The effect of using binary mixtures of zwitterionic and charged lipids on nanodisc formation and stability

Abstract

Nanodiscs are self-assembled $10 nm particles composed of lipid bilayer patches, stabilized by helical amphipathic belt proteins. The size, monodispersity and well-defined structure make the nanodiscs a popular model for the biological cell membrane, especially for structural and functional studies of membrane proteins. The structures and properties of nanodiscs made of zwitterionic lipids are well known. However, the biological cell membrane is negatively charged and thus nanodiscs containing anionic lipids should provide a better mimic of the native environment for membrane proteins. Despite the broad potential of charged nanodiscs, a systematic study of the influence of charged lipids on the nanodisc structure and stability has not yet been accomplished. In this paper, binary systems of zwitterionic DMPC mixed with the anionic lipids DMPG or DMPA or with the cationic synthetic DMTAP are used to prepare negatively and positively charged nanodiscs, respectively. Size exclusion chromatography analysis shows that nanodiscs can be prepared with high yield at all compositions of DMPC and DMPG, while mixtures of DMPC with either DMPA or DMTAP impair nanodisc formation. The presence of DMPG improves the stability of the nanodisc, both thermally and over time upon storage at À20 C, as compared to pure DMPC nanodiscs. This stabilization is attributed to favourable electrostatic interactions between the anionic head of DMPG and cationic charges of the belt protein and internanodisc repulsion that prevents aggregation of nanodiscs. In contrast, even small fractions of DMPA result in a faster degradation at À20 C. These results suggest that the mixing of DMPC and DMPG provides nanodiscs that are better suited for studies of the function and structure of membrane proteins not only due to their inherent charge but also due to their improved thermal and storage stability compared to pure DMPC nanodiscs.

Nanodiscs in Membrane Biochemistry and Biophysics

“…The stability of Nanodiscs with respect to thermal unfolding and prolonged storage was compared for discs with binary mixtures of zwitterionic DMPC, negatively charged DMPA or DMPG, and with the synthetic cationic lipid DMTAP. 189 A high yield of Nanodiscs could be obtained for all mixtures of DMPC and DMPG, but DMPA at higher than 25% fraction and DMTAP at more than 10%, destabilized Nanodiscs and precipitated at high concentrations. On the other hand, 25% DMPG stabilized Nanodiscs with respect to prolonged storage and to thermal denaturation probed as by DSC.…”

Structure and Dynamics of GPCRs in Lipid Membranes: Physical Principles and Experimental Approaches

“…This includes commonly used neutral detergents, as well as zwitterionic membrane lipids used in lipid nanodiscs or nanoparticles. With the ambition to mimic cellular membranes, it is reasonable to include negatively charged head groups in the membrane mimetic [ 88 , 89 ]. Ψ I comprises the electric potentials of the hydrocarbon core of the lipid membrane.…”

Self-assembly of polymer-encased lipid nanodiscs and membrane protein reconstitution

“…Thermal stability of nanodiscs is important for various applications including the biophysical and structural studies of membrane proteins [34,35] . Experimental studies have reported that peptide or MSP based nanodiscs can be stable up to ~ 60 C. [36] Thermal unfolding has been seen with further increase in temperature that destabilizes the nanodisc's size.…”