by Singer and Nicholson of simply providing a fluid bilayer matrix in which proteins reside. Current models of lipid organization in membranes postulate that lipid structural diversity enables nonrandom lipid mixing in each leaflet of the bilayer, resulting in regions with special physical and functional properties, i.e., microdomains. Central to understanding the tendencies of membrane lipids to mix nonrandomly in biomembranes is the identification and evaluation of structural features that control membrane lipid lateral mixing interactions in simple model membranes. The surface balance provides a means to evaluate the lateral interactions among different lipids at a most fundamental level--mixed in binary/ternary combinations that self-assemble at the air-water interface as monomolecular films, i.e., monolayers. Analysis of surface pressure and interfacial potential as a function of average cross-sectional molecular area provide insights into hydrocarbon chain ordering, lateral compressibility/elasticity, and dipole effects under various conditions including those that approximate one leaflet of a bilayer. Although elegantly simple in principle, effective use of the surface balance requires proper attention to various experimental parameters, which are described herein. Adequate attention to these experimental parameters ensures that meaningful insights are obtained into the lipid lateral interactions and enables lipid monolayers to serve as a basic platform for use with other investigative approaches.

Fig. 1

Langmuir-type surface balance for determining the surface pressure and surface potential as a function of average cross-sectional molecular area. γ is the surface tension of surface occupied by lipid amphiphile and γ₀ is the surface tension of clean aqueous subphase.

Fig. 2

Representative π-A isotherms for “Raft” Lipids. Dotted line shows the condensed isotherm of cholesterol. Dashed line shows the liquid-expanded (fluid) isotherm of POPC. Solid line shows isotherm of bovine brain SM. The discontinuity occurring near 64 Ų/mol (14 mN/m) represents the onset of a 2D-phase transition from liquid-expanded-to-condensed behavior with monolayer collapse occurring near 42 Ų/mol.

Fig. 3

The effect of temperature on 16:0 SM monolayers. Isotherms (from top to bottom by 2D-phase transition) were measured at 30, 24, 20, 15, and 10°C for SM monolayers containing palmitoyl acyl chains.

Fig. 4

16:0 SM-cholesterol average molecular area vs composition analysis. Plots are shown for three different surface pressures in mN/m (5, squares; 15, circles; 30 triangles). The linear plots show the average molecular area obtained by calculation using the molecular areas of pure 16:0 SM and cholesterol, each apportioned by mole fraction. The nonlinear curves represent the experimentally observed areas for the mixtures. Negative deviation from ideal additivity (linear plots) shows the condensing or ordering effect of cholesterol.

Fig. 5

Changes in surface compressional moduli (C_s⁻¹) induced on mixing of equimolar cholesterol with PC or SM. Lower symbols represent the C_s⁻¹ values of the pure lipids in the absence of cholesterol. Upper symbols represent the experimentally observed C_s⁻¹ values for binary mixtures with equimolar cholesterol. The X along each line represents the ideal C_s⁻¹ values calculated based on additivity of each pure lipid component in the binary mixtures. All C_s⁻¹ values were determined at a surface pressure of 30 mN/m. The notation X:Y refers to the acyl chain length in carbon atoms (X) and the number of cis-double bonds (Y) present in the different molecular species of PC or SM.