An acid-compatible co-polymer for the solubilization of membranes and proteins into lipid bilayer-containing nanoparticles

The fundamental importance of membrane proteins in drug discovery has meant that membrane mimetic systems for studying membrane proteins are of increasing interest. One such system has been the amphipathic, negatively charged poly(styrene-co-maleic acid) (SMA) polymer to form “SMA Lipid Particles” (SMALPs) which have been widely adopted to solubilize membrane proteins directly from the cell membrane. However, SMALPs are only soluble under basic conditions and precipitate in the presence of divalent cations required for many downstream applications. Here, we show that the positively charged poly(styrene-co-maleimide) (SMI) forms similar nanoparticles with comparable efficiency to SMA, whilst remaining functional at acidic pH and compatible with high concentrations of divalent cations. We have performed a detailed characterization of the performance of SMI that enables a direct comparison with similar data published for SMA. We also demonstrate that SMI is capable of extracting proteins directly from the cell membrane and can solubilize functional human G-protein coupled receptors (GPCRs) expressed in cultured HEK 293T cells. “SMILPs” thus provide an alternative membrane solubilization method that successfully overcomes some of the limitations of the SMALP method

Differences in SMA-like polymer architecture dictate the conformational changes exhibited by the membrane protein rhodopsin encapsulated in lipid nano-particles

Membrane proteins are of fundamental importance to cellular processes and nano-encapsulation strategies that preserve their native lipid bilayer environment are particularly attractive for studying and exploiting these proteins. Poly(styrene-co-maleic acid) (SMA) and related polymers poly(styrene-co-(N-(3-N′,N′-dimethylaminopropyl)maleimide)) (SMI) and poly(diisobutylene-alt-maleic acid) (DIBMA) have revolutionised the study of membrane proteins by spontaneously solubilising membrane proteins direct from cell membranes within nanoscale discs of native bilayer called SMA lipid particles (SMALPs), SMILPs and DIBMALPs respectively. This systematic study shows for the first time, that conformational changes of the encapsulated protein are dictated by the solubilising polymer. The photoactivation pathway of rhodopsin (Rho), a G-protein-coupled receptor (GPCR), comprises structurally-defined intermediates with characteristic absorbance spectra that revealed conformational restrictions with styrene-containing SMA and SMI, so that photoactivation proceeded only as far as metarhodopsin-I, absorbing at 478 nm, in a SMALP or SMILP. In contrast, full attainment of metarhodopsin-II, absorbing at 382 nm, was observed in a DIBMALP. Consequently, different intermediate states of Rho could be generated readily by simply employing different SMA-like polymers. Dynamic light-scattering and analytical ultracentrifugation revealed differences in size and thermostability between SMALP, SMILP and DIBMALP. Moreover, encapsulated Rho exhibited different stability in a SMALP, SMILP or DIBMALP. Overall, we establish that SMA, SMI and DIBMA constitute a ‘toolkit’ of solubilising polymers, so that selection of the appropriate solubilising polymer provides a spectrum of useful attributes for studying membrane proteins

A method for detergent-free isolation of membrane proteins in their local lipid environment.

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

Challenges of objective structured clinical examination as a tool in medical assessment

Medical education is witnessing changes across the globe to produce more competent and responsive medical graduates to meet patients' growing needs. Medical educators are aspiring for more objective and relevant assessment methods for the evaluation of medical graduates. Objective structured clinical examination (OSCE) introduced in the year 1975 for clinical evaluation has gone through many changes over the past 45 years and matured over time. The article describes the challenges of OSCE as a tool in medical assessment from students' and organizers' perspectives. We have also suggested a feasible solution to address the challenges while conducting OSCE to assess medical students

Structural analysis of a nanoparticle containing a lipid bilayer used for detergent-free extraction of membrane proteins

In the past few years there has been a growth in the use of nanoparticles for stabilizing lipid membranes that contain 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 previously described the use of an amphipathic polymer (poly(styrene-co-maleic acid), SMA) to produce discoidal nanoparticles with a lipid bilayer core containing the 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 unencapsulated 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 the structure of the SMALP forms the foundation for future development and applications of SMALPs in membrane protein production and analysis.[Figure not available: see fulltext.] © 2015, Tsinghua University Press and Springer-Verlag Berlin Heidelberg.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

BTN3A1 Discriminates γδ T Cell Phosphoantigens from Nonantigenic Small Molecules via a Conformational Sensor in Its B30.2 Domain

Human Vγ9/Vδ2 T-cells detect tumour cells and microbial infections by recognising small phosphorylated prenyl metabolites termed phosphoantigens (P-Ag). The type-1 transmembrane protein Butyrophilin 3A1 (BTN3A1) is critical to the P-Ag-mediated activation of Vγ9/Vδ2 T-cells, however, the molecular mechanisms involved in BTN3A1-mediated metabolite sensing are unclear, including how P-Ag are discriminated from non-antigenic small molecules. Here, we utilised NMR and X-ray crystallography to probe P-Ag sensing by BTN3A1. Whereas the BTN3A1 Immunoglobulin Variable domain failed to bind P-Ag, the intracellular B30.2 domain bound a range of negativelycharged small molecules, including P-Ag, in a positively-charged surface pocket. However, NMR chemical shift perturbations indicated BTN3A1 discriminated P-Ag from non-antigenic small molecules by their ability to induce a specific conformational change in the B30.2 domain that propagated from the P-Ag binding site to distal parts of the domain. These results suggest BTN3A1 selectively detects P-Ag intracellularly via a conformational antigenic sensor in its B30.2 domain, and have implications for rational design of antigens for Vγ9/Vδ2 -based T-cell immunotherapies

Structure and function of BamE within the outer membrane and the β-barrel assembly machine

Insertion of folded proteins into the outer membrane of Gram-negative bacteria is mediated by the essential β2-barrel assembly machine (Bam). Here, we report the native structure and mechanism of a core component of this complex, BamE, and show that it is exclusively monomeric in its native environment of the periplasm, but is able to adopt a distinct dimeric conformation in the cytoplasm. BamE is shown to bind specifically to phosphatidylglycerol, and comprehensive mutagenesis and interaction studies have mapped key determinants for complex binding, outer membrane integrity and cell viability, as well as revealing the role of BamE within the Bam complex

Genética molecular del virus del síndrome reproductor y respiratorio porcino : aspectos evolutivos, diagnósticos e inmunógenos

El síndrome reproductor y respiratorio porcino esta producido por un virus arn, encuadrado en una reciente familia: arteriviridae. Se han desarrollado tres objetivos en este trabajo: 1/ el desarrollo y puesta a punto de una técnica rt-pcr para el diagnostico directo de la enfermedad; 2/ la expresión de la proteína codificada por la orf-5 vírica tanto en sistemas procariotas como eucariotas; 3/ el estudio de variabilidad existente entre cepas europeas del virus. Los resultados del primer objetivo indican que la técnica puesta a punto es altamente sensible y especifica, permitiendo la detección de aproximadamente 100 di50ct en muestras de campo. Con respecto al segundo objetivo, se ha logrado la caracterización de la antes mencionada proteína, así como la purificación parcial de ella. Por ultimo, los estudios de variabilidad demostraron una elevada semejanza entre las cepas analizadas, aunque si bien mayor entre las de un mismo paí