RESUMEN
We report the preparation and performance of TEM grids bearing stabilized nonfouling lipid monolayer coatings. These films contain NTA capture ligands of controllable areal density at the distal end of a flexible poly(ethylene glycol) 2000 (PEG2000) spacer to avoid preferred orientation of surface-bound histidine-tagged (His-tag) protein targets. Langmuir-Schaefer deposition at 30 mN/m of mixed monolayers containing two novel synthetic lipids-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(5-amido-1-carboxypentyl)iminodiacetic acid]polyethylene glycolamide 2000) (NTA-PEG2000-DSPE) and 1,2-(tricosa-10',12'-diynoyl)-sn-glycero-3-phosphoethanolamine-N-(methoxypolyethylene glycolamide 350) (mPEG350-DTPE)-in 1:99 and 5:95 molar ratios prior to treatment with a 5 min, 254 nm light exposure was used for grid fabrication. These conditions were designed to limit nonspecific protein adsorption onto the stabilized lipid coating by favoring the formation of a mPEG350 brush layer below a flexible, mushroom conformation of NTA-PEG2000 at low surface density to enable specific immobilization and random orientation of the protein target on the EM grid. These grids were then used to capture His6-T7 bacteriophage and RplL from cell lysates, as well as purified His8-green fluorescent protein (GFP) and nanodisc solubilized maltose transporter, His6-MalFGK2. Our findings indicate that TEM grid supported, polymerized NTA lipid monolayers are capable of capturing His-tag protein targets in a manner that controls their areal densities, while efficiently blocking nonspecific adsorption and limiting film degradation, even upon prolonged detergent exposure.
Asunto(s)
Microscopía por Crioelectrón/instrumentación , Histidina/química , Ácido Nitrilotriacético/química , Oligopéptidos/química , Fosfatidiletanolaminas/química , Polietilenglicoles/química , Proteínas Recombinantes de Fusión/química , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Adsorción , Bacteriófago T7/química , Extractos Celulares/química , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Expresión Génica , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Histidina/genética , Microscopía Electrónica de Transmisión/instrumentación , Oligopéptidos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Propiedades de SuperficieRESUMEN
The synthesis and assembly properties of a new water-soluble deep-cavity cavitand are discussed. For a homologous series of alkanes, the host can form a range of approximately isoenergetic 1:1, 2:1, and 2:2 complexes. As a result of this 'confluence' of binding and assembly the host displays an unusual, nonmonotonic, assembly profile. Thus, no or limited assembly is observed with methane through butane, pentane triggers assembly, and hexane through octane again does not promote assembly, whereas nonane and a larger guest again induce assembly. This unusual behavior is discussed in the context of the diversity of nodes of chemical systems (networks).
Asunto(s)
Alcanos/química , Conformación Molecular , Solubilidad , Estereoisomerismo , Agua/químicaRESUMEN
We explored surface charge transfer interaction between a family of phthalocyanine (Pc) compounds and transition metal dichalcogenides (TMDs). Comparing the device characteristics of TMD field-effect transistors (FETs), we demonstrate both p-type and n-type doping of TMDs by tuning the work function of the metal substitution in the Pc compound. Our findings suggest a near linear correlation between the metal work function and doping level. Such doping predictability has yet to be achieved whereas we provide here the first report of its kind.
RESUMEN
We report the fabrication of transmission electron microscopy (TEM) grids bearing graphene oxide (GO) sheets that have been modified with Nα, Nα-dicarboxymethyllysine (NTA) and deactivating agents to block non-selective binding between GO-NTA sheets and non-target proteins. The resulting GO-NTA-coated grids with these improved antifouling properties were then used to isolate His6-T7 bacteriophage and His6-GroEL directly from cell lysates. To demonstrate the utility and simplified workflow enabled by these grids, we performed cryo-electron microscopy (cryo-EM) of His6-GroEL obtained from clarified E. coli lysates. Single particle analysis produced a 3D map with a gold standard resolution of 8.1 Å. We infer from these findings that TEM grids modified with GO-NTA are a useful tool that reduces background and improves both the speed and simplicity of biological sample preparation for high-resolution structure elucidation by cryo-EM.