Supplementary Figure 9: Covalent circularization enhances thermal stability of MSPs and embedded VDAC-1.
From: Covalently circularized nanodiscs for studying membrane proteins and viral entry
Thermal unfolding of MSP1D1 (black) and cNW11 (red) without (a) and with lipids (b) followed by circular dichroism (CD) spectroscopy at 222 nm, the wavelength most characteristic of helical secondary structure. A lipid mixture of POPC:POPG at a molar ratio of 3:2 was used to generate nanodiscs. (c) Reconstitution into MSP1D1 nanodiscs increases the melting temperature, Tm, of VDAC1 by 9.2°C over that of VDAC1 in an LDAO micelle environment, and covalent circularization of the scaffold protein (cNW11) raises the Tm by an additional 9.2°C. Thermal unfolding of human VDAC-1 was followed by CD spectroscopy at 218 nm, the wavelength most characteristic of β-sheet secondary structure. Orange: VDAC1 in 0.1% LDAO, black: VDAC1 reconstituted into conventional nanodiscs (assembled using MSP1D1), and red: VDAC1 reconstituted into circularized nanodiscs (assembled using cNW11). Nanodiscs were made with a 3:2 POPC:POPG mixture. (d) Thermal unfolding of VDAC1 reconstituted into circularized nanodiscs (assembled using cNW9) followed by CD spectroscopy at 218 nm. Nanodiscs were made with POPC/POPG lipids at a molar ratio of 3:2. All samples were in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl. (e) Analysis of the VDAC1 nanodisc assembly reaction. Top: size-exclusion chromatography and negative-stain EM of VDAC1 in cNW9 nanodiscs. The negative-stain EM shows nanodiscs containing a single channel. The stain-filled channels appear as dark spots inside the nanodiscs. Bottom: SDS-PAGE analysis of the nanodisc assembly. Fractions 1-6 were collected and analyzed.
