Using a recently described self-assembly process (Bayburt, T. H.; Grinkova, Y. V.; Sligar, S. G. Nano Letters 2002, 2, 853-856), we prepared soluble monodisperse discoidal lipid/protein particles with controlled size and composition, termed Nanodiscs, in which the fragment of dipalmitoylphosphatidylcholine (DPPC) bilayer is surrounded by a helical protein belt. We have customized the size of these particles by changing the length of the amphipathic helical part of this belt, termed membrane scaffold protein (MSP). Herein we describe the design of extended and truncated MSPs, the optimization of self-assembly for each of these proteins, and the structure and composition of the resulting Nanodiscs. We show that the length of the protein helix surrounding the lipid part of a Nanodisc determines the particle diameter, as measured by HPLC and small-angle X-ray scattering (SAXS). Using different scaffold proteins, we obtained Nanodiscs with the average size from 9.5 to 12.8 nm with a very narrow size distribution (+/-3%). Functionalization of the N-terminus of the scaffold protein does not perturb their ability to form homogeneous discoidal structures. Detailed analysis of the solution scattering confirms the presence of a lipid bilayer of 5.5 nm thickness in Nanodiscs of different sizes. The results of this study provide an important structural characterization of self-assembled phospholipid bilayers and establish a framework for the design of soluble amphiphilic nanoparticles of controlled size.

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.

Each species from bacteria to human has a distinct genetic fingerprint. Therefore, a mechanism that detects a single molecule of DNA represents the ultimate analytical tool. As a first step in the development of such a tool, we have explored using a nanometer-diameter pore, sputtered in a nanometer-thick inorganic membrane with a tightly focused electron beam, as a transducer that detects single molecules of DNA and produces an electrical signature of the structure. When an electric field is applied across the membrane, a DNA molecule immersed in electrolyte is attracted to the pore, blocks the current through it, and eventually translocates across the membrane as verified unequivocally by gel electrophoresis. The relationship between DNA translocation and blocking current has been established through molecular dynamics simulations. By measuring the duration and magnitude of the blocking current transient, we can discriminate single-stranded from double-stranded DNA and resolve the length of the polymer.