Reconstitution of Membrane Proteins into Platforms Suitable for Biophysical and Structural Analyses

Abstract

Integral membrane proteins have historically been challenging targets for biophysical research due to their low solubility in aqueous solution. Their importance for chemical and electrical signaling between cells, however, makes them fascinating targets for investigators interested in the regulation of cellular and physiological processes. Since membrane proteins shunt the barrier imposed by the cell membrane, they also serve as entry points for drugs, adding pharmaceutical research and development to the interests. In recent years, detailed understanding of membrane protein function has significantly increased due to high-resolution structural information obtained from single-particle cryo-EM, X-ray crystallography, and NMR. In order to further advance our mechanistic understanding on membrane proteins as well as foster drug development, it is crucial to generate more biophysical and functional data on these proteins under defined conditions. To that end, different techniques have been developed to stabilize integral membrane proteins in native-like environments that allow both structural and biophysical investigations-amphipols, lipid bicelles, and lipid nanodiscs. In this chapter, we provide detailed protocols for the reconstitution of membrane proteins according to these three techniques. We also outline some of the possible applications of each technique and discuss their advantages and possible caveats.

Keywords: Amphipol; Bicelles; Lipids; Membrane protein biophysics; Membrane proteins; Membrane scaffold; Nanodisc; Reconstitution.

Figure 1:. Reconstitution of purified membrane proteins into native-like environments.

Outline of the possibilities to transfer purified, integral membrane proteins solubilized in detergent into more native-like environments. For each platform exemplary applications are listed.

Figure 2:. Characteristics of detergents, amphipols and bicelles.

Chemical characteristics of hydrophilic headgroups and hydrophobic tails are present in detergent as shown for (A) n-Decyl-α-D-Maltopyranoside - DM, (B) Lauryl Maltose Neopentyl Glycol - LMNG, and amphipols as shown for (C) amphipol A8-35, and (D) PMAL-C12. Chemical structures were adapted from www.anatrace.com. (E) Cartoon of a lipid bicelle with a protein (KcsA ion channel, PDB: 1BL8) incorporated into a lipid bicelle. Long-chain lipids in blue, short-chain lipids in orange, KcsA in magenta surface representation

Figure 3:. Structural features of nanodiscs.

(A) Gel filtration profiles (Superose 6 16/600) used to assess the reconstitution quality of the cyclic nucleotide-gated K⁺ channel SthK into nanodiscs formed with different MSPs. Molar ratios of SthK:MSP2N2:POPG 1:1:125 (black) and SthK:MSP1E3:POPG 1:1:75 (blue) are shown. The bigger MSP (MSP2N2) shows an increased peak for empty nanodiscs. For the smaller MSP1E3, the SDS-PAGE clearly resolves the assembly of nanodiscs with aggregated SthK in the void peak (1), a peak for SthK inserted in MSP1E3 in nanodiscs (2) and empty nanodiscs (3). (B) Uranyl-acetate negative stain EM image of SthK in MSP1E3 (peak 2 in panel (A)) recorded on a JEM-1400 with 100 kV and a magnification of 150000. (C) Apolipoprotein A-I (PDB: 2N5E[49]) is shown in stick representation (colored by atom) and (D) colored by the surface electrostatics. (E) top view and (F) side view of the GTPase K-RAS4B tethered to an apolipoprotein A-I nanodisc (PDB: 2MSD[50]) with the nanodisc colored by surface electrostatics, lipids shown in stick representation and the GTPase as cartoon. (G) Top view and (H) side view of the density from single particle cryoEM of a ligand-gated ion channel (yellow, EMDB: 7484) incorporated into MSP1E3 nanodiscs (blue)[51]. Panels C-F were prepared using Pymol (www.pymol.org), panels (G) and (H) were prepared using UCSF Chimera[52].