RECONSTITUTION AND IMAGING OF A MEMBRANE-PROTEIN IN A NANOMETER-SIZE PHOSPHOLIPID-BILAYER

A phospholipid bilayer of nanometer dimension has been used as a suppo rt for the study of reconstituted functional single-membrane proteins. This nanobilayer consists of an approximately 10-nm-diameter circular phospholipid domain stabilized by apolipoprotein A1. As a demonstrati on of this methodology, we formed the nanobilayers in the presence of hepatic microsomal NADPH-cytochrome P450 reductase. Incubation of a so lution of enzyme-containing nanobilayers with a freshly cleaved mica s ubstrate resulted in the spontaneous formation of a fully oriented sup ported monolayer of discoidal phospholipid domains. The P450-reductase in the oriented monolayer retains its catalytic activity. Characteriz ation by scanning force microscopy revealed isolated single-membrane p roteins that could be stably imaged over time. These results define a novel technique for the study of single-membrane proteins in a bilayer environment. (C) 1998 Academic Press.