Self-Assembly of Discoidal Phospholipid Bilayer Nanoparticles with Membrane Scaffold Proteins

Abstract

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.

Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties

“…2a by using densitometry and standard curves of purified proteins for which concentrations had been determined by quantitative amino acid analysis. We could use the Tar-6H per scaffold protein ratio to calculate the Tar-6H per disk ratio for each purified preparation because of previous studies (12,13,16,17) of the properties of membrane scaffold proteins and the Nanodiscs formed by them. Determinations of lipid content, protein content, and diameters of Nanodiscs made with membrane scaffold proteins of different lengths, with or without inserted transmembrane protein, have been performed (12,13,16,17).…”

“…Here, we used Nanodiscs (Fig. 1b), an emerging technology for manipulating membrane proteins (12,13), to prepare small particles of lipid bilayer containing one or only a few chemoreceptor dimers ( Fig. 1 c-e).…”

Coarse Grained Protein−Lipid Model with Application to Lipoprotein Particles

“…28 To test the ability of the model to reproduce nanodisc assembly, we performed a series of simulations. The first self-assembly simulation, referred to as SimND, started from a system in which the scaffold proteins were left in a circular shape, but shifted from their equilibrium position (Figure 4) on the nanodisc.…”

“…28 The assembly process by which homogeneous nanodisc particles form is initiated by the removal of cholate from a starting mixture of cholate, scaffold protein, and lipids. Although the assembly mechanisms of native HDL particles and of nanodisc particles are different, the information gathered from studying the assembly of nanodiscs should shed light on HDL particle formation.…”

An amphipathic polypeptide derived from poly‐γ‐glutamic acid for the stabilization of membrane proteins

“…1,2 A nanometer-sized discoidal structure, nanodisc, consisting of lipids and membrane scaffold proteins, [3][4][5] and a disc-shaped lipid-detergent mixture, bicelle, [6][7][8] have been developed to stabilize membrane proteins. An alternative method is to use amphipathic polymers containing both of hydrophobic and hydrophilic groups.…”