We have developed the Nanodisc system, a soluble nanobilayer for studying protein-membrane interactions while allowing strict control over local membrane composition [4
]. Nanodiscs consist of a nanoscale discoidal bilayer ringed and stabilized by “Membrane Scaffold Protein” (MSP). Nanodiscs self-assemble from mixtures of phospholipid, MSP and detergent [5
], yielding monodisperse preparations whose bilayer size and phospholipid composition are under strict experimental control. The physical properties of Nanodisc bilayers have been extensively documented and are comparable to conventional liposomes, including phospholipid bilayer thickness, surface area, phase transition temperature, metal ion interactions and ability to support protein-membrane interactions [4
]. A “standard” Nanodisc encompasses an ~8 nm-diameter phospholipid bilayer (with ~70 phospholipids per leaflet), while longer versions of MSP have been engineered to allow formation of larger Nanodiscs [5
Integral membrane proteins included in Nanodisc self-assembly reactions embed into these nanoscale bilayers just as they do in conventional liposomes [6
]. We successfully incorporated TF into Nanodiscs in which we varied the bilayer composition from 0 to 90% PS, with the balance being phosphatidylcholine (PC). TF:FVIIa complexes were assembled on these nanobilayers and had catalytic activities very similar to TF:FVIIa on conventional liposomes [15
]. In surface plasmon resonance studies using Nanodiscs, we found that the affinity of FX for membranes increased monotonically with PS content, reaching maximal affinities at about 80% PS. The number of FX binding sites per nanobilayer also increased with PS content, with one FX binding site per 7 to 8 PS molecules. This is consistent with the notion that FX binds to a nanocluster of PS molecules on the bilayer surface.
Maximal rates of FX activation by TF:FVIIa on Nanodiscs required about 70% PS [15
]; by comparison, TF:FVIIa complexes on liposomes require ~30% PS for maximal activity [16
]. This is also consistent with the notion that FX activation by TF:FVIIa occurs preferentially on PS-rich nanoclusters on the membrane surface. Solution-phase FX has been proposed to bind directly to TF:FVIIa; alternatively, membrane-bound FX has been proposed to laterally diffuse (or skip) on the membrane surface in order to encounter TF:FVIIa [3
]. The Nanodisc surface can bind at most 5 or 6 FX molecules, which the TF:FVIIa complex will activate to FXa within a few seconds. However, we observed linear rates of FX activation over 20-minute time courses [15
], demonstrating that TF:FVIIa is not dependent on a large, preexisting pool of membrane-bound FX substrate.