Heterogeneous structure of silk fibers from Bombyx mori resolved by 13C solid-state NMR spectroscopy

The molecular conformation of silk fibrion is characterized by solid-state 13C NMR before spinning (silk I structure) and after spinning (silk II structure). We compare native silk fibers with the quasi-crystalline Cp-fraction and a synthetic model peptide (Ala-Gly)15, both of which can be converted either into silk I by dialysis from 9 M LiBr or into silk II by treatment with formic acid. Our results demonstrate that silk II fibers are intrinsically heterogeneous, consisting of beta-sheets, distorted beta-turns, and distorted beta-sheets. This higher-order heterogeneity is revealed by the 13C-NMR Cbeta-peak of Ala, indicating that the Ala side chains are stacked partially in parallel and partially face-to-face, at a ratio of 1:2.     

DEMONSTRATION THAT PHOSPHORESCENT 6-BROMO-2-NAPHTHYL SULFATE CAN BE USED TO PROBE HEME ACCESSIBILITY IN HEME PROTEINS

The phosphorescence properties of 6-bromo-2-naphthyl sulfate (BNS) in aqueous solution were studied. The phosphorescence lifetime is several hundred microseconds and is self-quenched. Although a fluorescent photoproduct is formed from BNS, it does not interfere with the decay properties of triplet-state BNS and its utility as a probe of the accessibility of the heme group in heme proteins. Quenching of BNS phosphorescence does not occur for the non-heme protein lysozyme and apomyoglobin but occurs by a dynamic mechanism with a quenching constant of 1-2 x 10(9) M-1 s-1 for cytochrome c and myoglobin and with a quenching constant of 6.2 x 10(9) M-1 s-1 for protoporphyrin IX. The phosphorescence of an inclusion complex of 1-bromonaphthalene and beta-cyclodextrin is not quenched by heme-containing proteins. The temperature and viscosity dependencies of the rate with which BNS phosphorescence is quenched by microperoxidase-11 are consistent with unit quenching efficiency. These results indicate that quenching of BNS phosphorescence occurs only upon contact with the quencher, and the quenching constant can be used to assess the degree of accessibility of the heme group.     

A general strategy for the diastereoselective synthesis of 2,6-diaryl-3,7-dioxabicyclo[3.3.0]octane lignans

A strategy for the stereoselective synthesis of all the possible diastereoisomers of the 2,6-diaryl-3,7-dioxabicyclo[3.3.0]octane (furofuran) lignans from a single dihydrofuran precursor is described. The key steps involve a diastereocontrolled templated cationic cyclization followed by stereoselective reduction of the resulting methyl glycoside.     

Reactivity of DNA guanyl radicals with phenolate anions

Guanine bases are the most easily oxidized sites in DNA. Electron-deficient guanine species are major intermediates produced in DNA by the direct effect of ionizing radiation (ionization of the DNA itself) because of preferential hole migration within DNA to guanine bases. By using thiocyanate ions to modify the indirect effect (ionization of the solvent), we are able to produce these single-electron-oxidized guanine radical species in dilute aqueous solutions of plasmid DNA where the direct effect is negligible. The guanyl radical species produce stable modified guanine products. They can be detected in the plasmid by converting them to strand breaks after incubation with a DNA repair enzyme. If a phenol is present during irradiation, the yield of modified guanines is decreased. The mechanism is reduction of the guanine radical species by the phenol. It is possible to derive a rate constant for the reaction of the phenol with the guanyl radical. The pH dependence shows that phenolate anions are more reactive than their conjugate acids, although the difference for guanyl radicals is smaller than with other single-electron-oxidizing agents. At physiological pH values, the reduction of a guanyl radical entails the transfer of a proton in addition to the electron. The relatively small dependence of the rate constant on the driving force implies that the electron cannot be transferred before the proton. These results emphasize the potential importance of acidic tyrosine residues and the intimate involvement of protons in DNA repair.     

Impact of prefractionation using Gradiflow on two-dimensional gel electrophoresis and protein identification by matrix assisted laser desorption/ionization-time of flight-mass spectrometry

Prefractionation of protein samples prior to two-dimensional electrophoresis (2-DE) has the potential to increase the dynamic detection range for proteomic analysis. We evaluated a membrane-based electrophoretic separation technique (Gradiflow) for its ability to fractionate an exoproteome sample from the filamentous fungus Trichoderma reesei. The sample was separated on the basis of size and charge. Buffer optimization was found to be necessary for successful size fractionation. Fractionation by charge was used to resolve the sample into four fractions that were subjected to analysis by two-dimensional electrophoresis (2-DE). Enhanced detection of low-abundance proteins with selective removal of high-abundance species was achieved. Fractionated and unfractionated samples were examined for differences in the ability to identify proteins following 2-DE using trypsin in-gel digestion followed by peptide mass fingerprinting using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Fractionated samples showed marked improvement in protein identification ability and sequence coverage. This study demonstrates the utility of the Gradiflow for fractionation, resulting in an enhancement of resolution and characterization of a moderately complex proteome.     

Effect of cerium, holmium and samarium ions on the thermal and structural properties of the HZSM-12 zeolite

The study of the incorporation of rare earth elements as additives in Y zeolites is a very interesting field of research, mainly by its potential application as additives in catalytic cracking process. In this work was studied the thermal and structural properties of cerium, holmium and samarium supported on HZSM-12 zeolite. The obtained materials were characterized by X-ray diffraction (XRD), infrared spectroscopy (FTIR), nitrogen adsorption, thermogravimetry (TG/DTG), differential scanning calorimetry (DSC) and differential thermal analysis (DTA). TG/DSC/DTA analyses showed that the dehydration temperatures of RE/HZSM-12 zeolites (RE=Ce, Ho, Sm) increase in relation to pure HZSM-12. The acid properties were investigated by pyridine thermo desorption via TG. The results showed two events of mass loss attributed to elimination of pyridine adsorbed on the weak+medium acid sites and on the strong acid sites.     

Nonequilibrium synthesis and assembly of hybrid inorganic-protein nanostructures using an engineered DNA binding protein

We show that a protein with no intrinsic inorganic synthesis activity can be endowed with the ability to control the formation of inorganic nanostructures under thermodynamically unfavorable (nonequilibrium) conditions, reproducing a key feature of biological hard-tissue growth and assembly. The nonequilibrium synthesis of Cu(2)O nanoparticles is accomplished using an engineered derivative of the DNA-binding protein TraI in a room-temperature precursor electrolyte. The functional TraI derivative (TraIi1753::CN225) is engineered to possess a cysteine-constrained 12-residue Cu(2)O binding sequence, designated CN225, that is inserted into a permissive site in TraI. When TraIi1753::CN225 is included in the precursor electrolyte, stable Cu(2)O nanoparticles form, even though the concentrations of [Cu(+)] and [OH(-)] are at 5% of the solubility product (K(sp,Cu2O)). Negative control experiments verify that Cu(2)O formation is controlled by inclusion of the CN225 binding sequence. Transmission electron microscopy and electron diffraction reveal a core-shell structure for the nonequilibrium nanoparticles: a 2 nm Cu(2)O core is surrounded by an adsorbed protein shell. Quantitative protein adsorption studies show that the unexpected stability of Cu(2)O is imparted by the nanomolar surface binding affinity of TraIi1753::CN225 for Cu(2)O (K(d) = 1.2 x 10(-)(8) M), which provides favorable interfacial energetics (-45 kJ/mol) for the core-shell configuration. The protein shell retains the DNA-binding traits of TraI, as evidenced by the spontaneous organization of nanoparticles onto circular double-stranded DNA.     

Mechanism of azole antifungal activity as determined by liquid chromatographic/mass spectrometric monitoring of ergosterol biosynthesis

A liquid chromatography/mass spectrometry (LC/MS) method for separation and characterization of ergosterol biosynthetic precursors was developed to study the effect of Posaconazole on sterol biosynthesis in fungi. Ergosterol biosynthetic precursors were characterized from their electron ionization mass spectra acquired by a normal-phase chromatography, particle beam LC/MS method. Fragment ions resulting from cleavage across the D-ring and an abundant M - 15 fragment ion were diagnostic for methyl substitution at C-4 and C-14. Comparison of the sterol profile in control and treated Candida albicans incubations showed depletion of ergosterol and accumulation of C-4 and C-14 methyl-substituted sterols following treatment with Posaconazole. These C-4 and C-14 methyl sterols are known to be incapable of sustaining cell growth. The results demonstrate that Posaconazole exerts its antifungal activity by inhibition of ergosterol biosynthesis. Furthermore, Posaconazole appears to disrupt ergosterol biosynthesis by inhibition of lanosterol 14alpha-demethylase.     

Nanoparticle-cored dendrimers: synthesis and characterization

The synthesis and characterization of a group of new dendrimers-namely, nanoparticle-cored dendrimers (NCDs)-are described. These materials were obtained by the reduction of hydrogen tetrachloroaurate phase-transferred into toluene in the presence of Fréchet-type polyaryl ether dendritic disulfide wedges of generation 1-5. These materials, possessing nanometer-sized gold clusters at the core and dendritic wedges radially connected to the core by Au-S bonds, were analyzed by TEM and TGA, and by UV, IR, and NMR spectroscopies. The number of branching units connected to the core decreased with the generation of the dendritic wedge, and this number changed from 2.18/nm(2) for Au-G-2 to 0.27/nm(2) for Au-G-5. This result suggests that, in the higher-generation NCDs, a large fraction of the surface area of the metal cluster is not passivated and is therefore available for catalytic activity.     

The anionic alkylation of easily reducible arenes. A photochemical route to nucleophilic aromatic substitution of anthracene by organolithiums

High yield nucleophilic addition ensues upon mixing tetrahydrofuran solutions of benzyllithium or cyclooctadienyllithium and anthracene at low temperatures At least part of this addition proceeds by a single electron transfer pathway. Photolysis of the resulting adducts leads to the elimination of lithium hydride, giving net nucleophilic substitution in approximately 50% yield. The analogous reaction fails with naphthalene, where photolysis of the organolithium/ arene mixture leads to dimeric products derived from the organolithium.