Nanoparticulate phospholipid bilayer disks were assembled from phospholipid and a class of amphipathic helical proteins termed membrane scaffold proteins (MSP). Several different MSPs were produced in high yield using a synthetic gene and a heterologous expression system and purified to homogeneity by a one-step purification. The self-assembly process begins with a mixture of the phospholipid and MSP in the presence of a detergent. Upon removal of detergent, 10-nm diameter particles form containing either saturated or unsaturated phospholipid. The ratio of components in the initial mixture was found to be crucial for formation of a monodisperse population of nanoparticles. Exploration of the phase diagram of the lamellar to phospholipid−detergent mixed micelle transition reveals that self-assembly proceeds from the mixed micellar phase. In this case a homogeneous and monodisperse population is formed. In contrast, particle formation from the detergent− phospholipid lamellar phase results in altered size, yield, composition, and heterogeneity of the resultant particles. The nanodisks contain approximately 160 saturated or 125 unsaturated lipids and can be formed from designed amphipathic r-helical scaffold proteins. The 10-nm particles can thus contain two molecules of MSP1 or a single molecule of an MSP1 fusion (MSP2). The phospholipid bilayer main phase transition temperature is preserved in the nanodisks as determined by fluorescence spectroscopy. Scanning probe microscopy shows a monolayer of nanodisks on a mica surface with a diameter of 10 nm and the thickness of a single phospholipid bilayer (5.7 nm), confirming the presence of a bilayer domain. The gentle method of self-assembly and robustness of the resulting nanodisks provides a means for generating soluble lipid bilayer membranes on the nanometer scale and opens the possibility of using these nanostructures to incorporate single membrane proteins into a native-like environment.

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 bacteriorhodpsin 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.

Nanodiscs are soluble nanoscale phospholipid bilayers which can self-assemble integral membrane proteins for biophysical, enzymatic or structural investigations. This means for rendering membrane proteins soluble at the single molecule level offers advantages over liposomes or detergent micelles in terms of size, stability, ability to add genetically modifiable features to the Nanodisc structure and ready access to both sides of the phospholipid bilayer domain. Thus the Nanodisc system provides a novel platform for understanding membrane protein function. We provide an overview of the Nanodisc approach and document through several examples many of the applications to the study of the structure and function of integral membrane proteins.

Nanodiscs are nanometer scale planar membranes of controlled size that are rendered soluble in aqueous solution via an encircling amphipathic membrane scaffold protein "belt" (Bayburt, T. H., Grinkova, Y. V., and Sligar, S. G. (2002) Nano. Lett. 2, 853-856). Integral membrane proteins can be self-assembled into the Nanodisc bilayer with defined stoichiometry, which allows an unprecedented opportunity to investigate the nature of the oligomerization state of a G-protein-coupled receptor and its coupling to heterotrimeric G-proteins. We generated Nanodiscs having one and two rhodopsins present in the 10-nmdiameter lipid bilayer domain. Efficient transducin activation and isolation of a high affinity transducin-metarhodopsin II complex was demonstrated for a monodisperse and monomeric receptor. A population of Nanodiscs containing two rhodopsins was generated using an increased ratio of receptor to membrane scaffold protein in the self-assembly mixture. The two-rhodopsin population was isolated and purified by density gradient centrifugation. Interestingly, in this case, only one of the two receptors present in the Nanodisc was able to form a stable metarhodopsin II-G-protein complex. Thus there is clear evidence that a monomeric rhodopsin is capable of full coupling to transducin. Importantly, presumably due to steric interactions, it appears that only a single receptor in the Nanodiscs containing two rhodopsins can interact with G-protein. These results have important implications for the stoichiometry of receptor-G-protein coupling and cross talk in signaling pathways.A growing pool of evidence suggests the facile homo-and heterodimerization of G-protein-coupled receptors (GPCRs) 2 with potential functional implications for cellular signaling. These conclusions are based largely on coimmunoprecipitation, resonance energy transfer between labeled GPCRs in cells, functional complementation, and in vitro analysis of isolated receptors (1). For example, purified leukotriene receptor BLT1 has been shown to exist as a dimer in detergent, which forms a pentameric complex of dimer with heterotrimeric G-protein and demonstrates cooperative interaction with ligand (2, 3). Oligomeric arrays of rhodopsin, a class A GPCR of the visual transduction system, have been observed in the atomic force microscope using native disk membranes (4 -6). Rhodopsin dimers have been observed in uranyl formate-stained preparations by electron microscopy (8) and have also been studied in detergent-solubilized preparations (9, 10). Recent evidence for self-association of rhodopsin in liposomes has been obtained by fluorescence techniques (11) consistent with earlier work (12). Other investigations, using detergents with varying properties, isolated and characterized rhodopsin in different oligomeric states, with the conclusion that oligomers activate transducin more efficiently (13). In contrast to these studies, rhodopsin has long been thought to function as a monomer based on extensive biophysical evidence, as reviewed in Ref. 14.To resolve t...

The rat mast cell line RBL-2H3.1 contains an 85-kDa cytosolic phospholipase A2 (cPLA2) that is very likely involved in liberating arachidonate from membrane phospholipid for the synthesis of eicosanoids following stimulation with either calcium ionophore or IgE/antigen. In this study, the intracellular location of cPLA2 was determined using immunofluorescence microscopy and immuno-gold electron microscopy. In nonstimulated cells, cPLA2 is distributed throughout the cytosol and is excluded from the nucleoplasm. Following cell activation with calcium ionophore, most of the cPLA2 translocates to the nuclear envelope, and the enzyme remains there during the entire period that ionophore is present. With IgE/antigen stimulation for 5 min, approximately 20-30% of the cPLA2 translocates to the nuclear envelope, and after 30 min of stimulation, most of the enzyme returns to the cytosol. Measurement of intracellular calcium using the dye Fura-2/AM shows that the level of calcium rises immediately after antigen is added, remains high for about 30 s, and then declines back to resting levels. Activation with calcium ionophore produces a 10-fold larger release of arachidonate than does stimulation with IgE/antigen. Thus, the results suggest that the extent of membrane binding of cPLA2 correlates with the release of arachidonate and that the site of arachidonate liberation is the nuclear envelope where many of the enzymes that oxygenate this fatty acid are located.