Monolayer Technique

Amphiphilic molecules spontaneously adsorb to the air-water interface forming a two-dimensional monolayer [140-142]. The free energy of the system is minimized by reducing interactions between nonpolar groups and water molecules. Lipids, as one type of amphiphiles, arrange at an air-water interface in a way that the headgroup points toward the aqueous subphase and the chains toward the air. The lipid monolayer can be used as a model for one leaflet of a cell membrane [143-145]. Although cell membranes are composed of lipid bilayers, the Langmuir lipid monolayers are two-dimensional surface films that have been extensively used to model biological membranes.

Monolayer techniques have been widely employed as potent tools to study interactions of peptides with lipid monolayers at the molecular level [8]. Two types of monolayer experiments have to be distinguished: the time dependent adsorption of the peptide from the subphase to a preformed lipid monolayer at the air-water interface, and the determination of a surface pressure-area isotherm of a co-spread lipid peptide mixture upon compression of the mixed film.

To determine the adsorption process and the phase behavior during compression, the surface pressure is observed in dependence of time or molecular area, respectively. The surface pressure π itself is defined as difference between the surface tension of the modified water surface γ and the surface tension of pure water γ0.

π = γ - γ0 (2.8)

The surface pressure can be measured by the Wilhelmy plate-method. The force F on a plate, which is partially immersed into the subphase, is measured. The plate is often very thin and made of platinum, but even plates made of glass, quartz, mica and filter paper can be used.

The forces acting on the plate consist of downward forces due to gravity and surface tension, and an upward force due to buoyancy of the displaced water. For a rectangular plate with the length lp, the width wp and the thickness tp, of material density ρp, immersed to a depth hi in a liquid of density ρi, the net downward force is given by the following equation:

F = ρp · g · lp · wp · tp + 2γ · (tp· wp) (cos q) - ρi · g · ti · wi · hi (2.9)

where q is the contact angle of the liquid on the solid plate and g is the gravitational constant.

The surface pressure is then determined by measuring the change in F for a stationary plate between a clean surface and the same surface with a monolayer present. If the plate is completely wetted by the liquid (i.e. q = 0 => cos q = 1) the surface pressure is obtained from the following equation

Methods and Theory

This force is then converted into surface tension (mN m^-1) with the help of the dimensions of the plate.

2.6.3.1 Monolayer Adsorption Experiments

The adsorption process of an interaction partner to a phospholipid monolayer is typically measured by the constant area method [10, 146-150] or the constant surface pressure method [9, 151]. In the constant area method used here, the initial surface pressure π_ini of the phospholipid monolayer is adjusted by spreading different amounts of lipids (see Figure 2.6.1). Membrane insertion of injected compound is accompanied by a change in the surface pressure, since the area of the trough surface is kept constant. Upon adsorption, the gain of the Gibbs free energy has enthalpic and entropic contributions. Coulomb-, van der Waals forces, and hydrogen bonds have an effect on the enthalpy of the system, whereas the release of water molecules and counter ions and the rearrangement of the molecule, especially of peptides, higher surface excess, not all molecules reaching the surface are adsorbed caused by a present energy barrier. To ensure that the adsorption maximum is reached, a concentration dependent adsorption isotherm is measured first. There are three main energy barriers, one associated with diffusion from the bulk, another related to the interfacial pressure, and a third to the interfacial electrical potential. It was found that the rate of adsorption depends on the surface pressure of the lipid and the sequence of the peptide. The molecule must do work against the surface pressure in order to create a hole of area A for itself to move into. This amount of work is equal to





G dA (2.12)

The required energy to adsorb to the interface is smaller for small molecules as they tend to adsorb to a greater extent [152].

0 1 2 3 4 5 adsorption experiments of a peptide solution to a lipid monolayer. The fixed through area is covered with a lipid monolayer, the surface pressure is recorded with a Wilhelmy plate, and the peptide stock solution is injected into the aqueous subphase through a channel in the trough. Bottom left: time dependent development of the surface pressure π of a lipid monolayer after injection of a ligand or peptide solution into the subphase at a starting surface pressure πini. Bottom right: Difference of surface pressure Δπ after peptide adsorption to a lipid monolayer with different initial surface pressure.

Adsorption experiments of peptides to a lipid monolayer at different initial surface pressures were performed to study the effect of the peptide on physical state of the monolayer and the lipid density at the air-water interface. The kinetics of protein binding onto phospholipid monolayers were monitored until the equilibrium surface pressure π0 was reached. The kinetic curves show an overlay of two effects, the decrease of π upon lipid condensation and the increase of π upon peptide incorporation into the monolayer. To analyze the kinetics of adsorption quantitatively the experimental adsorption curves were fitted using the following bi-exponential equation: surface pressure at equilibrium after 5 hours. The amplitude of both effects corresponds to the properties of the peptides describing the relative strength of the electrostatic and hydrophobic interaction with the lipid monolayer.

Methods and Theory

The change in surface pressure Δπ = π₀ - π_ini observed after adsorption of a protein or peptide to a lipid monolayer as a function of the initial surface pressure π_ini shows in many cases a linear relationship, from which the maximum insertion pressure MIP is determined, i.e. the value of π_ini, where Δπ = 0 mN m^-1. Per definition MIP is the value above which no incorporation into the lipid bilayer occurs anymore [8, 147-149]. However, for the binding of cationic pentapeptides to DPPG monolayers that this relation was already observed before and the interpretation cannot be used for the electrostatic binding of peptides, as negative surface pressure changes and changes in slope were observed [123].

2.6.3.2 Surface Pressure - Area Isotherms

Monolayer studies with phospholipids and peptides co-spread at an air-water interface can be performed in a well-defined way. The two-dimensional molecular density and composition can be varied, as well as the temperature and the ionic strength conditions of the subphase.

Using surface pressure-area (π - A) isotherms [141], one can observe that decreasing the surface area at the interface of the lipid or peptide molecule induces a series of two-dimensional phase transitions due to changes in the molecular packing. The compression rate must be slow enough to ensure that changes occur under thermodynamic equilibrium conditions [8]. Other useful characteristics of monolayers are a planar arrangement over macroscopic dimensions and at least one symmetry axis, the plane normal. This technique is well-known [143] to study the two-dimensional alignment of the molecules with additional techniques, e.g. fluorescence microscopy, infrared reflection absorption spectroscopy (IRRAS), Brewster angle microscopy and X-ray reflectivity and diffraction.