Reconstitution of membrane proteins into vesicular membranes

Abstract

Membrane-associated functions such as energy transduction, transport of nutrients, recognition and transmission of external signals, and cell-cell interactions are catalysed by an appropriate specific protein(s) embedded in the membrane. These membrane proteins have been identified, solubilized and purified from membranes by various procedures as described in the preceding chapters. Their activities can be divided into two essential categories; one is non-vectorial catalytic activity such as electron transport, ATP hydrolysis and binding of substrates, and the other is vectorial activity such as translocation of ions and substrates across the membrane. The former is analysed with the respective proteins under conditions similar to those applied to soluble proteins when appropriate lipids or detergents are used. However, the latter requires a strictly enclosed membrane structure that partitions the aqueous internal space from its surroundings. To meet this requirement for isolated membrane proteins, they are introduced into lipid bilayer vesicles, liposomes, to form proteoliposomes. For example, a purified H+ -translocating ATPase from membranes of thermophilic bacteria, which catalyses ATP synthesis in oxidative phosphorylation, manifests its ATP hydrolysing activity when a small amount of phospholipids is added to form non-vesicular protein-lipid complexes. However, an ATP-dependent translocation of H+ can be observed only after reconstituting it into proteoliposomes.