The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy

Abstract

Despite the great progress recently made in resolving their structures, investigation of the structural biology of membrane proteins still presents major challenges. Even with new technical advances such as lipidic cubic phase crystallisation, obtaining well-ordered crystals remains a significant hurdle in membrane protein X-ray crystallographic studies. As an alternative, electron microscopy has been shown to be capable of resolving > 3.5 Å resolution detail in membrane proteins of modest (~ 300 kDa) size, without the need for crystals. However, the conventional use of detergents for either approach presents several issues, including the possible effects on structure of removing the proteins from their natural membrane environment. As an alternative, it has recently been demonstrated that membrane proteins can be effectively isolated, in the absence of detergents, using a styrene maleic acid co-polymer (SMA). This approach yields SMA lipid particles (SMALPs) in which the membrane proteins are surrounded by a small disk of lipid bilayer encircled by polymer. Here we use the Escherichia coli secondary transporter AcrB as a model membrane protein to demonstrate how a SMALP scaffold can be used to visualise membrane proteins, embedded in a near-native lipid environment, by negative stain electron microscopy, yielding structures at a modest resolution in a short (days) timeframe. Moreover, we show that AcrB within a SMALP scaffold is significantly more active than the equivalent DDM stabilised form. The advantages of SMALP scaffolds within electron microscopy are discussed and we conclude that they may prove to be an important tool in studying membrane protein structure and function.

Overcoming bottlenecks in the membrane protein structural biology pipeline

“…During the formation of SMALPs from a biological membrane, membrane proteins can become trapped inside these discs (as shown in Figure 1D), solubilising them into small particles (much like nanodiscs when using membrane scaffolding proteins), but without the need for detergent at any stage. The proteins within a SMALP can be effectively purfied using affinity chromatography [39,[42][43][44]. The particles are stable once formed so there is no need to supplement buffers during purification as there is when using detergents; this is a major cost advantage.…”

“…The use of SMALPs has facilitated improved thermostability and homogeneity of membrane proteins such as P-glycoprotein (P-gp) and the adenosine A2a receptor [39,42,44]. To date no crystal structure has been reported using SMALPs but both negative stain and cryo-EM low resolution structural maps have been determined [39,43]. Unlike MSP-nanodiscs, the polymer does not show significant extra-electron density; EM shows a very small annulus of lipids and polymer.…”

The potential use of single-particle electron microscopy as a tool for structure-based inhibitor design

“…New methodologies for extracting membrane proteins in more native states have been reported, for example styrene maleic acid lipid particles (SMALPs), which extract membrane proteins with their native lipids rather than in detergent micelles, and have been used for EM analysis (Postis et al, 2015;Lee et al, 2016). Furthermore, saposonin-lipoprotein nanoparticles have shown promise in high-resolution cryo-EM studies on membrane proteins (Frauenfeld et al, 2016).…”

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

“…The biological sources include bacteria (Dörr et al 2014;Paulin et al 2014;Postis et al 2015;Prabudiansyah et al 2015;Swainsbury et al 2014) and yeast (Gulati et al 2014;Jamshad et al 2015a;Long et al 2013;Skaar et al 2015) as well as cultures of insect (Gulati et al 2014) and human cells (Gulati et al 2014;Jamshad et al 2015a), which together account for all major biosystems that are used for recombinant MP production. The proteins that have thus been incorporated into native nanodiscs span a wide variety of MPs of different sizes (see e.g., forthcoming Table 1), ranging from those with a single membrane spanning α-helix (Paulin et al 2014) to oligomeric complexes comprising up to 36 transmembrane helices (Postis et al 2015). The extraction of MPs in native nanodiscs hence seems to be 1 3 independent from the host membrane and to be generically applicable to all members of this class of proteins.…”