Polymer nanodiscs: Advantages and limitations

Abstract

There is considerable interest in the development of membrane mimetics to study the structure, dynamics and function of membrane proteins. Polymer nanodiscs have been useful as a membrane mimetic by not only providing a native-like membrane environment, but also have the ability to extract the desired membrane protein directly from the cell membrane. In spite of such great potential, polymer nanodiscs have their disadvantages including lack of size control and instability at low pH and with divalent metals. In this review, we discuss how these limitations have been overcome by simple modifications of synthetic polymers commonly used to form nanodiscs. Recently, size control has been achieved using an ethanolamine functionalization of a low molecular weight polymer. This size control enabled the use of polymer-based lipid-nanodiscs in solution NMR and macro-nanodiscs in solid-state NMR applications. The introduction of quaternary ammonium functional groups has been shown to improve the stability in the presence of low pH and divalent metal ions, forming highly monodispersed nanodiscs. The polymer charge has been shown to play a significant role on the reconstitution of membrane proteins due to the high charge density on the nanodisc's belt. These recent developments have expanded the applications of polymer nanodiscs to study the membrane proteins using wide variety of techniques including NMR, Cryo-EM and other biophysical techniques.

Keywords: Membrane proteins; Polymer charge; Polymer nanodiscs; Size control; Solid-state NMR; pH resistant.

Figure 1.. Characterization SMA- EA:DMPC nanodisc:

A) Reaction scheme used for the synthesis of SMA-EA. b) Characterization of SMA-EA by ¹³C CP- MAS NMR: SMA (black) and SMA- EA (red); spectra were obtained under 8 kHz spinning speed. TEM images of nanodiscs obtained from DMPC:SMAEA (1:1 w/w) (d) and DMPC:SMAEA( 1:3 w/w) (e). (f) Normalized Static light scattering showing the solubilization of large DMPC MLVs in to smaller particles after the addition of SMA-EA polymer, inset showing the increase in the transparency of the solution. (g) Schematic showing the formation of nanodiscs. (h) ³¹P NMR spectra showing the appearance of an isotropic peak after the polymer addition indicating the formation of small size nanodiscs that tumble fast on the NMR time scale. (i) TIRF images showing the solubilization of 1 mol % of rhodamine- functionalized DMPE (1,2- Dimyristoyl- sn- glycero- 3- phosphoethanolamine) containing DMPC MLVs by the addition of the SMA-EA polymer: before (left) and after (right) the addition of SMA-EA. (This Figure was adapted with permission from Ravula et al., 2017b)

Figure 2.. pH resistant polymer nanodiscs.

(a) Reaction scheme used in the synthesis of SMA-QA. (b) Characterization of SMA-QA using ¹³C CP-MAS NMR experiment: SMA (black) and SMA-QA (red). (c) SLS profile showing the solubilization of DMPC MLVs as a function of the added polymer concentration. (d) Size exclusion chromatograms (SEC) showing the size variance with respect to polymer:lipid ratio. (e) DLS profiles of the nanodiscs made from different polymer:lipid ratios. SLS profiles showing the stability of nanodiscs against pH (f) and divalent metal ions (g). (h-k) TEM images of nanodiscs that were prepared from the indicated polymer to lipid ratio. (This Figure was adapted with permission from Ravula et al., 2018c).

Figure 3.. Schematic overview of polymer nanodiscs.

Nanodiscs are formed spontaneously after the addition of the polymer. The size of nanodiscs can be controlled by changing the lipid: polymer ratio. The small nanodiscs were found to be isotropic and therefore can be used in solution NMR experiments, whereas the large nanodiscs called as macro-nanodiscs align in the magnetic field enabling structural studies on membrane proteins using solid state NMR spectroscopy.