New structural insights and molecular-modelling studies of 4-methyl-5-beta-hydroxyethylthiazole kinase from Pyrococcus horikoshii OT3 (PhThiK)

4-Methyl-5-beta-hydroxyethylthiazole kinase (ThiK) catalyses the phosphorylation of the hydroxyl group of 4-methyl-5-beta-hydroxyethylthiazole. This work reports the first crystal structure of an archaeal ThiK: that from Pyrococcus horikoshii OT3 (PhThiK) at 1.85 angstrom resolution with a phosphate ion occupying the position of the beta-phosphate of the nucleotide. The topology of this enzyme shows the typical ribokinase fold of an alpha/beta protein. The overall structure of PhThiK is similar to those of Bacillus subtilis ThiK (BsThiK) and Enterococcus faecalis V583 ThiK (EfThiK). Sequence analysis of ThiK enzymes from various sources indicated that three-quarters of the residues involved in interfacial regions are conserved. It also revealed that the amino-acid residues in the nucleotide-binding, magnesium ion-binding and substrate-binding sites are conserved. Binding of the nucleotide and substrate to the ThiK enzyme do not influence the quaternary association (trimer) as revealed by the crystal structure of PhThiK.

Structural studies of analgesics and their interactions. Part 4. Crystal structures of phenylbutazone and a 2 : 1 complex between phenylbutazone and piperazine

The X-ray crystal structures of 4-butyl-1,2-diphenylpyrazolidine-3,5-dione (phenylbutazone)(I). and its 2 : 1 complex (II) with piperazine have been determined by direct methods and the structures refined to R 0.096 (2 300 observed reflections measured by diffractometer) and 0.074 (2 494 observed reflections visuallyestimated). Crystals are monoclinic, space group P21/c; for (I)a= 21.695(4), b= 5.823(2), c= 27.881(4)Å, = 108.06 (10)°, Z= 8, and for (II)a= 8.048(4), b= 15.081(4), c= 15.583(7)Å, = 95.9(3)°, Z= 2. The two crystallographically independant molecules in the structure of (I) are similar except for the conformation of the butyl group, which is disordered in one of the molecules. In the pyrazolidinedione group, the two C–C bonds are single and the two C–O bonds double. The two nitrogen atoms in the five-membered ring are pyramidal with the attached phenyl groups lying on the opposite sides of the mean plane of the ring. The phenylbutazone molecule in (II) exists as a negative ion owing to deprotonation of C-4. C-4 is therefore trigonal and the orientation of the Bu group with respect to the pyrazolidinedione group is considerably different from that in (I); there is also considerable electron delocalization along the C–O and C–C bonds. These changes in geometry and electronic structure may relate to biological activity. The doubly charged cationic piperazine molecule exists in the chair form with the nitrogen atoms at the apices. The crystal structure of (II) is stabilized by ionic interactions and N–H O hydrogen bonds.

N. m. r. and infrared spectroscopic studies of hydrogen bonding in hindered alcohols. An instance of a true hydrogen bonded dimer in an alcohol

Hydrogen bonding in the highly hindered alcohol 2,4-dimethyl-3-ethyl-3-pentanol has been studied by proton n.m.r. and infrared spectroscopy. This alcohol associates to form a dimer but no higher hydrogen bonded species; hence the monomer–dimer equilibrium can be studied without interference from competing processes. Spectral and thermodynamic properties for the hydrogen bonding are reported.

Genetic studies of the PRP17 gene of Saccharomyces cerevisiae: a domain essential for function maps to a nonconserved region of the protein

The PRP17 gene product is required for the second step of pre-mRNA splicing reactions. The C-terminal half of this protein bears four repeat units with homology to the beta transducin repeat. Missense mutations in three temperature-sensitive prp17 mutants map to a region in the N-terminal half of the protein. We have generated, in vitro, 11 missense alleles at the beta transducin repeat units and find that only one affects function in vivo. A phenotypically silent missense allele at the fourth repeat unit enhances the slow-growing phenotype conferred by an allele at the third repeat, suggesting an interaction between these domains. Although many missense mutations in highly conserved amino acids lack phenotypic effects, deletion analysis suggests an essential role for these units. Only mutations in the N-terminal nonconserved domain of PRP17 are synthetically lethal in combination with mutations in PRP16 and PRP18, two other gene products required for the second splicing reaction. A mutually allele-specific interaction between Prp17 and snr7, with mutations in U5 snRNA, was observed. We therefore suggest that the functional region of Prp17p that interacts with Prp18p, Prp16p, and U5 snRNA is the N terminal region of the protein.

Synthesis, characterization, thermal degradation, and comparative chain dynamics studies of weak-link polysulfide polymers

Synthesis, spectroscopic and thermal characterization of two new classes of polysulfide polymers: poly[1(phenoxymethyl) ethylene polysulfide] (PPMEP), and poly [1-(phenoxy) ethylene polysulfide] (PPEP) is presented. The direct pyrolysis mass spectrometry (DP-MS) technique, used to study the thermal degradation behavior of these polysulfide polymers, indicated that the polymers underwent degradation through the weak-links scission. The thermal stability of the polysulfide polymers decreased as the ``rank'' (number of sulfur atoms in the polysulfide linkage; n=1, 2, 4) increased. The main-chain flexibility of these polysulfide polymers in terms of their C-13 NMR spinlattice relaxation time (T-1) measurements on the backbone methine (-CH-) and methylene (-CH2-) carbons are reported here for the first time. A comparative study of the solution chain dynamics indicated that it increased as ``rank'' of the polysulfide linkages decreased as well as by introducing side chain spacers such as, ether (-O-) or methyleneoxy (-CH2O-) groups.

Electrical Percolation studies of Polystyrene-multi wall carbon nanotubes Composites

Composites of Polystyrene-multi wall carbon nanotubes (PS-MWNTs) were prepared with loading up to 7 wt% of MWNTs by simple solvent mixing and drying technique. MWNTs with high aspect ratio similar to 4000 were used to make the polymer composites. A very high degree of dispersion of MWNTs was achieved by ultrasonication technique. As a result of high dispersion and high aspect ratio of the MWNTs electrical percolation was observed at rather low weight fraction similar to 0.0021. Characterization of the as prepared PS-MWNTs composites was done by Electron microscopy (EM), X-ray diffraction technique (XRD) and Thermogravimetery analysis (TGA).

Conformational studies of the triphenylphosphazenyl side chain in cyclophosphazenes. II. Crystal and molecular structure of 2,4,4,6,6,8,8-heptachloro-2-triphenylphosphazenylcyclotetra(phosphazene), N4P4Cl7(NPPh3)

Abstract is not available.

X-ray spectroscopic studies of amorphous materials

Measurement of the chemical shifts ΔE of the K-absorption edge in both crystalline and amorphous states of several solids shows that ΔE is generally smaller in the amorphous state. More covalent solids appear to be associated with small values of ΔE.

Optical, dielectric and morphological studies of sol-gel derived nanocrystalline TiO2 films

Nanocrystalline TiO2 films have been synthesized on glass and silicon substrates by sol-gel technique. The films have been characterized with optical reflectance/transmittance in the wavelength range 300-1000nm and the optical constants (n, k) were estimated by using envelope technique as well as spectroscopic ellipsometry. Morphological studies have been carried Out using atomic force microscope (AFM). Metal-Oxide-Silicon (MOS) capacitor was fabricated using conducting coating on TiO2 film deposited on silicon. The C-V measurements show that the film annealed at 300 degrees C has a dielectric constant of 19.80. The high percentage of transmittance, low surface roughness and high dielectric constant suggests that it can be used as an efficient anti-reflection coating on silicon and other optical coating applications and also as a MOS capacitor.

Solution NMR studies of acetohydroxy acid synthase I: Identification of the sites of inter-subunit interactions using multidimensional NMR methods

The novel multidomain organization in the multimeric Escherichia coli AHAS I (ilvBN) enzyme has been dissected to generate polypeptide fragments. These fragments when cloned, expressed and purified reassemble in the presence of cofactors to yield a catalytically competent enzyme. Structural characterization of AHAS has been impeded due to the fact that the holoenzyme is prone to dissociation leading to heterogeneity in samples. Our approach has enabled the structural characterization using high-resolution nuclear magnetic resonance methods. Near complete sequence specific NMR assignments for backbone H-N, N-15, C-13 alpha and C-13(beta) atoms of the FAD binding domain of ilvB have been obtained on samples isotopically enriched in H-2, C-13 and N-15. The secondary structure determined on the basis of observed C-13(alpha) secondary chemical shifts and sequential NOEs indicates that the secondary structure of the FAD binding domain of E. coli AHAS large Subunit (ilvB) is similar to the structure of this domain in the catalytic subunit of yeast AHAS. Protein-protein interactions involving the regulatory subunit (ilvN) and the domains of the catalytic subunit (ilvB) were studied using circular dichroic and isotope edited solution nuclear magnetic resonance spectroscopic methods. Observed changes in circular dichroic spectra indicate that the regulatory subunit (ilvN) interacts with ilvB alpha and ilvB beta domains of the catalytic subunit and not with the ilvB gamma domain. NMR chemical shift mapping methods show that ilvN binds close to the FAD binding site in ilvB beta and proximal to the intrasubunit ilvB alpha/ilvB beta domain interface. The implication of this interaction on the role of the regulatory subunit oil the activity of the holoenzyme is discussed. NMR studies of the regulatory domains show that these domains are structured in solution. Preliminary evidence for the interaction of ilvN with the metabolic end product of the pathway, viz., valine is also presented.

Microwave absorption studies of iron encapsulating carbon-polymer nanocomposite films

Iron encapsulated carbon nanoparticle polyvinyl chloride composite films have been prepared by solvent mixing and drying method. The films were characterized by scanning electron microscope (SEM) and high resolution transmission electron microscope (HRTEM). A 5 nm thin graphitic carbon coating is observed on cubic Fe nanoparticles. The microwave absorption studies by wave guide technique in the Ka band range showed highest electromagnetic interference shielding efficiency of 18dB on a 300 micron thick film. The shielding efficiency depends on weight % of the filler in the composite. The data obtained for different films indicate that these lightweight materials are good candidates for potential electromagnetic interference shielding applications.

X-ray crystallographic and NMR studies of pantothenate synthetase provide insights into the mechanism of homotropic inhibition by pantoate

The structural basis for the homotropic inhibition of pantothenate synthetase by the substrate pantoate was investigated by X-ray crystallography and high-resolution NMR spectroscopic methods. The tertiary structure of the dimeric N-terminal domain of Escherichia coli pantothenate synthetase, determined by X-ray crystallography to a resolution of 1.7 Å, showed a second molecule of pantoate bound in the ATP-binding pocket. Pantoate binding to the ATP-binding site induced large changes in structure, mainly for backbone and side chain atoms of residues in the ATP binding HXGH(34–37) motif. Sequence-specific NMR resonance assignments and solution secondary structure of the dimeric N-terminal domain, obtained using samples enriched in 2H, 13C, and 15N, indicated that the secondary structural elements were conserved in solution. Nitrogen-15 edited two-dimensional solution NMR chemical shift mapping experiments revealed that pantoate, at 10 mm, bound at these two independent sites. The solution NMR studies unambiguously demonstrated that ATP stoichiometrically displaced pantoate from the ATP-binding site. All NMR and X-ray studies were conducted at substrate concentrations used for enzymatic characterization of pantothenate synthetase from different sources [Jonczyk R & Genschel U (2006) J Biol Chem 281, 37435–37446]. As pantoate binding to its canonical site is structurally conserved, these results demonstrate that the observed homotropic effects of pantoate on pantothenate biosynthesis are caused by competitive binding of this substrate to the ATP-binding site. The results presented here have implications for the design and development of potential antibacterial and herbicidal agents.

Fluorescent probe studies of biomembranes and model systems. The design and use of anionic site reporters

The fluorescence properties of a homologous series of fluorescent alkylamines are described. The binding of the probes to crythrocyte membranes increases with the length of the alkyl chain. The probes are shown to interact more strongly with membranes than with protein and lipid model systems. The binding of the probes to the membrane is sensitive to the cation concentration of the medium.

13C nuclear magnetic resonance studies of the binding of alkali and alkaline earth metal salts to amides

3C resonances of carbonyl and methyl groups in amides are shifted down-field on interaction with alkali and alkaline earth metal salts. The magnitude of the shift depends on the ionic potential of the cation. Ions like Li+ bind to the amide carbonyl group both in neat amide solutions as well as in concentrated salt solutions in water.