RESUMO
Electron crystallography is well suited for studying the structure of membrane proteins in their native lipid bilayer environment. This technique relies on electron cryomicroscopy of two-dimensional (2D) crystals, grown generally by reconstitution of purified membrane proteins into proteoliposomes under conditions favoring the formation of well-ordered lattices. Growing these crystals presents one of the major hurdles in the application of this technique. To identify conditions favoring crystallization a wide range of factors that can lead to a vast matrix of possible reagent combinations must be screened. However, in 2D crystallization these factors have traditionally been surveyed in a relatively limited fashion. To address this problem we carried out a detailed analysis of published 2D crystallization conditions for 12 ß-barrel and 138 α-helical membrane proteins. From this analysis we identified the most successful conditions and applied them in the design of new sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Using these matrices we have run 19 crystallization screens for 16 different membrane proteins totaling over 1300 individual crystallization conditions. Six membrane proteins have yielded diffracting 2D crystals suitable for structure determination, indicating that these new matrices show promise to accelerate the success rate of membrane protein 2D crystallization.
Assuntos
Proteínas de Membrana/química , Cristalização , Detergentes/química , Concentração de Íons de Hidrogênio , Lipídeos/químicaRESUMO
Various nanostructures were fabricated by ion irradiation on large area (100) Si surfaces covered by colloidal Langmuir-Blodgett films as nanolithographic masks. The ordered structure of the Langmuir-Blodgett monolayer composed from spherical Stöber silica particles of 200 nm and 450 nm diameter offer the possibility to form local surface swelling patterns during the ion bombardment step. Utilizing the dependence of the surface morphology on the irradiation parameters the tunability of nanostructuring was studied for 40 keV Ar+ and 500 keV Xe2+ ions. We show that the periodicity of the resulted surface pattern is determined by the size of the masking particles, while the height of nanostructures can be tuned by the ion fluence. The quality of projection of the nanomask contours to the substrate-the contrast of masking-can be set by choosing appropriate ion energy, thereby determining the curvature of the surface pattern. Moreover, deformation of the nanomask due to ion-nanoparticle interactions should be taken into account since these effects can be also utilized for tailoring various structures. The silica masking layers before and after ion irradiation and the resulting Si surface patterns were investigated by field emission scanning electron microscopy and atomic force microscopy analysis.
RESUMO
Ion beam mixing has been used to produce a silicon carbide (SiC)-rich nanolayer for protective coating. Different C/Si/C/Si/C/Si(substrate) multilayer structures (with individual layer thicknesses falling in the range of 10-20 nm) have been irradiated by Ar+ and Xe+ ions at room temperature in the energy and fluence ranges of 40-120 keV and 1-6 × 1016 ion/cm2, respectively. The effects of ion irradiation, including the in-depth distribution of the SiC produced, was determined by Auger electron spectroscopy depth profiling. The thickness of the SiC-rich region was only some nanometers, and it could be tailored by changing the layer structure and the ion irradiation conditions. The corrosion resistance of the layers was investigated by potentiodynamic electrochemical test in 4 M KOH solution. The measured corrosion resistance of the SiC-rich layers was orders of magnitude better than that of pure silicon, and a correlation was found between the corrosion current density and the effective areal density of the SiC.
RESUMO
To design custom magnetic nanostructures, it is indispensable to acquire precise knowledge about the systems in the nanoscale range where the magnetism forms. In this paper we present the effect of a curved surface on the evolution of magnetism in ultrathin iron films. Nominally 70 Å thick iron films were deposited in 9 steps on 3 different types of templates: (a) a monolayer of silica spheres with 25 nm diameter, (b) a monolayer of silica spheres with 400 nm diameter and (c) for comparison a flat silicon substrate. In situ iron evaporation took place in an ultrahigh vacuum chamber using the molecular beam epitaxy technique. After the evaporation steps, time differential nuclear forward scattering spectra, grazing incidence small angle X-ray scattering images and X-ray reflectivity curves were recorded. In order to reconstruct and visualize the magnetic moment configuration in the iron cap formed on top of the silica spheres, micromagnetic simulations were performed for all iron thicknesses. We found a great influence of the template topography on the onset of magnetism and on the developed magnetic nanostructure. We observed an individual magnetic behaviour for the 400 nm spheres which was modelled by vortex formation and a collective magnetic structure for the 25 nm spheres where magnetic domains spread over several particles. Depth selective nuclear forward scattering measurements showed that the formation of magnetism begins at the top region of the 400 nm spheres in contrast to the 25 nm particles where the magnetism first appears in the region where the spheres are in contact with each other.
RESUMO
A new isotope-assisted cross-relaxation editing experiment, [1H-13C]DINE-NOESY[1H-15N]HSQC (DINE=Double INEPT Edited), is proposed. It is based on the selective inversion of CH/CH3 or CH2 protons in the middle of the mixing time. The experiment sorts out the spin diffusion paths according to the principal mediators, either the CH/CH3 or the CH2 protons. This is useful in the structure refinement process, as it enables proper alignment of the aliphatic protons in the vicinity of NH protons.
Assuntos
Conformação Proteica , Proteínas/química , Sequência de Aminoácidos , Isótopos de Carbono , Humanos , Hidrogênio , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Modelos Teóricos , Ubiquitinas/químicaRESUMO
We have analyzed cross-relaxation in fractionally deuterated molecules and showed that the full matrix analysis fails except when the dilution is extreme. This is because the isotopic dilution alters the matrix exponential relationship between the observed spectrum and the cross-relaxation rate constants sought. Consequently, an average of the spectra of various isotopomers differs from the matrix exponential of an average relaxation matrix. We have derived a series expansion that allows the determination of the cross-relaxation rate constants in arbitrarily deuterated molecules.
RESUMO
The solution structures of staphylococcal nuclease (nuclease) H124L and its ternary complex, (nuclease-H124L).pdTp.Ca2+, were determined by ab initio dynamic simulated annealing using 1925 NOE, 119 phi, 20 chi 1 and 112 hydrogen bond constraints for the free protein, and 2003 NOE, 118 phi, 20 chi 1 and 114 hydrogen bond constraints for the ternary complex. In both cases, the final structures display only small deviations from idealized covalent geometry. In structured regions, the overall root-mean-square deviations from mean atomic coordinates are 0.46 (+/- 0.05) A and 0.41 (+/- 0.05) A for the backbone heavy atoms of nuclease and its ternary complex, respectively. The backbone conformations of residues in the loop formed by Arg81-Gly86, which is adjacent to the active site, are more precisely defined in the ternary complex than in unligated nuclease. Also, the protein side chains that show NOEs and evidence for hydrogen bonds to pdTp (Arg35, Lys84, Tyr85, Arg87, Tyr113, and Tyr115) are better defined in the ternary complex. As has been observed previously in the X-ray structures of nuclease-WT, the binding of pdTp causes the backbone of Tyr113 to change from an extended to a left-handed alpha-helical conformation. The NMR structures reported here were compared with available X-ray structures: nuclease-H124L [Truckses et al. (1996) Protein Sci., 5, 1907-1916] and the ternary complex of wild-type staphylococcal nuclease [Loll and Lattman (1989) Proteins Struct. Funct. Genet., 5, 183-201]. Overall, the solution structures of nuclease-H124L are consistent with these crystal structures, but small differences were observed between the structures in the solution and crystal environments. These included differences in the conformations of certain side chains, a reduction in the extent of helix 1 in solution, and many fewer hydrogen bonds involving side chains in solution.
Assuntos
Cálcio/química , Nuclease do Micrococo/química , Nucleotídeos de Timina/química , Simulação por Computador , Cristalografia por Raios X , Variação Genética , Ligação de Hidrogênio , Nuclease do Micrococo/antagonistas & inibidores , Nuclease do Micrococo/genética , Modelos Moleculares , Conformação Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade da EspécieRESUMO
A method for quantitative determination of magnetization exchange rate constants (cross-relaxation and chemical exchange) from a series of two-dimensional exchange spectra is presented. The method, the least error matrix analysis (LEMA), combines a series of full matrix calculations at different mixing times in a least-squares manner. LEMA embodies the principal advantages of full-relaxation matrix analysis (FMA) and initial rate buildup (BU) analysis. Like FMA, it takes into account all the relations among the spectral matrix elements and in analogy to BU makes use of their time evolution. By means of calculations, simulations, and experiments, we have shown that LEMA provides the dynamic matrix from a given set of experimental data with errors that are smaller than in either FMA or BU calculations.