Chapter 11 - Reconstitution of membrane proteins in phospholipid bilayer nanodiscs

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.

Figure 1

Structure of Nanodiscs, modeled with POPC as lipid. Lipid bilayer fragment (white space filling) is encircled by two amphipathic helices of MSP (gray ribbon). The graphic was generating using the PyMOL Molecular Graphics system.

Figure 2

Number of DPPC molecules per Nanodisc experimentally determined using tritiated lipids. Panel A, number of lipids in Nanodiscs formed with extended MSP proteins. Panel B, number of lipids in Nanodiscs formed with truncated MSP proteins. For the description of MSP constructs see Table I.

Figure 3

Elution profile from Nanodisc reconstitutions. Panel A, elution profile of MSP1E3 bR trimer Nanodiscs after assembly. After detergent removal the sample was injected onto a Superdex 200 prep grade column at a flow rate of 0.5 mL/min. The main peak corresponds to Nanodiscs containing three bR. Panel B, elution profile of MSP1E3 rhodopsin Nanodisc assembly mixture produced from solubilized rod outer segments. The sample was injected onto a Superdex 200 HR 10/30 column run at a flow rate of 0.5 mL/min.

Figure 4

Crystal structure of mouse P-gp (PDB: 3G5U) in the nucleotide-free state, as seen from the plane of the membrane (Aller et al., 2009). The TMDs are embedded in the membrane, while the NBDs protrude into the interior of the cell. The graphic was generated using the PyMOL Molecular Graphics system.

Figure 5

ATPase activity of P-gp in MSP1E3D1 Nanodiscs as compared to proteoliposomes. Squares represent activity of P-gp in MSP1E3D1 Nanodiscs and circles represent activity in liposomes. Open symbols show basal activity in the absence of drug and filled symbols show activity in the presence of 50 μM nicardipine.