Rapid preparation of nanodiscs for biophysical studies

Abstract

Nanodiscs, which are disc-shaped entities that contain a central lipid bilayer encased by an annulus of amphipathic helices, have emerged as a leading native-like membrane mimic. The current approach for the formation of nanodiscs involves the creation of a mixed-micellar solution containing membrane scaffold protein, lipid, and detergent followed by a time consuming process (3-12 h) of dialysis and/or incubation with sorptive beads to remove the detergent molecules from the sample. In contrast, the methodology described herein provides a facile and rapid procedure for the preparation of nanodiscs in a matter of minutes (<15 min) using Sephadex® G-25 resin to remove the detergent from the sample. A panoply of biophysical techniques including analytical ultracentrifugation, dynamic light scattering, gel filtration chromatography, circular dichroism spectroscopy, and cryogenic electron microscopy were employed to unequivocally confirm that aggregates formed by this method are indeed nanodiscs. We believe that this method will be attractive for time-sensitive and high-throughput experiments.

Keywords: Analytical ultracentrifugation; Circular dichroism spectroscopy; Cryogenic electron microscopy; Dynamic light scattering; Nanodiscs; Reconstitution.

Figure 1.

Cartoon image of a nanodisc. Membrane scaffolding protein is shown in blue. Dimyristoylphosphatidylcholine lipid is shown in yellow.

Figure 2.

Pictorial representation of hypothetical nanodisc formation using Sephadex® G-25 resin.

Figure 3.

Elution profiles of mixed micellar solutions passed over Sephadex® G-25 resin. Spherical data points represent elution fractions. Red traces indicate the elution profile of detergent. Absorbances are normalized for facile viewing. 10 mM KH₂PO₄ pH 7.4, 50 mM Na₂SO₄ was used to equilibrate the column and collect fractions.

Figure 4.

Elution profiles of particles on a HiLoad™ 16/60 Superdex™ 200 column. vo is void volume and v_e is elution volume. 10 mM KH₂PO₄ pH 7.4, 50 mM Na₂SO₄ was used to equilibrate the column and collect fractions.

Figure 5.

DLS autocorrelation functions, R_h, and ${\mu }_2/{\overline{\Gamma }}^2$ values of the two highest intensity fractions from the Superdex™ 200 elution profile of particles produced on the Sephadex® G-25 column using 1× phosphate-buffered saline (PBS). Data was collected for 5 mins at 25° C using values of viscosity and refractive index of 1.0200 cP and 1.335 respectively for PBS.[35]

Figure 6.

Sedimentation equilibrium profiles of particles produced on the Sephadex® G-25 column using sodium cholate (a-c), CHAPS (d-f), and octyl glucoside (g-i) in 10 mM KH₂PO₄ pH 7.4, 50 mM Na₂SO₄ at 25°C. Residuals are displayed underneath each equilibrium profile.

Figure 7.

Circular dichroism spectra of particles in 10 mM KH₂PO₄ pH 7.4, 50 mM Na2SO4 at 25°C. [Θ] is mean residue ellipticity.

Figure 8.

Cryogenic electron microscopy images of particles produced using Sephadex® G-25 resin with 10 mM KH₂PO₄ pH 7.4, 50 mM Na₂SO₄.

Figure 9.

Particle reconstruction images of particles produced using Sephadex® G-25 resin with 10 mM KH₂PO₄ pH 7.4, 50 mM Na₂SO₄.