Understanding the Structural Pathways for Lipid Nanodisc Formation: How Styrene Maleic Acid Copolymers Induce Membrane Fracture and Disc Formation

Abstract

Lipid nanodiscs formed by mixtures of styrene maleic acid (SMA) copolymers and lipid membranes are important tools for studying membrane proteins in many biotechnological applications. However, molecular interactions leading up to their formation are not well understood. Here, we elucidate the nanodisc formation pathways for SMA/lipid vesicle mixtures using small-angle X-ray scattering (SAXS) that allows detailed in situ nanostructural information. SMA copolymer that is initially aggregated in solution inserts its styrene units into the lipid bilayer hydrocarbon region, leading to fractures in the membrane. The initial copolymer–lipid interactions observed in the vesicles are also present in the formed discs, with excess copolymer distributed along the normal of the bilayer. The size and SMA distribution in the resulting discs strongly depend on the temperature, lipid/copolymer ratio, and lipid type. We find that the solubilization limit increases for membranes above the melting point, suggesting that defects in gel-like lipid membranes play a significant role in membrane fracturing and nanodisc formation. These findings provide unique insights into the formation of nanodiscs as well as into the microscopic mechanism of solubilization, which plays an important role in many applications and products ranging from household goods to biotechnology and medicine.

Mechanisms of Formation, Structure, and Dynamics of Lipoprotein Discs Stabilized by Amphiphilic Copolymers: A Comprehensive Review

“…Amphiphilic polymers form aggregates in aqueous solution. These aggregates correspond to globular, collapsed copolymer conformation identified for SMA by SAXS experiments [ 65 ] and coarse-grained simulations [ 40 ]. Nile red fluorescence experiments indicated that this conformation containing hydrophobic domains allows for the most effective solubilization as opposed to the random coil conformation [ 20 ].…”

“…Nile red fluorescence experiments indicated that this conformation containing hydrophobic domains allows for the most effective solubilization as opposed to the random coil conformation [ 20 ]. A number of factors may influence the morphology and the extensiveness of polymer aggregates such as the charge and concentration of polymers, the pH and ionic strength of the solution, and the degree of polymer blockiness [ 40 , 65 ]. In turn, all of these factors may eventually affect the effectiveness of solubilization [ 3 ].…”

Current Developments in Native Nanometric Discoidal Membrane Bilayer Formed by Amphipathic Polymers

“…On the other hand, the encapsulation event perturbs the membrane curvature (by forming a bulge) and planarity, and allows formation of toroidal pores [ 56 ] so that water and water-soluble molecules such as fluorescein can permeate inside ( Figure 6 A) Intriguingly, after encapsulation, the distribution of Na+ ions undergo remarkable changes, as well, and that compensates the repulsion between anionic carboxyl moieties in the lipid-water interface. Small angle X-ray scattering (SAXS) analysis unraveled the details of membrane (composed of either DMPC or POPC) fractionation pathways by SMA 3:1 [ 55 ] in which, two−four times more SMA(3:1) is required for lipids in the liquid crystalline phase than in the gel phase to get the lipid vesicles to form lipid-SMA nanodiscs. Under this condition, mixed lipid/SMA(3:1) vesicles coexist with nanodiscs.…”

“…In silico approaches have already been utilized to simulate the behavior of dendrimers [50], polymermediated fusion, micelle-lipid interfaces [51], and lipoprotein complexes [52]; therefore molecular dynamic (MD) simulations have proved to be useful to shed light on molecularscale interaction of SMA and solubilization of biomembrane [53,54]. Coarse-grained (CG) field molecular dynamics simulations and experimental data confirmed the self-aggregation of polyanionic SMA copolymers in solution resulting in globular aggregates [55]. Due to considerable affinity of polymer molecules to membrane (DDPC lipid molecules) that is driven by primary interaction of styrenes with hydrophobic acyl chains, SMA polymers spontaneously (within 20 msec of simulation) and cooperatively insert into adjacent lipid bilayer, bend the membrane at the site of adsorption, then slowly penetrate to the lipid bilayer and localize in the acyl chains of lipids apart from phosphate headgroups, hence leaving styrene groups in perpendicular orientation to lipid acyl chains.…”

Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells

“…It utilizes styrene maleic acid (SMA) copolymers to extract integral membrane proteins directly from the lipid bilayer, into nanodiscs with diameters ranging from approximately 10 to 30 nm, together with annular lipids ( Jamshad et al, 2015b ; Cuevas Arenas et al, 2016 ; Ravula et al, 2017 ; Overduin et al, 2021 ). Mechanistically, it has been proposed that SMA is aggregated in solution, disaggregating upon contact with membranes where hydrophobic styrenes insert into the core of the membrane leading to fracture and nanodisc formation ( Bjornestad et al, 2021 ). SMA extraction entirely negates the use of detergents while retaining proteins in their surrounding lipidic environment.…”