Vibrational spectra of hydrazine carbothioamide and its 15N- and D-labelled species

Raman and infrared spectra of hydrazine carbothioamide (HCTA) and its three 15N-labelled molecules (H2N NH CS15NH2, H2 15N15NHCSNH2 and H2 15N15NHCS15NH2) and their deuterated compounds have been obtained. A complete normal coordinate analysis of HCTA has been made and revised assignments are presented. The factor group splittings of HCTA have been interpreted.

High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.

Influence of cross-correlation between dipolar and chemical shift anisotropy relaxation mechanisms upon the transverse relaxation rates of 15N in macromolecules

Heteronuclear multiple-quantum coherence relaxation rate are calculated for the individual transitions of the S spin in an AIS nuclear spin system assuming that the heteronucleus (S spin) has relaxation contributions from both intramolecular dipole-dipole and chemical shift anisotropy relaxation. The individual multiplet components of the heteronuclear zero- and double-quantum coherences are shown to have different transverse relaxation rates. The cross-correlation between the two relaxation mechanisms is shown to be the dominant cause of the calculated differential line broadening. Experimental data are presented using as an example a uniformly 15N labelled sample of human epidermal growth factor.

Conformational Dynamics in Alkyl Chains of an Anchored Bilayer: A Molecular Dynamics Study

Molecular dynamics (MD) simulations are reported for an anchored bilayer formed by the intercalation of cetyl trimethyl ammonium (CTA) and CH3(CH2)15N+(CH3) ions in a layered solid, CdPS3. The intercalated CTA ions are organized with the cationic headgroups tethered to the inorganic sheet and the hydrocarbon tails arranged as bilayers. Simulations were performed at three temperatures, 65, 180, and 298 K, using an isothermal−isobaric ensemble that was subsequently switched once macroscopic parameters had converged to a canonical isothermal−isochoric ensemble. The simulations are able to reproduce the experimental features of this system, including the formation of the bilayer and layer-to-layer separation distance. An analysis of the conformation of the chains showed that at all three temperatures a fraction of the alkyl chains retained a planar all-trans conformation, and that gauche bonds occurred as part of a “kink” (gauche+−trans−gauche−) sequence and not as isolated gauche bonds. Trans−gauche isomerization rates for the alkyl chains in the anchored bilayer are slower than those in lipid bilayers at the same temperature and show a progressive increase as the torsion numbers approach the tail. A two-dimensional periodic Voronoi tessellation analysis was performed to obtain the single-molecular area of an alkyl chain in the bilayer. The single-molecular area relaxation times are an order of magnitude longer than the trans−gauche isomerization times. The results indicate that the trans−gauche isomerization is associated with the creation and annihilation of a kink defect sequence. The results of the present MD simulation explain the apparent conflicting estimates of the gauche disorder in this system as obtained from infrared and 13C nuclear magnetic resonance measurements.

On the Maximum Cloud Zone and the ITCZ over Indian, Longitudes during the Southwest Monsoon

An investigation is presented of the daily variation of the maximum cloud zone (MCZ) and the 7W mb trough in the Northern Hemisphere over the Indian longitudes 70–90°E during April–October for 1973–77. It is found that during June–September there are two favorable locations for a MCZ over these longitudes–on a majority of days the MCZ is present in the monsoon zone north of 15°N, and often a secondary MCZ occurs in the equatorial region (0–10°N). The monsoon MCZ gets established by northward movement of the MCZ occurring over the equatorial Indian ocean in April and May. The secondary MCZ appears intermittently, and is characterized by long spells of persistence only when the monsoon MCZ is absent. In each of the seasons studied, the MCZ temporarily disappeared from the mean summer monsoon location (15–28°N) about four weeks after it was established near the beginning of July. It is reestablished by the northward movement of the secondary MCZ, which becomes active during the absence of the monsoon MCZ, in a manner strikingly similar to that observed in the spring to summer transition. A break in monsoon conditions prevails just prior to the temporary disappearance of the monsoon MCZ. Thus we conclude that the monsoon MCZ cannot survive for longer than a month without reestablishment by the secondary MCZ. Possible underlying mechanisms are also discussed.

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.

A new Fuzzy-LOGIC based Model for Chlorophyll-a in Pulicat Lagoon, India

Coastal lagoons are complex ecosystems exhibiting a high degree of non-linearity in the distribution and exchange of nutrients dissolved in the water column due to their spatio-temporal characteristics. This factor has a direct influence on the concentrations of chlorophyll-a, an indicator of the primary productivity in the water bodies as lakes and lagoons. Moreover the seasonal variability in the characteristics of large-scale basins further contributes to the uncertainties in the data on the physico-chemical and biological characteristics of the lagoons. Considering the above, modelling the distributions of the nutrients with respect to the chlorophyll-concentrations, hence requires an effective approach which will appropriately account for the non-linearity of the ecosystem as well as the uncertainties in the available data. In the present investigation, fuzzy logic was used to develop a new model of the primary production for Pulicat lagoon, Southeast coast of India. Multiple regression analysis revealed that the concentrations of chlorophyll-a in the lagoon was highly influenced by the dissolved concentrations of nitrate, nitrites and phosphorous to different extents over different seasons and years. A high degree of agreement was obtained between the actual field values and those predicted by the new fuzzy model (d = 0.881 to 0.788) for the years 2005 and 2006, illustrating the efficiency of the model in predicting the values of chlorophyll-a in the lagoon.