One-third of eukaryotic proteins are integrated within membranes, as are the targets of 40% of approved drugs. However, the lack of a general means of solubilizing, stabilizing and structurally characterizing active membrane proteins has frustrated efforts to understand their mechanisms and exploit their potential value. Here we report that bilayer disks formed by phospholipids and styrene maleic anhydride copolymer preserve the functional and structural integrity of alpha-helical and beta-barrel transmembrane proteins. They form 11 nm particles that are monodispersed, biocompatible, thermostable, and water-soluble, allowing diverse membrane proteins to be simply and rapidly presented for virtually any in vitro analysis.

Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.

The folding of transmembrane proteins into the outer membrane presents formidable challenges to Gram-negative bacteria. These proteins must migrate from the cytoplasm, through the inner membrane and into the periplasm, before being recognized by the beta-barrel assembly machinery, which mediates efficient insertion of folded beta-barrels into the outer membrane. Recent discoveries of component structures and accessory interactions of this complex are yielding insights into how cells fold membrane proteins. Here, we discuss how these structures illuminate the mechanisms responsible for the biogenesis of outer membrane proteins.

In the past few years there has been a growth in the use of nano-particles for stabilizing lipid membranes with embedded proteins. These bionanoparticles provide a solution to the challenging problem of membrane protein isolation by maintaining a lipid bilayer essential to protein integrity and activity. We have described the use of an amphipathic polymer (Poly(styrene-co-maleic acid); SMA) to produce discoidal nanoparticles that contain a lipid bilayer with embedded protein. However the structure of the nanoparticle itself has not yet been determined. This leaves a major gap in understanding how the SMA stabilizes the encapsulated bilayer and how the bilayer relates physically and structurally to an unecapsulated lipid bilayer. In this paper we address this issue by describing the structure of the SMA Lipid Particle (SMALP) using data from small angle neutron scattering (SANS), electron microscopy (EM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC) and nuclear magnetic resonance spectroscopy (NMR). We show that the particle is disc shaped containing a polymer “bracelet” encircling the lipid bilayer. The structure and orientation of the individual components within the bilayer and polymer are determined showing that styrene moieties within SMA intercalate between the lipid acyl chains. The dimensions of the encapsulated bilayer are also determined and match those measured for a natural membrane. Taken together, the description of structure of the SMALP forms the foundation of future development and applications of SMALPs in membrane protein production and analysis.

Vg9Vd2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vg9Vd2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vg9Vd2 TCR-mediated responses. Surface plasmon resonance experiments established Vg9Vd2 TCRs used germline-encoded Vg9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vg9Vd2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vg9Vd2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.