Preparation of Lipid Nanodiscs with Lipid Mixtures.

Lipid nanodiscs provide a native-like lipid environment for membrane proteins, and they have become a valuable platform for the study of membrane biophysics. A range of biophysical and biochemical analyses are enabled when membrane proteins are captured in lipid nanodiscs. Two parameters that can be controlled when capturing membrane proteins in lipid nanodiscs are the radius, and hence the surface area of the lipid surface, and the composition of the lipid bilayer.

Conformational dynamics of P-glycoprotein in lipid nanodiscs and detergent micelles reveal complex motions on a wide time scale.

P-glycoprotein (P-gp) is a highly substrate-promiscuous efflux transporter that plays a critical role in drug disposition. P-gp utilizes ATP hydrolysis by nucleotide-binding domains (NBDs) to drive transitions between inward-facing (IF) conformations that bind drugs and outward-facing (OF) conformations that release them to the extracellular solution. However, the details of the protein dynamics within either macroscopic IF or OF conformation remain uncharacterized, and the functional role of local dynamics has not been determined.

Differential Coupling of Binding, ATP Hydrolysis, and Transport of Fluorescent Probes with P-Glycoprotein in Lipid Nanodiscs.

The ATP binding cassette transporter P-glycoprotein (ABCB1 or P-gp) plays a major role in cellular resistance to drugs and drug interactions. Experimental studies support a mechanism with nucleotide-dependent fluctuation between inward-facing and outward-facing conformations, which are coupled to nucleotide hydrolysis. However, detailed insight into drug-dependent modulation of these conformational ensembles is lacking.

Applications of Lipid Nanodiscs for the Study of Membrane Proteins by Surface Plasmon Resonance.

Methods for the initial steps of surface plasmon resonance analysis of membrane proteins incorporated in lipid nanodiscs are described. Several types of Biacore sensor chips are available and require distinct strategies to immobilize proteonanodiscs on the chip surface. The procedures for immobilization on three of these chips (NTA, antibody coupled CM5, and L1) are described in this unit and results are demonstrated for a model system with cytochrome P4503A4 (CYP3A4) in nanodiscs binding to a polyclonal anti-CYP3A4 antibody.