Conformational changes in a photosensory LOV domain monitored by time-resolved NMR spectroscopy

Phototropins are light-activated kinases from plants that utilize light-oxygen-voltage (LOV) domains as blue light photosensors. Illumination of these domains leads to the formation of a covalent linkage between the protein and an internally bound flavin chromophore, destabilizing the surrounding protein and displacing an alpha-helix from its surface. Here we use a combination of spectroscopic tools to monitor the kinetic processes that spontaneously occur in the dark as the protein returns to the noncovalent ground state. Using time-resolved two-dimensional (2D) NMR methods, we measured the rate of this process at over 100 independent sites throughout the protein, establishing that regeneration of the dark state occurs cooperatively within a 1.6-fold range of observed rates. These data agree with other spectroscopic measurements of the kinetics of protein/FMN bond cleavage and global conformational changes, consistent with these processes experiencing a common rate-limiting step. Arrhenius analyses of the temperature dependence of these rates suggest that the transition state visited during this regeneration has higher energy than the denatured form of this protein domain despite the fact that there is no global unfolding of the domain during this process.     

Use of modifiers in on-line and off-line supercritical fluid extraction

For many applications using supercritical fluid extraction (SFE), modifiers may be required.This paper will present some findings regarding the use of various modifiers including methanol, hexane, acetone, chloroform, dichloromethane, toluene, and tributylphosphate, in on-line and off-line SFE with cryogenic adsorbent trapping. The specific applications involved the extractions of petroleum hydrocarbons and pesticides from naturally incurred soils.     

Structural,luminescence, and NMR studies of the reversible binding of acetate,lactate, citrate,and selected amino acids to chiral diaqua ytterbium,gadolinium, and europium complexes

The nature of the ternary complexes formed in aqueous media at ambient pH on reversible binding of acetate, lactate, citrate, and selected amino acids and peptides to chiral diaqua europium, gadolinium, or ytterbium cationic complexes has been examined. Crystal structures of the chelated ytterbium acetate and lactate complexes have been defined in which the carboxylate oxygen occupies an "equatorial" site in the nine-coordinate adduct. The zwitterionic adduct of the citrate anion with [EuL1] was similar to the chelated lactate structure, with a 5-ring chelate involving the apical 3-hydroxy group and the alpha-carboxylate. Analysis of Eu and Yb emission CD spectra and lifetimes (H2O and D2O) for each ternary complex, in conjunction with 1H NMR analyses of Eu/Yb systems and 17O NMR and relaxometric studies of the Gd analogues, suggests that carbonate, oxalate, and malonate each form a chelated (q = 0) square-antiprismatic complex in which the dipolar NMR paramagnetic shift (Yb, Eu) and the emission circular polarization (gem for Eu) are primarily determined by the polarizability of the axial ligand. The ternary complexes with hydrogen phosphate, with fluoride, and with Phe, His, and Ser at pH 6 are suggested to be monoaqua systems with Eu/Gd with an apical bound water molecule. However, for the ternary complexes of simple amino acids with [YbL1]3+, the enhanced charge demand favors a chelate structure with the amine N in an apical position. Crystal structures of the Gly and Ser adducts confirm this. In peptides and proteins (e.g. albumin) containing Glu or Asp residues, the more basic side chain carboxylate may chelate to the Ln ion, displacing both waters.     

Analysis of Mammalian Cell Cytoplasm with Electrophoresis in Nanometer Inner Diameter Capillaries

Capillary electrophoresis in 770 nanometer inner diameter capillaries coupled to electrochemical detection with an etched electrode matching an etched capillary (etched electrochemical detection) has been used with ultrasmall sampling to inject subcellular samples from intact single mammalian cells. Separations of cytoplasmic samples taken from rat pheochromocytoma cells have been achieved. As little as 8% of the total volume of a single cell has been sampled and analyzed. Dopamine has been identified and quantified in these PC12 cells using this technique. The average cytoplasmic level of dopamine in rat pheochromocytoma cells has been determined to be 240 ± 60 μM. The use of electrophoresis in 770 nanometer inner diameter capillaries with electrochemical detection to monitor cytoplasmic neurotransmitters at the single cell level can provide information about complex cellular functions such as neurotransmitter storage and synthesis.     

Fused s-triazino heterocycles. XIII. 1,3,7,10,11c-Pentaazabenz[de]-anthracene and 1,3,7,10,11,13,13d-heptaazabenz[de]cyclopenta[H]-anthracene,two new ring systems

The synthesis of the title compounds 5a-c and 6a-c is described using, 2,6-diaminopyridine as starting material. The key intermediates are 2-t-butyl-4-cyano-5(2-dimethylaminoethenyl)-1,3,6,9b-tetraazaphenalene 4 and 10-amino-2-t-butyl-10,11-dihydro-11-imino-1,3,7,10,11c-pentaazabenz[de]anthracene 5c .     

The enthalpy of formation of tungsten azide pentafluoride

The enthalpy of hydrolysis of solid tungsten azide pentafluoride in alkaline aqueous solution (1.O mol dm^?3 KOH; 298.2K) is ?578 kJ mol^?1. Hence its enthalpy of formation is ?1170 kJ mol^?1.