Directed Self-Assembly of Monodisperse Phospholipid Bilayer Nanodiscs with Controlled Size

Abstract

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.

Structures of the calcium-activated, non-selective cation channel TRPM4

“…Briefly, TRPM4, MSP1 46 and lipid (POPC: POPG: POPE = 3:1:1) were mixed at a molar ratio of 1:4:30, and incubated on ice for 30 minutes. Detergents were removed by adding Bio-beads SM2 (Bio-Rad) to a concentration of 100 mg/ml followed by gentle agitation.…”

Coarse Grained Protein−Lipid Model with Application to Lipoprotein Particles

“…The diameters of the nanodiscs, 9.5 nm at 300 K, 10.5 nm at 323 K, and 10.6 nm at 353 K (as measured from the CG simulations) did, in fact, increase with temperature and correlated well with experimental SAXS measurements (see Table 4). 25,26 The gaps between the ends of membrane scaffold protein strands increase with temperature suggesting that perhaps disassembly of nanodiscs results from the loss of favorable salt-bridging (represented in our CG model through CG particle charges) between the two scaffold protein strands (Figure 5d, e, and f).…”

“…Membrane scaffold proteins are engineered based on the structure of the lipid binding domain of apo A-1. Nanodiscs have been well characterized 25,26,27 and in addition to acting as platforms for studying membrane proteins, their well-defined size and composition provide an ideal system for studying the HDL lipid-binding domain.…”

Cryo-EM structure of SNAP-SNARE assembly in 20S particle

“…Using the lipid nanodisc technology [29][30][31][32], we successfully reconstituted functional 20S particles with the transmembrane helix of SNARE complex's VAMP protein integrated into the nanodisc (Supplementary information, Figure S2A and S2B). These nanodisc-tethered 20S (ND-20S) particles behaved much better than mono-dispersed molecules under EM for both negatively stained and frozen-hydrated specimens (Figure 1A and Supplementary information, Figure S2C).…”