Chapter3

Tools for Studying Membranes

• Enrichment of membrane factions

• Isolation of membrane components

• Characterization of membrane components

• Reconstitution of membrane components

Enrichment of Membrane Fractions

• Disruption of source material

• Tool = Homogenization

• Separation/enrichment

• Tool = Centrifugation

• Purification

• Tool = Detergents

• Reconstitution

• Tool = lipid bilayer model system

Subcellular Composition

Disrupt - Enrich - Isolate

Disruption of Source Material

Enrichment

Detergents

• Water soluble surfactants (reduce surface tension)

• Amphiphilic in nature

• Effect in solubilizing membrane components

• More soluble in water compared to lipids

• Grouped by function (ionic, zwitterionic, nonionic)

• Impurities are common in detergents (!!!!)

Ionic detergents

Nonionic detergents

Action of Detergents

• Act through micelle formation (spherical assembly)

• Partial exposure of hydrophobic moieties

• Occurs at a defined concentration (and Temperature)

• CMC critical micellar concentration

• Below CMC - all monomers

The Critical Micellar Concentration (CMC)

Stages in detergent solubilization

Practical Aspects

• Detergents enable to bring membrane proteins and lipids in the aqueous phase

• Steps of actions are concentration dependent

• Intercalation to mixed micelles (above CMC necessary)

• Detergent concentration and detergent-to-protein ratio influence extraction

• Membrane solubilization is roughly at CMC

Membrane Reconstitution

• Monolayer model systems provided insight how lipid components and physical parameters (pH, ion strength, T) influence one leaflet of a membrane

• Planar bilayer model systems provided insight into electrochemical aspects, transport events, and membrane protein behavior

• Curved, closed-in bilayer model systems (liposomes) provided insight mostly into membrane protein behavior

Lipid Monolayers

• Amphipathic molecules (lipids) line up at an air-water interface

• Access to inside of lipid bilayer• Control of composition and amount of lipid• Model system to study constraints of membrane

behavior by lipid components

A black film = lipid bilayer

Figure 3.18 Montal Mueller technique for making bilayers

Planar Lipid Bilayer

• Separation of two compartments

• Studies of electrical properties, transport, osmosis, and smaller proteins

• Electrophysiological studies per-patch clamp

Patch clamp technique

Liposomes

Variation in size and lamellar content

Liposomes

• Extrusion through polycarbonate filters (SUVs)

• Control of pore size influences vesicle size

• Purification by size exclusion chromatography

• Freeze-thaw/dialysis (LUVs)

• LUVs usefull for protein insertion (control of membrane curvature)