Reconstitution and imaging of a membrane protein in a nanometer-size phospholipid bilayer.
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.
Department of Biochemistry, University of Illinois, Urbana, Illinois, 61801, USA.
T H
TH
Bayburt
J W
JW
Carlson
S G
SG
Sligar
D000538:Aluminum Silicates / Q000378:metabolism; D016632:Apolipoprotein A-I / Q000378:metabolism; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism; D008075:Lipoproteins, HDL / Q000648:ultrastructure; D008565:Membrane Proteins / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force; D008862:Microsomes, Liver; D009247:NADH, NADPH Oxidoreductases / Q000648:ultrastructure*; D009251:NADPH-Ferrihemoprotein Reductase; D010743:Phospholipids / Q000378:metabolism; D013499:Surface Properties; D014357:Trypsin / Q000378:metabolism
D000538:Aluminum Silicates; D016632:Apolipoprotein A-I; D008051:Lipid Bilayers; D008075:Lipoproteins, HDL; D008565:Membrane Proteins; D010743:Phospholipids; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D014357:Trypsin; C011934:mica