Model membrane systems

One of the primary hurdles in the study of membrane proteins is the provision in

vitro of an appropriate environment to satisfy the complex structural requirements

of molecules that normally reside within a fluid milieu enriched with an intricate array of amphiphilic and hydrophobic lipids. A number of model membrane systems have been developed in order to study the structures and functions of membrane proteins. The major systems in use are micelles, bicelles, planar lipid bilayers, lipid monolayers and liposomes. Micelles are small, roughly spherical structures formed from the self-association of detergent monomers above a critical concentration threshold called the critical micellar concentration (CMC) (Garavito and Ferguson-Miller, 2001). The size and shape of micelles depends on the size, type and stereochemistry of the detergent monomer and the solvent environment (Wennerström and Lindman, 1979; Mitchell et al., 1983). A number of different detergents can be used for micelle formation, the main categories of which are: a) ionic detergents; b) non-ionic detergents; and c) zwitterionic detergents (Seddon

et al., 2004). Ionic detergents have a head group with either a net cationic of

anionic charge attached to a hydrophobic hydrocarbon chain (as in SDS) or steroidal backbone (as in bile acid salts such as sodium cholate). Ionic detergents can be relatively denaturing. Non-ionic detergents such as Triton® X-100 are mild and relatively non-denaturing. They contain uncharged hydrophilic head groups with hydrophobic tails. Zwitterionic detergents such as dodecyldimethyl-

N-amineoxide (DDAO) combine the properties of ionic and non-ionic detergents.

The advantage of using micelle-forming detergents as model membrane systems is that the detergents can both solubilise membrane proteins from their lipid environment and then replace that environment without too much disruption to the native structure of the protein. Mixed detergent-lipid micellar systems are also in use for membrane-protein study, and often provide a more stabilising environment

than detergent micelles alone (reviewed in Seddon et al., 2004). Despite their small size and high surface curvature, micelles have been used extensively as biomimetic systems for membrane proteins and peptides, utilising techniques such as high-resolution nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy and circular dichroism (CD) (Jelinek and Kolusheva, 2005).

Bicelles are a form of mixed detergent-lipid micelle, but have a much higher lipid component (Sanders and Prosser, 1998). Usually short chain lipids such as dimyristoylphosphatidylcholine (DMPC) are used with detergents such as dihexanoylphosphatidylcholine (DHPC; Sanders and Schwonek, 1992) or a zwitterionic bile salt derivative, 3-[(3-cholamidopropyl)dimethylammonio]-2- hydroxyl-1-propanesulfonate (CHAPSO; Sanders and Prestegard, 1990). Mixed in the correct composition and the correct temperature, the detergent-lipid mixture forms edge-stabilised, bilayered, discoidal structures known as bicelles (Figure 1.4). They are used primarily in NMR studies because of the fact that they can be magnetically aligned (Sanders and Landis, 1995).

Planar lipid systems such as bilayers and monolayers aim to model the phospholipid ordering within cellular membranes and to mimic the lateral organisation of cell surfaces (Greenhall et al., 1998). Phospholipid monolayers deposited at the air-water interface of aqueous solutions (Langmuir monolayers) are used as model membrane systems for studying the interactions of certain peptides and membrane proteins with membranes. Thermodynamic and microscopy techniques such as pressure-area isotherms and fluorescence microscopy can be used to investigate, for example, structural disruption and phase separation caused by peptides incorporated into the monolayers (Chen et

al., 2003). Phospholipid monolayers can also be used in so-called “tip-dip”

electrophysiology experiments, whereby a monolayer is spread on the surface of an aqueous bath, and a glass pipette passed repeatedly through the monolayer until the resistance reaches a certain level (e.g. in Harrop et al., 2001; Warton et al., 2002). The relevant membrane-insertion-competent protein is then added to the bath and electrophysiological recordings made. The most common use of planar

Figure 1.4. Schematic structure of a bicelle.

Cross-section of a bicelle composed of a mixture of phospholipids (blue) and detergent (red). The detergent molecules stabilise the edge of the disc.

lipid bilayers is as black lipid membranes. These consist of thin lipid films placed across small apertures separating baths containing ionic solutions (Winterhalter, 2000). Conductance measurements are then undertaken to study the formation of pores and ion channels in the membrane, their relative permeability and their ion selectivity.

Liposomes are bilayered lipid vesicles which enclose an aqueous space separate from the external solution. There are four major classes of liposomes: small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs) and multilamellar vesicles (MLVs). SUVs range in size up to about 100 nm, averaging between 30-70 nm, and are generally prepared by sonication of MLVs (Papahadjopoulos and Miller, 1967). LUVs have hydrodynamic diameters of 100-1000 nm, and can be prepared by a number of methods, including sonication (Lasch et al., 2003), reverse-phase evaporation (Szoka and Papahadjopoulos, 1978), extrusion (Olsen et al., 1979; Hope et al., 1985; MacDonald et al., 1991) and detergent dialysis (Mimms et al., 1981) (Figure 1.5). GUVs can range up to 100 µm, but are generally of the order of up to 10 µm. They are prepared by electroporation (Angelova and Dimitrov, 1986). MLVs consist of multiple concentric bilayers and are formed by hand-shaking of a dried lipid film hydrated with aqueous buffer (Lasch et al., 2003). The surfaces and properties or liposomes can be modified by the choice of phospholipid, as

well as by the incorporation of proteins such as lectins and glycoproteins, or even synthetic polymers (Jones, 1995).

Figure 1.5. Schematic structure of an LUV.

Large unilamellar vesicle showing enclosed aqueous compartment enclosed by phospholipid bilayer.

An enormous amount of research has gone into the formation, structure, properties and applications of liposomes, and a search of the PubMed database at NCBI retrieves 33 433 results of articles with the word “liposomes” in their text. This intense interest is driven by the fact that liposomes so closely resemble the biological cell and as such can be used to study cellular processes such as transport phenomena, and their biological compatibility also means that they are particularly useful as drug delivery systems (Jones, 1995). Liposomes have been used to deliver chemotherapeutic agents (Leyland-Jones, 1993), antiviral drugs (Phillips, 1992), antibacterials (Škalko et al., 1992), and have also been used for gene therapy (Li and Huang, 2006; Karmali and Chaudhuri, 2007). They have

Aqueous compartment

Phospholipid bilayer

also been used as cosmetic agents for the delivery of moisturisers or anti- inflammatory agents to the skin (Puglia et al., 2004; Betz et al., 2005; Nasr et al., 2008). Generally, larger vesicles are preferred for structural and functional studies of membrane-incorporated proteins because of the closer resemblance of the surface curvature of these vesicles to that of biological cells, and because of the larger encapsulated volume inside these vesicles (Matsuda et al., 1997; Kahya

et al., 2000).