Detergent-Free Incorporation of a Seven-Transmembrane Receptor Protein into Nanosized Bilayer Lipodisq Particles for Functional and Biophysical Studies

Abstract

SMA-Lipodisq nanoparticles, with one bacteriorhodopsin (bR) per 12 nm particle on average (protein/lipid molar ratio, 1:172), were prepared without the use of detergents. Using pulsed and continuous wave nitroxide spin label electron paramagnetic resonance, the structural and dynamic integrity of bR was retained when compared with data for bR obtained in the native membrane and in detergents and then with crystal data. This indicates the potential of Lipodisq nanoparticles as a useful membrane mimetic.

Bacterial Reaction Centers Purified with Styrene Maleic Acid Copolymer Retain Native Membrane Functional Properties and Display Enhanced Stability

“…A recently developed alternative is the use of an amphipathic styrene maleic acid (SMA) copolymer (Figure 1 A) that is able to remove the protein from a membrane with its associated lipids intact in the form of a protein/lipid nanodisc bound by the polymer. [2][3][4][5] This approach has been used to successfully solubilize membrane proteins from artificial liposomes [2,6] and native membranes, [7,8] and is one of a number of alternatives being developed for housing integral membrane proteins outside the native membrane. [9] Figure 1.…”

Effect of Polymer Composition and pH on Membrane Solubilization by Styrene-Maleic Acid Copolymers

“…This has opened options to purify membrane proteins that are unstable in detergent micelles and to study native protein-lipid interactions by biochemical methods. A further advantage of these so-called native nanodisks is that they are small, with sizes in the range of 10-25 nm (4)(5)(6)(9)(10)(11)(12)(13)(14)(15). This makes them suitable to be characterized by a variety of biophysical approaches including UV/Vis-and fluorescence spectroscopy (5,6,8), as well as light scattering techniques (6,14).…”

The Styrene-Maleic Acid Copolymer: A Versatile Tool in Membrane Research

“…However, it is also possible to insert membrane proteins into SMALPs, as was initially shown for PagP and bacteriorhodopsin (Knowles et al 2009;Orwick-Rydmark et al 2012) and later for the potassium channel modulator protein KCNE1 (Sahu et al 2013). To achieve this, proteins can either first be solubilized with detergent and then reconstituted into liposomes by conventional techniques or native membranes containing the protein can be supplemented with synthetic lipid after which SMA is added to obtain SMALPs (see e.g., Goddard et al 2015).…”

“…Although many reports suggest that the presence of incorporated proteins increases the particle size (Knowles et al 2009;Long et al 2013;Orwick-Rydmark et al 2012), a comparison of different studies does not show a clear systematic correlation of the disc size and the dimensions of the membranespanning domain of the protein (see Table 1). For instance, nanodiscs containing a KcsA tetramer with eight transmembrane (TM) helices have an average size of 10 nm, whereas the smaller bacteriorhodopsin (7 TM helices) yields slightly bigger particles with diameters of 12 nm.…”

“…For such proteins, a potential strategy to improve incorporation into nanodiscs might be the addition of synthetic DMPC lipid. This lipid is very efficiently solubilized by SMA (Scheidelaar et al 2015) and it has been used in early studies for assisted solubilization of proteins from native membranes (Knowles et al 2009;Orwick-Rydmark et al 2012). Reconstitution into DMPC liposomes also allowed solubilization of a protein of the β-barrel type (PagP) by SMA (Knowles et al 2009).…”