Solution structure by 1H and dynamics by natural abundance 13C NMR of a receptor recognising peptide derived from a C-terminal fragment of neuropeptide Y

Summary A peptide consisting of 20 amino acid residues, derived from a C-terminal fragment of neuropeptide Y (NPY) and showing high affinity to NPY receptors, was synthesised. Its sequence is PAADLARYRHYIN-LITRQRY-NH₂, and the solution structure was calculated from NMR-derived distance and torsion angle restraints, obtained at 15°C in a solvent mixture of water and 30% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol, by using DIANA and restrained energy minimisation. The structure was found to consist of a well-defined -helix in the centre, with a few residues at the termini having less well defined conformations. The spinlattice and spin-spin relaxation rates of -carbons have been determined on ¹³C at natural abundance. From 1D experiments the global rotational correlation time was determined and from 2D experiments the dynamics of each individual residue was obtained. The results demonstrate that the C-H vectors in the -helix essentially follow the global motion. Towards the termini, contributions from local dynamics increase. This tendency is correlated to the increasing uncertainty of the structure towards the peptide ends. An effective molecular volume was calculated from the temperature dependence of the global rotational correlation time. This is well compatible with a monomeric peptide, solvated by water and 1,1,1,3,3,3-hexafluoro-2-propanol. The presence of peptide dimers was ruled out as being inconsistent with the relaxation data.Supplementary material available from the authors: Two data tables and 10 PDB coordinate files of the calculated NMR structures of P7. One data table contains all detected and integrated NOE intensities; the other connects each proton and pseudoatom to an atom number used in the NOE table. The table contents served as input data files for CALIBA.Currently on leave from the Institute of Chemical Physics and Biophysics, Tallinn, Estonia.     

Synthesis and characterization of novel symmetrical phthalocyanines substituted with four benzo [d] [1, 3] dioxol-5-ylmethoxy groups

Metal (Zn, Cu, Co, Mg) phthalocyanines substituted with four benzo [d] [1, 3] dioxol-5-ylmethoxy unit through oxy-bridges have been synthesized from phthalonitrile derivative, 4-(benzo [d] [1, 3] dioxol-5-ylmethoxy) phthalonitrile. Benzo [d] [1, 3] dioxol-5-ylmethoxy substituted Pcs showed the enhanced solubility in organic solvents. The newly prepared compounds have been characterized by elemental analyses, IR, UV-Vis, ¹H NMR spectra.     

C-terminal amino acids are essential for human heat shock protein 70 dimerization

The human inducible heat shock protein 70 (hHsp70), which is involved in several major pathologies, including neurodegenerative disorders and cancer, is a key molecular chaperone and contributes to the proper protein folding and maintenance of a large number of protein structures. Despite its role in disease, the current structural knowledge of hHsp70 is almost exclusively based on its Escherichia coli homolog, DnaK, even though these two proteins only share ~50 % amino acid identity. For the first time, we describe a complete heterologous production and purification strategy that allowed us to obtain a large amount of soluble, full-length, and non-tagged hHsp70. The protein displayed both an ATPase and a refolding activity when combined to the human Hsp40. Multi-angle light scattering and bio-layer interferometry analyses demonstrated the ability of hHsp70 to homodimerize. The role of the C-terminal part of hHsp70 was identified and confirmed by a study of a truncated version of hHsp70 that could neither dimerize nor present refolding activity.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-014-0526-3) contains supplementary material, which is available to authorized users.     

Activity and growth efficiency of heterotrophic bacteria in a salt marsh (Ria de Aveiro, Portugal)

Bacterial utilization of monomers is recognized as an important step in the biogeochemical cycling of organic matter. In this study we have compared the heterotrophic activity of bacterial communities from different micro-habitats within a salt marsh environment (Ria de Aveiro, Portugal) in order to establish spatial patterns of bacterial abundance, monomer turnover rates (Tr) and bacterial growth efficiency (BGE). Differences in bacterial abundance and activity could be found between distinct plant rhizospheres. BGE tended to be lower at Halimione portulacoides banks, when compared to Sarcocornia perennis subsp. perennis banks which, on the contrary, showed the highest bacterial densities. Experiments of amendment of natural samples with organic and inorganic supplements indicated that salt marsh bacteria are not strongly regulated by salinity but the increased availability of labile organic matter causes a significant metabolic shift towards mineralization.     

Insights from modelling the 3D structure of the extracellular domain of alpha7 nicotinic acetylcholine receptor

Based on the crystal structure of acetylcholine-binding protein, the three-dimensional structures of the extracellular domain, or the ligand-binding domains, of the monomer, homodimer, and homopentamer of the alpha7 nicotinic acetylcholine receptor were derived. The interface between two subunits, where the ligand-binding site is located, was investigated. Furthermore, an explicit definition of the ligand-binding pocket was illustrated that might provide useful clues for conducting various mutagenesis studies for finding drugs against schizophrenia and Alzheimer's disease.     

Preparation and characterization of ultra violet (UV) radiation cured bio-degradable films of sago starch/PVA blend

Polymer films of sago starch/polyvinyl alcohol (PVA) were prepared by casting and cured under ultra violet (UV) radiation. Different blends were made varying the concentration of sago starch and PVA. Tensile strength (TS) and elongation at break (Eb) of the prepared films were studied. Films made up of sago starch and PVA with a ratio of 1:2 showed the highest TS and Eb. The physico-mechanical properties of prepared films were improved by grafting with acrylic monomers with the aid of UV radiation. A series of formulations was prepared with two monomers 2-ethyl 2-hydroxymethyl 1,3 methacrylate (EHMPTMA) and 2-ethylhexylacrylate (EHA) and a photoinitiator. Monomer concentration, soaking time and radiation dose were optimized in terms of grafting and mechanical properties. The highest TS was at 50% EHMPTMA and 48% EHA and 2% photo initiator at 5 min soaking time and recorded value was 6.58 MPa. The prepared films were further characterized with NMR spectroscopy and scanning electron microscope (SEM).     

Strong light-induced reorganization of pigment-protein complexes of thylakoid membranes in rye (spectroscopic study)

The supramolecular reorganization of LHCII complexes within the thylakoid membrane in Secale cereale leaves under low and high light condition was examined. Rye seedlings were germinated hydroponically in a climate chamber with a 16 h daylight photoperiod, photosynthetic photon flux density (PPFD) of 150 μmol m⁻² s⁻¹ and 24/16 °C day/night temperature. The influence of pre-illumination of the plants with high light intensity on the PSII antenna complexes was studied by comparison of the structure and function of the LHCII complexes and organization of thylakoid membranes isolated from 10-day-old plants illuminated with low (150 μmol m⁻² s⁻¹) or high (1200 μmol m⁻² s⁻¹) light intensity. Aggregated and trimeric with monomeric forms of LHCII complexes were separated from the whole thylakoid membranes using non-denaturing electrophoresis. Analyses of fluorescence emission spectra of these different LHCII forms showed that the monomer was the most effective aggregating antenna form. Moreover, photoprotection connected with LHCII aggregation was more effective upon LHCII monomers in comparison to trimer aggregation. Light stress induced specific organization of neighboring LHCII complexes, causing an increase in fluorescence yield of the long-wavelength bands (centered at 701 and 734 nm). The changes in the organization of the thylakoid membrane under light stress, observed by analysis of absorbance spectra obtained by Fourier transform infrared spectroscopy, also indicated light-induced LHCII aggregation.     

Evaluation of chromium(VI) removal behaviour by two isolates of Synechocystis sp. in terms of exopolysaccharide (EPS) production and monomer composition

Chromium(VI) removal and its association with exopolysaccharide (EPS) production in cyanobacteria were investigated. Synechocystis sp. BASO670 produced higher EPS (548 mg L⁻¹) than Synechocystis sp. BASO672 (356 mg L⁻¹). While the EC₅₀ of the Cr(VI) for Synechocystis sp. BASO670 and Synechocystis sp. BASO672 were determined as 11.5 mg L⁻¹, and 2.0 mg L⁻¹, respectively, there was no relation between Cr(VI) removal and EPS production. Synechocystis sp. BASO672, which has higher EPS value, removed (33%) more Cr(VI) than Synechocystis sp. BASO670. Monomer compositions of EPS of each of the isolates were determined differently. Synechocystis sp. BASO672 which removed higher Cr(VI), had higher values of uronic acid and glucuronic acid (192 μg/mg and 89%, respectively). Our results showed that EPS might play a role in Cr(VI) tolerance. Monomer composition, especially uronic acid and glucuronic acid content of EPS may have enhanced Cr(VI) removal.     

Theoretical studies on the molecular structure and vibrational spectra of some dimethyl substituted pyridine derivatives

The molecular geometries, normal mode frequencies, intensities and corresponding infrared assignments of monomeric and dimeric 2,3-dimethylpyridine, 2,4-dimethylpyridine, 3,4-dimethylpyridine, 3,5-dimethylpyridine and monomeric 2,6-dimethylpyridine in the ground state were investigated at the density functional theory (DFT)-B3LYP level using the 6-311+G(d, p) basis set. The vibrational frequencies and geometric parameters of C–H stretching and bending in the fundamental region were calculated and compared to the Fourier transform infrared (FT-IR) data obtained. In the studied monomeric and dimeric dimethyl substituted pyridine derivatives, the C–H stretching and bending frequency shifts that occur between the dimer and the monomer may be diagnostic of the magnitude of dimerization energy. As supported by data in the literature, the most stable dimeric form was obtained for the 3,4-dimethylpyridine molecule. Figure Molecular model and numbering scheme of the studied dimeric dimethylpyridinederivatives     

Implications of oligomeric forms of POD-1 and POD-2 proteins isolated from cell walls of the biocontrol agent Pythium oligandrum in relation to their ability to induce defense reactions in tomato

The cell wall protein fraction (CWP) isolated from the biocontrol agent Pythium oligandrum induces defense reactions in tomato. CWP contains two novel elicitin-like proteins, POD-1 and POD-2, both with seven cysteines. To determine the essential structure in the defense-eliciting components of CWP, five fractions (F1, F2, F3, F4 and F5) were fractionated from CWP using cation chromatography and their components and disulfide bond compositions were analyzed. The expression levels of three defense-related genes (PR-6, LeCAS and PR-2b) were determined in tomato roots treated with each of the five fractions. Of the five fractions, F4 containing a heterohexamer of POD-1 and POD-2, and F5 containing a homohexamer of POD-1, both with disulfide bonds formed between all cysteine residues, induced the expression of three genes. F4 treatment also induced the accumulation of ethylene in tomato. The predicted three-dimensional structures of POD-1 and POD-2, and the results of SEC and MALDI-TOF MS analyses suggest that F4 consists of three POD-1 and POD-2 disulfide-bonded heterodimers that interleave into a hexameric ring through noncovalent association. These results suggest that this structure, which F5 also appears to form, is essential for stimulating defense responses in tomato.