Hydrogen/deuterium exchange on yeast ATPase supramolecular protein complex analyzed at high sensitivity by MALDI mass spectrometry

To evaluate the ability of hydrogen/deuterium exchange of amide protons followed by mass spectrometry (HXMS) to yield topological information about supramolecular protein complexes, this approach has been tested with the 370 kDa hetero-oligomeric complex of yeast F1-ATPase. The study was focused on the epsilon subunit (6612 Da) of the complex. Deuterium back exchange due to the chromatographic isolation step of this subunit was strongly reduced by means of fast micro-chromatography, and MALDI-MS was used to analyze either the intact subunit or peptide mixtures resulting from its proteolytic cleavage. A deuterium labeling kinetic study was conducted with epsilon subunit being a part of the F1 native complex. The effect of a secondary structure was also investigated by means of HXMS on the isolated epsilon subunit. Finally, to determine which regions of epsilon subunit are accessible to solvent in F1-ATPase during exchange, the complex was submitted to hydrogen/deuterium exchange, the epsilon subunit was purified by micro-chromatography, digested by pepsin, and resulting peptide fragments were analyzed by MALDI-MS. The combination of hydrogen/deuterium exchange, fast micro-chromatography and MALDI-MS was shown to be a fast and efficient way to obtain detailed topological information for the epsilon subunit when it is engaged in the ATPase complex.     

Synthesis,characterization, and catalytic performance of single-site iron(III) centers on the surface of SBA-15 silica

A new molecular precursor strategy has been used to prepare a series of single-site catalysts that possess isolated iron centers supported on mesoporous SBA-15 silica. The iron centers were introduced via grafting reactions of the tris(tert-butoxy)siloxy iron(III) complex Fe[OSi(O(t)Bu)(3)](3)(THF) with SBA-15 in dry hexane. This complex reacts cleanly with the hydroxyl groups of SBA-15 to eliminate HOSi(O(t)Bu)(3) (as monitored by (1)H NMR spectroscopy) with formation of isolated surface species of the type identical with SiO-Fe-[OSi(O(t)Bu)(3)](2)(THF). In this way, up to 21% of the hydroxyl sites on SBA-15 were derivatized (0.23 Fe nm(-)(2)), and iron loadings in the range of 0.0-1.90% were achieved. The structure of the surface-bound iron species, as determined by spectroscopic methods (electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), UV-vis, and in situ infrared measurements) and by elemental analyses, contains a pseudotetrahedral iron(III) center. The THF ligand of this surface-bound complex was quantitatively displaced by acetonitrile (by (1)H NMR spectroscopy). Calcination of these materials at 300 degrees C for 2 h under oxygen resulted in removal of all organic matter and site-isolated iron surface species that are stable to condensation to iron oxide clusters. Spectroscopic data (UV-vis and EPR) suggest that the iron centers retain a mononuclear, pseudotetrahedral iron(III) structure after calcination. The calcinated, iron-grafted SBA-15 materials exhibit high selectivities as catalysts for oxidations of alkanes, alkenes, and arenes, with hydrogen peroxide as the oxidant.     

Rate constants from the reaction path Hamiltonian. II. Nonseparable semiclassical transition state theory

For proton transfer reactions, the tunneling contributions to the rates are often much larger than thermally activated rates at temperatures of interest. A number of separable tunneling corrections have been proposed that capture the dependence of tunneling rates on barrier height and imaginary frequency size. However, the effects of reaction pathway curvature and barrier anharmonicity are more difficult to quantify. The nonseparable semiclassical transition state theory (TST) of Hernandez and Miller [Chem. Phys. Lett. 214, 129 (1993)] accounts for curvature and barrier anharmonicity, but it requires prohibitively expensive cubic and quartic derivatives of the potential energy surface at the transition state. This paper shows how the reaction path Hamiltonian can be used to approximate the cubic and quartic derivatives used in nonseparable semiclassical transition state theory. This enables tunneling corrections that include curvature and barrier anharmonicity effects with just three frequency calculations as required by a conventional harmonic transition state theory calculation. The tunneling correction developed here is nonseparable, but can be expressed as a thermal average to enable efficient Monte Carlo calculations. For the proton exchange reaction NH2 + CH4 <==> NH3 + CH3, the nonseparable rates are very accurate at temperatures from 300 K up to about 1000 K where the TST rate itself begins to diverge from the experimental results.     

Comprehensive DFT study of nitrous oxide decomposition over Fe-ZSM-5

The reaction mechanism for nitrous oxide decomposition has been studied on hydrated and dehydrated mononuclear iron sites in Fe-ZSM-5 using density functional theory. In total, 46 different surface species with different spin states (spin multiplicity M(S) = 4 or 6) and 63 elementary reactions were considered. Heats of adsorption, activation barriers, reaction rates, and minimum energy pathways were determined. The approximate minimum energy pathways and transition states were calculated using the "growing string method" and a modified "dimer method". Spin surface crossing (e.g., O(2) desorption) was considered. The minimum potential energy structure on the seam of two potential energy surfaces was determined with a multiplier penalty function algorithm by Powell and approximate rates of spin surface crossings were calculated. It was found that nitrous oxide decomposition is first order with respect to nitrous oxide concentration and zero order with respect to oxygen concentration. Water impurities in the gas stream have a strong inhibiting effect. In the concentration range of 1-100 ppb, the presence of water vapor influences the surface composition and the apparent rate coefficient. This is especially relevant in the temperature range of 600-700 K where most experimental kinetic studies are performed. Apparent activation barriers determined over this temperature range vary from 28.4 (1 ppb H(2)O) to 54.8 kcal/mol (100 ppb H(2)O). These results give an explanation why different research groups and different catalyst pretreatments often result in very different activation barriers and preexponential factors. Altogether perfect agreement with experimental results could be achieved.     

System-wide investigation of ErbB4 reveals 19 sites of Tyr phosphorylation that are unusually selective in their recruitment properties

The first three members of the ErbB family of receptor tyrosine kinases activate a wide variety of signaling pathways and are frequently misregulated in cancer. Much less is known about ErbB4. Here we use tandem mass spectrometry to identify 19 sites of tyrosine phosphorylation on ErbB4, and protein microarrays to quantify biophysical interactions between these sites and virtually every SH2 and PTB domain encoded in the human genome. Our unbiased approach highlighted several previously unrecognized interactions and led to the finding that ErbB4 can recruit and activate STAT1. At a systems level, we found that ErbB4 is much more selective than the other ErbB receptors. This suggests that ErbB4 may enable ErbB2 and ErbB3 to signal independently of EGFR under normal conditions, and provides a possible explanation for the protective properties of ErbB4 in cancer.     

Epitope mapping on bovine prion protein using chemical cross-linking and mass spectrometry

An analytical strategy for the analysis of antigen epitopes by chemical cross-linking and mass spectrometry is demonstrated. The information of antigen peptides involved in the binding to an antibody can be obtained by monitoring the antigen peptides modified by a partially hydrolyzed cross-linker in the absence and in the presence of an antibody. This approach was shown to be efficient for characterization of the epitope on bovine prion protein bPrP(25-241) specifically recognized by a monoclonal antibody, 3E7 (mAb3E7), with only a small amount of sample (200 picomoles) needed. After cross-linking of the specific immuno complex, a matrix-assisted laser desorption/ionization (MALDI) mass spectrometer equipped with an ion conversion dynode (ICD) high-mass detector was used to optimize the amount of cross-linked complex formed at 202 kDa before proteolytic digestion. To identify the cross-linked peptides after proteolysis without ambiguity, isotope-labeled cross-linkers, disuccinimidyl suberate (DSS-d0/d12) and disuccinimidyl glutarate (DSG-d0/d6), together with high-resolution Fourier transform ion-cyclotron resonance mass spectrometry (FTICR-MS) were used. As a result, a complete fading of the peak intensities corresponding to the peptides representing the epitope was observed when bPrP/mAb3E7 complexes were formed.     

Hydrogen-transfer reactions from phenols to TEMPO prefluorescent probes in micellar systems

A nitroxide prefluorescent probe has been used to evaluate local reactivity of antioxidants in micellar systems. An apparent rate constant that directly reflects the relevance of antioxidant hydrophobicity on the reaction toward nitroxide radicals has been defined. Dramatic increases in this parameter for quercetin are shown on moving from methanol to micellar media: 90 and 230 fold enhancements for SDS and Triton X100 micelles, respectively. This is a clear consequence of the favorable partition of reactants in the micelles.     

Copper-catalyzed cyclization of iodo-tryptophans: a straightforward synthesis of pyrroloindoles

Pyrroloindoles are a key structural motif found in a wide number of biologically active alkaloids. Intramolecular copper-catalyzed coupling of readily accessible 2-iodo-tryptophan derivatives occurs in excellent yield, affording a wide range of polysubstituted, enantioenriched tetrahydropyrrolo[2,3- b]indoles. Diketopiperazines are also suitable substrates for this cyclization reaction, which affords a straightforward entry to tetra- to hepta-polycyclic systems.     

TMC-95A–D and analogues: Chemistry and biology

The proteasome regulates diverse intracellular processes, including cell-cycle progression, cell adhesion and migration, apoptosis and antigen presentation: selective inhibitors of the proteasome, therefore, have great therapeutic potential for the treatment of cancer. TMC-95A–D are unique natural products and represent a new class of noncovalent, reversible, and selective proteasome inhibitors with exceptionally strong bioactivity profiles and interesting structural properties. Significant recent advances in the syntheses of these natural products have led to intense interest in the development of related compounds as potential anticancer agents: the chemistry and biology of these natural products and analogues will be described in this review article.     

The Biological Activity of Monarda didyma L. Essential Oil and Its Effect as a Diet Supplement in Mice and Broiler Chicken

The use of growth-promoting antibiotics in livestock faces increasing scrutiny and opposition due to concerns about the increased occurrence of antibiotic-resistant bacteria. Alternative solutions are being sought, and plants of Lamiaceae may provide an alternative to synthetic antibiotics in animal nutrition. In this study, we extracted essential oil from Monarda didyma, a member of the Lamiaceae family. We examined the chemical composition of the essential oil and then evaluated the antibacterial, antioxidant, and anti-inflammatory activities of M. didyma essential oil and its main compounds in vitro. We then evaluated the effectiveness of M. didyma essential oil in regard to growth performance, feed efficiency, and mortality in both mice and broilers. Carvacrol (49.03%) was the dominant compound in the essential oil extracts. M. didyma essential oil demonstrated antibacterial properties against Escherichia coli (MIC = 87 µg·mL⁻¹), Staphylococcus aureus (MIC = 47 µg·mL⁻¹), and Clostridium perfringens (MIC = 35 µg·mL⁻¹). Supplementing the diet of mice with essential oil at a concentration of 0.1% significantly increased body weight (+5.4%) and feed efficiency (+18.85%). In broilers, M. didyma essential oil significantly improved body weight gain (2.64%). Our results suggest that adding M. didyma essential oil to the diet of broilers offers a potential substitute for antibiotic growth promoters.