Covalently circularized nanodiscs for studying membrane proteins and viral entry

Abstract

We engineered covalently circularized nanodiscs (cNDs) which, compared with standard nanodiscs, exhibit enhanced stability, defined diameter sizes and tunable shapes. Reconstitution into cNDs enhanced the quality of nuclear magnetic resonance spectra for both VDAC-1, a β-barrel membrane protein, and the G-protein-coupled receptor NTR1, an α-helical membrane protein. In addition, we used cNDs to visualize how simple, nonenveloped viruses translocate their genomes across membranes to initiate infection.

Figure 1

Producing covalently circularized NW11 and nanodiscs. (a) A general outline of the constructs that are used for making covalently circularized nanodiscs. (b) Outline of the procedure for creating circularized proteins over a Cu²⁺ chip. Immobilizing NW11 on the Cu²⁺ chip for circularization reduces the chances for head-to-tail linkage of two neighboring NW11 molecules and also offers a quick reaction time. (c) SDS-PAGE analysis of NW11 before (lane 1) and after (lane 2) circularization. (d) MS/MS spectrum of a tryptic peptide of cNW11 confirming the ligation of the N-terminal residues (GSTFSK) to the C-terminal LPGTG motif. The b and y ions that were identified in the MS/MS spectrum are highlighted in blue and red. MS/MS and intact mass data for other NWs constructs (cNW9, cNW30, cNW50) are provided in Supplementary Table 1 and Supplementary Figs. 1–3. (e) Diameter distribution for nanodiscs made using circularized NW11 (top) and non-circularized NW11 (bottom) and representative negative-stain EM images. In the box-and-whisker plots, center lines show the means; box limits indicate the 25th and 75th percentiles; whiskers g o down to 5 percentile and up to 95 percentile. Raw data (jittered along x for clarity) are shown next to its representative plot. There is less variance in the lengths of cNW11 compared to NW11 nanodiscs (p < 0.001). Diameter distributions for NW30, cNW30 and cNW50 nanodiscs are provided in Supplementary Fig. 4. Scale bars, 100 nm (e).

Figure 2

Analysis of membrane proteins in different size nanodiscs. (a) 2D ¹H-¹⁵N TROSY HSQC spectrum of monomeric [U-²H,¹⁵N] labeled VDAC-1 in cNW9 nanodiscs. (b) Representative image of negatively stained cNW9 nanodiscs containing single VDAC-1 channel (see also supplementary Fig. 11). The stain-filled channels appear as dark spot inside the nanodisc. Right: SDS-PAGE analysis of cNW9 nanodiscs containing VDAC-1. (c) 2D ¹H-¹⁵N TROSY HSQC spectrum of dimeric [U-²H,¹⁵N] labeled VDAC-1 in cNW11 nanodiscs. (d) Representative image of negatively stained cNW11 nanodiscs containing two VDAC-1 channels. Right: SDS-PAGE analysis of cNW11 nanodiscs containing VDAC-1. (e) ¹H-¹⁵N-TROSY HSQC spectrum of 40uM ¹⁵N-labeled NTR1 in cNW9 nanodisc acquired at 45°C on a Bruker 800MHz spectrometer. (f) Superimposed ¹H-¹⁵N-TROSY HSQC spectra of 40uM ¹⁵N-labeled NTR1 in cNW9 nanodisc before (red) and after (blue) addition of purified heterotrimeric G protein composed of Gα_i1, Gβ₁, and Gγ₁. The integrity of the cNW9 nanodiscs was not affected even though the G protein was added with a small amount of DDM (the final DDM concentration was less than 10% of its critical micellar concentration (CMC)). Scale bars, 50 nm (b, d).

Figure 3

Poliovirus caught in the act. (a) Negative-stain EM of 50-nm circularized nanodiscs plus poliovirus. A control (nanodiscs without CD155) is shown to illustrate the relative dimensions of the 30-nm poliovirus and the 50-nm nanodisc. (b) Outline of the procedure used to initiate poliovirus bridging and fusion with nanodiscs decorated with CD155. (c) Negative-stain EM images showing individual viruses tethered to nanodiscs. (d) Cryo-EM image of 50-nm nanodiscs plus poliovirus. (e) Cryo-EM image showing a tilted view of 50-nm nanodisc. (f) Cryo-EM images showing individual viruses tethered to nanodiscs. (g, h) Cryo-EM images showing the creation of a putative pore in the nanodisc by the poliovirus. (i) Cryo-EM image showing three viral particles around a 15-nm nanodisc. (j) Cryo-EM image showing individual viruses ejecting RNA after incubation with CD155-decorated 15-nm nanodiscs. Scale bars, 100 nm (a, d, e, f, j) and 50 nm (g, h, i).