Incorporation and analysis of bilayer constituents in nanodiscs

Abstract

Nanodiscs, engineered lipoparticles composed of a lipid bilayer and two surrounding amphipathic proteins, have been utilized to house and solubilize integral membrane proteins and other molecules with large hydrophobic groups which limit their aqueous solubility. In the experiments described herein, lipids from the Nanodiscs’ bilayer, gadolinium chelates and integral membrane proteins were analyzed for their incorporation and stability within the Nanodisc structure. Four techniques are described for quantification of the exchange of lipids between Nanodiscs over time, including: scintillation counting, imaging proximity assay, NMR and TLC MALDI-MS. Additionally, the extent and longevity of inclusion of the hydrophobic gadolinium-chelate “C3lipo” doped into the Nanodisc bilayer was analyzed and preliminary cell uptake studies were performed. Finally, the incorporation of proteins from rat liver and fetal bovine brain membranes into Nanodiscs to create soluble membrane protein libraries is discussed.