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 >3.5Å 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.

Keywords: AcrB; Electron microscopy; Membrane protein; SMALP.

Graphical abstract

Fig. 1

(A) Biochemical characterisation of purified AcrB SMALPs. Coomassie blue-stained SDS-polyacrylamide gel of purified SMALPs. The mobilities of marker proteins of known molecular mass are shown on the left. (B) Representative fluorescence polarization of AcrB SMALP with R6G. Binding isotherm of AcrB SMALP with R6G, shows a KD of 52 ± 6.6 nM, in buffer containing 50 mM Tris (pH 8) 10 mM NaCl and 5% glycerol.

Fig. 2

(A) Representative negative stain AcrB classes. The classes are predominantly of a “side” view, equivalent to that viewed from the plane of the bilayer, with an example of a “base” view from the cytoplasmic surface of the protein shown in the far right panel (vii). B) Representative AcrB singlet classum on the left, with some of the raw particles which make up the class shown. C) Classes of the AcrB trimer doublets (top panel), with the orientations shown below (bottom panel) using the AcrB reconstruction. Scale bar represents 15 nm.

Fig. 3

Sedimentation velocity AUC profiles of AcrB SMALP at 10 mM and 500 mM NaCl in 50 mM Tris pH 8. Two distinctive populations are seen, indicating AcrB SMALP as singlet and doublet trimers, with sedimentation coefficients of 4.8 and 8.5 which correspond to Molecular Masses of 405 kDa and 810 kDa of the particles, respectively.

Fig. 4

(A) Surface view of the AcrB reconstruction. Clear 3-fold symmetry can be seen from the base ((B) (cytoplasmic face)) and top ((D) (periplasmic face)) of the structure. Fitting of the AcrB crystal structure (PDB ID : 1IWG; [24]) into the reconstruction showing the excellence of fit as seen from the side (E) and top (F). Extra density can be seen surrounding the transmembrane domain of the AcrB structure, which is accounted for by the SMA/phospholipid envelope.