title: A step-by-step method for the reconstitution of an ABC transporter into nanodisc lipid particles.
abstract: The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia.
authors.forename: Huan
authors.initials: H
authors.lastname: Bao
authors.forename: Franck
authors.initials: F
authors.lastname: Duong
authors.forename: Catherine S
authors.initials: CS
authors.lastname: Chan
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D004586:Electrophoresis; D029968:Escherichia coli Proteins / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D008027:Light; D008055:Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D049329:Nanostructures / Q000737:chemistry*; D033902:Periplasmic Binding Proteins / Q000737:chemistry; D012542:Scattering, Radiation
chemical_list: D018528:ATP-Binding Cassette Transporters; D029968:Escherichia coli Proteins; D008055:Lipids; C461619:MalE protein, E coli; D008565:Membrane Proteins; D033902:Periplasmic Binding Proteins; C461599:maltose transport system, E coli