Assessing the size, stability, and utility of isotropically tumbling bicelle systems for structural biology

Abstract

Aqueous phospholipid mixtures that form bilayered micelles (bicelles) have gained wide use by molecular biophysicists during the past 20 years for spectroscopic studies of membrane-bound peptides and structural refinement of soluble protein structures. Nonetheless, the utility of bicelle systems may be compromised by considerations of cost, chemical stability, and preservation of the bicelle aggregate organization under a broad range of temperature, concentration, pH, and ionic strength conditions. In the current work, 31P nuclear magnetic resonance (NMR) and atomic force microscopy (AFM) have been used to monitor the size and morphology of isotropically tumbling small bicelles formed by mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (DIOMPC) with either 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) or 1,2-di-O-hexyl-sn-glycero-3-phospho-choline (DIOHPC), testing their tolerance of variations in commonly used experimental conditions. 1H-15N 2D NMR has been used to demonstrate the usefulness of the robust DMPC-DIOHPC system for conformational studies of a fatty acid-binding protein that shuttles small ligands to and from biological membranes.

Reconstitution of integral membrane proteins into isotropic bicelles with improved sample stability and expanded lipid composition profile

“…Additional minor lysolipid and fatty acid components indicate some lipid hydrolysis; however, they are still only minor components after 6-months. If hydrolysis is a serious problem, a bicelle with ether-linked lipids, such as DMPC/DIOHPC, can be utilized [9]. However, it is preferable to use ester-linked lipids, as using the non-native ether-lipids may alter the structure and dynamics of peptides in the membrane [10].…”

“…It is difficult to determine shape from DLS data [13] alone and difficult to interpret R H quantitatively. Electron microscopy [13] and AFM [9], have been used to visually assess the morphology of these mixtures. Discoidal structures were observed with both methods, with larger discs and a few wormlike micelles also being observed via AFM under more physiological conditions.…”

“…Improved bicelle stability would allow for more widespread use. Previous attempts at increasing bicelle stability and sample lifetime have included using ether-linked lipids and/or adding up to 10% of long- and short-chain lipids with charged headgroups such as PS and PE-DTPA [4, 8, 9]. However, recent studies have indicated that non-native ether-linked lipids alter the structure and dynamics of antimicrobial peptides [10], making this a less desirable substitution.…”

NMR of liquid crystals and micellar solutions

“…1 H-15 N 2D NMR has been used to demonstrate the usefulness of the robust DMPC-DIOHPC system for conformational studies of a fatty acid-binding protein that shuttles small ligands to and from biological membranes. 170 Other authors focused on the formation of bicelles from a mixture of DMPC and the chelator-lipid di myristoylphosphatidylethanolamine-diethylenetriaminepentaacetate (DMPE-DTPA) with complexed lanthanides, either thulium (Tm 3 þ ) or lanthanum (La 3 þ ). The system was studied with SANS in a magnetic field, cryo-TEM, and 31 P NMR spectroscopy.…”

Ice breaking in GPCR structural biology

“…The other methods, such as the newly developed bicelles, nanodiscs or lipidic cubic phase (LCP), seek to trap each membrane protein molecule within a lipid bilayer before crystallization and take advantage of a native-like environment to preserve their integrity [62] . Compared to the in surfo method, the bicelle method uses a lipid bilayer of finite size and maintains the ability to diffuse in three dimensions in the process of forming a three-dimensional lattice ( Figure 4A).…”