RESUMEN
Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties. Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-ß-D-maltoside ('C12-b-M') as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors and the human Patched protein receptor by approximately 10°C compared to C12-b-M. Another surfactant yielded the highest stabilization of the human Patched protein receptor compared to C12-b-M (13°C) but was inferior for the G protein-coupled receptors. In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b(6 )f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M.
Asunto(s)
Cristalización/métodos , Proteínas de la Membrana/química , Tensoactivos/química , Agua/química , Chlamydomonas reinhardtii/química , Complejo de Citocromo b6f/química , Glucósidos/química , Humanos , Receptores Patched , Receptores de Superficie Celular/química , Receptores Acoplados a Proteínas G/química , SolubilidadRESUMEN
Neutron reflection has been used to study the interaction of cationic lipoplexes with different model membrane systems. The model membranes used are prepared as "floating" phospholipid bilayers deposited at a silicon/water interface and separated from the solid substrate either by an adsorbed phospholipid bilayer, polymer cushions composed of polyethylene glycol lipids, or a lipid monolayer adsorbed onto a chemically grafted hydrocarbon layer. The cationic lipoplexes studied are those formed by the complexation of calf thymus DNA with dimethyl-dioctadecylammonium bromide (DDAB), with either cholesterol or dioleoyl-L-alpha-phosphatidylethanolamine (DOPE) incorporated as "helper" lipid. The cationic lipoplexes are found to destroy three of the four types of (negatively charged) floating bilayers, with the rate of destruction dependent on the nature of the layer separating the floating bilayer from the silicon substrate. The only bilayers to remain intact after exposure to the lipoplexes were those fabricated above the chemically grafted (octadecyl) hydrocarbon layer. This supports the hypothesis that the high negative charge density of the SiO2 layer on the silicon surface may influence, by way of electrostatic interaction with the cationic lipid, the interaction of the lipoplexes with the model bilayer. It is concluded that the floating bilayer supported on a chemically grafted hydrocarbon layer lends itself perfectly to the study of lipoplex-membrane interactions and, with sufficient exposure time, would allow a detailed characterization of the structures formed at the membrane interface during the interaction.
Asunto(s)
Membrana Dobles de Lípidos/química , Adsorción , Animales , Bovinos , ADN/metabolismo , Membrana Dobles de Lípidos/metabolismo , Difracción de Neutrones , Fosfatidilcolinas/química , Polietilenglicoles/química , Dióxido de Silicio/química , Propiedades de SuperficieRESUMEN
We describe methods that have been developed within the ILL-EMBL Deuteration Laboratory for the production of maltose binding protein (MBP) that has been selectively labelled either with deuterated tryptophan or deuterated methionine (single labelling), or both (double labelling). MBP is used as an important model system for biophysical studies, and selective labelling can be helpful in the analysis of small-angle neutron scattering (SANS) data, neutron reflection (NR) data, and high-resolution neutron diffraction data. The selective labelling was carried out in E. coli high-cell density cultures using auxotrophic mutants in minimal medium containing the required deuterated precursors. Five types of sample were prepared and studied: (1) unmodified hydrogenated MBP (H-MBP), (2) perdeuterated MBP (D-MBP), (3) singly labelled MBP with the tryptophan residues deuterated (D-trp MBP), (4) singly labelled MBP with methionine residues deuterated (D-met MBP) and (5) doubly labelled MBP with both tryptophan and methionine residues deuterated (D-trp/met MBP). Labelled samples were characterised by size exclusion chromatography, gel electrophoresis, light scattering and mass spectroscopy. Preliminary small-angle neutron scattering (SANS) experiments have also been carried out and show measurable differences between the SANS data recorded for the various labelled analogues. More detailed SANS experiments using these labelled MBP analogues are planned; the degree to which such data could enhance structure determination by SANS is discussed.
Asunto(s)
Proteínas Portadoras/química , Deuterio/química , Metionina/química , Difracción de Neutrones/métodos , Dispersión del Ángulo Pequeño , Triptófano/química , Proteínas de Unión a Maltosa , Unión Proteica , Coloración y Etiquetado/métodosRESUMEN
Troponin is a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle by participating in a series of conformational events within the actin-based thin filament. Troponin is a heterotrimeric complex consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT). Ternary troponin complexes have been produced by assembling recombinant chicken skeletal muscle TnC, TnI and the C-terminal portion of TnT known as TnT2. A full set of small-angle neutron scattering data has been collected from TnC-TnI-TnT2 ternary complexes, in which all possible combinations of the subunits have been deuterated, in both the +Ca2+ and -Ca2+ states. Small-angle X-ray scattering data were also collected from the same troponin TnC-TnI-TnT2 complex. Guinier analysis shows that the complex is monomeric in solution and that there is a large change in the radius of gyration of TnI when it goes from the +Ca2+ to the -Ca2+ state. Starting with a model based on the human cardiac troponin crystal structure, a rigid-body Monte Carlo optimization procedure was used to yield models of chicken skeletal muscle troponin, in solution, in the presence and in the absence of regulatory calcium. The optimization was carried out simultaneously against all of the scattering data sets. The optimized models show significant differences when compared to the cardiac troponin crystal structure in the +Ca2+ state and provide a structural model for the switch between +Ca2+ and -Ca2+ states. A key feature is that TnC adopts a dumbbell conformation in both the +Ca2+ and -Ca2+ states. More importantly, the data for the -Ca2+ state suggest a long extension of the troponin IT arm, consisting mainly of TnI. Thus, the troponin complex undergoes a large structural change triggered by Ca2+ binding.