ABSTRACT
Self-assembled phospholipid bilayer Nanodiscs have become an important and versatile tool among model membrane systems to functionally reconstitute membrane proteins. Nanodiscs consist of lipid domains encased within an engineered derivative of apolipoprotein A-1 scaffold proteins, which can be tailored to yield homogeneous preparations of disks with different diameters, and with epitope tags for exploitation in various purification strategies. A critical aspect of the self-assembly of target membranes into Nanodiscs lies in the optimization of the lipid:protein ratio. Here we describe strategies for performing this optimization and provide examples for reconstituting bacteriorhodopsin as a trimer, rhodopsin, and functionally active P-glycoprotein. Together, these demonstrate the versatility of Nanodisc technology for preparing monodisperse samples of membrane proteins of wide-ranging structure.
Subject(s)
Lipid Bilayers/chemistry , Membrane Proteins/chemistry , Models, Biological , Nanostructures/chemistry , Phospholipids/chemistry , ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry , Animals , Bacteriorhodopsins/chemistry , Crystallography, X-Ray , Mice , Phosphatidylcholines/chemistryABSTRACT
A phospholipid bilayer of nanometer dimension has been used as a support 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 demonstration of this methodology, we formed the nanobilayers in the presence of hepatic microsomal NADPH-cytochrome P450 reductase. Incubation of a solution of enzyme-containing nanobilayers with a freshly cleaved mica substrate resulted in the spontaneous formation of a fully oriented supported monolayer of discoidal phospholipid domains. The P450-reductase in the oriented monolayer retains its catalytic activity. Characterization by scanning force microscopy revealed isolated single-membrane proteins that could be stably imaged over time. These results define a novel technique for the study of single-membrane proteins in a bilayer environment.
