Membrane phospholipids as regulators of tetraspanin oligomerisation

Tetraspanins are integral membrane proteins that play a key role in organising multi-molecular complexes. Tetraspanin CD81 is involved in cellular processes such as cell adhesion, cell proliferation, and mediating infection of medically important pathogens, including the hepatitis C virus. Despite these important roles, the comprehensive structural organisation, membrane distribution, and CD81 interaction with surrounding proteins and lipids are not known. To enable these studies, CD81 was solubilised and purified from Pichia pastoris membranes in poly (styrene-co-maleic-acid) lipid particles or SMALPs, to retain its surrounding membrane environment. Biophysical characterisation was conducted by circular dichroism spectroscopy and antigen-antibody ELISA. This indicated that SMALP-CD81 retains its secondary structure and is functionally stable, even at higher temperatures, in marked contrast with detergent-purified CD81. Subsequently, gel filtration conditions have been optimised to isolate functionally active SMALP-CD81 fractions. The native CD81 membrane distribution in HEK 293 and Huh-7 cell-lines was also studied using electron microscopy (EM). The EM images of cell sections indicated that CD81 is organised in isolated monomers as well as in clusters of potentially higher-order structures in both cell-lines. This data agrees with the general consensus that tetraspanins exist as tetraspanin-rich microdomains that modulate the function of interacting proteins

GPCRs in the round:SMA-like copolymers and SMALPs as a platform for investigating GPCRs

G-protein-coupled receptors (GPCRs) are the largest family of membrane proteins, regulate a plethora of physiological responses and are the therapeutic target for 30–40% of clinically-prescribed drugs. They are integral membrane proteins deeply embedded in the plasma membrane where they activate intracellular signalling via coupling to G-proteins and β-arrestin. GPCRs are in intimate association with the bilayer lipids and that lipid environment regulates the signalling functions of GPCRs. This complex lipid ‘landscape’ is both heterogeneous and dynamic. GPCR function is modulated by bulk membrane properties including membrane fluidity, microdomains, curvature, thickness and asymmetry but GPCRs are also regulated by specific lipid:GPCR binding, including cholesterol and anionic lipids. Understanding the molecular mechanisms whereby GPCR signalling is regulated by lipids is a very active area of research currently. A major advance in membrane protein research in recent years was the application of poly(styrene-co-maleic acid) (SMA) copolymers. These spontaneously generate SMA lipid particles (SMALPs) encapsulating membrane protein in a nano-scale disc of cell membrane, thereby removing the historical need for detergent and preserving lipid:GPCR interaction. The focus of this review is how GPCR-SMALPs are increasing our understanding of GPCR structure and function at the molecular level. Furthermore, an increasing number of ‘second generation’ SMA-like copolymers have been reported recently. These are reviewed from the context of increasing our understanding of GPCR molecular mechanisms. Moreover, their potential as a novel platform for downstream biophysical and structural analyses is assessed and looking ahead, the translational application of SMA-like copolymers to GPCR drug discovery programmes in the future is considered

Detergent-Free Membrane Protein Purification Using SMA Polymer

One of the big challenges for the study of structure and function of membrane proteins is the need to extract them from the membrane. Traditionally this was achieved using detergents which disrupt the membrane and form a micelle around the protein, but this can cause issues with protein function and/or stability. In 2009 an alternative approach was reported, using styrene maleic acid (SMA) copolymer to extract small discs of lipid bilayer encapsulated by the polymer and termed SMALPs (SMA lipid particles). Since then this approach has been shown to work for a range of different proteins from many different expression systems. It allows the extraction and purification of a target protein while maintaining a lipid bilayer environment. Recently this has led to several new high-resolution structures and novel insights to function. As with any method there are some limitations and issues to be aware of. Here we describe a standard protocol for preparation of the polymer and its use for membrane protein purification, and also include details of typical challenges that may be encountered and possible ways to address those

CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups

Tetraspanins exert a wide range of cellular functions of broad medical importance. Despite this, their biophysical characteristics are incompletely understood. Only two high-resolution structures of full-length tetraspanins have been solved. One is that of human CD81, which is involved in the infectivity of human pathogens including influenza, HIV, the malarial plasmodium parasite and hepatitis C virus (HCV). The CD81 crystal structure identifies a cholesterol-binding pocket, which has been suggested to be important in the regulation of tetraspanin function. Here we investigate the use of styrene-maleic anhydride co-polymers (SMA) for the solubilisation and purification of CD81 within a lipid environment. When CD81 was expressed in the yeast Pichia pastoris, it could be solubilised and purified using SMA2000. This SMALP-encapsulated CD81 retained its native folded structure, as determined by the binding of two conformation-sensitive anti-CD81 antibodies. Analysis by size exclusion chromatography revealed two distinct populations of CD81, only one of which bound the HCV glycoprotein, E2. Optimization of expression and buffer conditions increased the proportion of E2-binding competent CD81 protein. Mass spectrometry analysis indicated that the lipid environment surrounding CD81 is enriched with negatively charged lipids. These results establish a platform to study the influence of protein-lipid interactions in tetraspanin biology

Detergent-Free Membrane Protein Purification Using SMA Polymer

One of the big challenges for the study of structure and function of membrane proteins is the need to extract them from the membrane. Traditionally this was achieved using detergents which disrupt the membrane and form a micelle around the protein, but this can cause issues with protein function and/or stability. In 2009 an alternative approach was reported, using styrene maleic acid (SMA) copolymer to extract small discs of lipid bilayer encapsulated by the polymer and termed SMALPs (SMA lipid particles). Since then this approach has been shown to work for a range of different proteins from many different expression systems. It allows the extraction and purification of a target protein while maintaining a lipid bilayer environment. Recently this has led to several new high-resolution structures and novel insights to function. As with any method there are some limitations and issues to be aware of. Here we describe a standard protocol for preparation of the polymer and its use for membrane protein purification, and also include details of typical challenges that may be encountered and possible ways to address those