Identification of initiation sites for T4 lysozyme folding using CD and NMR spectroscopy of peptide fragments

Using CD and 2D H-1 NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the H-1 NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Her chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and IID-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.

Solution structures in aqueous SDS micelles of two amyloid beta peptides of A beta(1-28) mutated at the alpha-secretase cleavage site (K16E, K16F)

NMR solution structures are reported for two mutants (K16E, K16F) of the soluble amyloid beta peptide A beta(1-28). The structural effects of these mutations of a positively charged residue to anionic and hydrophobic residues at the alpha-secretase cleavage site (Lys16-Leu17) were examined in the membrane-simulating solvent aqueous SDS micelles. Overall the three-dimensional structures were similar to that for the native A beta(1-28) sequence in that they contained an unstructured N-terminus and a helical C-terminus. These structural elements are similar to those seen in the corresponding regions of full-length A beta peptides A beta(1-40) and A beta(1-42), showing that the shorter peptides are valid model systems. The K16E mutation, which might be expected to stabilize the macrodipole of the helix, slightly increased the helix length (residues 13-24) relative to the K16F mutation, which shortened the helix to between residues 16 and 24. The observed sequence-dependent control over conformation in this region provides an insight into possible conformational switching roles of mutations in the amyloid precursor protein from which A beta peptides are derived. In addition, if conformational transitions from helix to random coil to sheet precede aggregation of A beta peptides in vivo, as they do in vitro, the conformation-inducing effects of mutations at Lys16 may also influence aggregation and fibril formation. (C) 2000 Academic Press.

Conductivity, NMR and crystallographic study of N,N,N,N-tetramethylammonium dicyanamide plastic crystal phases: an archetypal ambient temperature plastic electrolyte material

N,N,N,N-Tetramethylammonium dicyanamide (Me(4)NDCA) has been examined via differential scanning calorimetry (DSC), thermogravimetric analysis, conductivity, single crystal X-ray diffraction and H-1 nuclear magnetic resonance (NMR) analyses, and was found to be highly conductive in the solid state (sigma = 10(-3) S cm(-2) at 420 K) and to also exhibit unusual plastic crystal behaviour. To investigate the correlation between such behaviour and the occurrence of molecular rotations in the crystal, H-1 NMR second moment measurements are compared with calculated values predicted from the crystal structure. While DSC analysis indicates a number of solid-solid transitions at ambient temperatures, subsequent H-1 NMR analysis of the Me4N+ cation shows that a variety of rotational motions become active at low (

NMR characterization of blends of poly(hydroxybutyrate-co-hydroxyvalerate) with poly(vinyl acetate)

The extent of mixing in blends of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) (27% HV) and poly(vinyl acetate) (PVAc) has been measured using a number of different techniques, principally solid-state NMR. Differential scanning calorimetry DSC measurements indicated effective mixing of the polymer chains on a scale of several nanometres. The results of H-1 T-1 and H-1 T-1rho. measurements confirm intimate mixing of the chains. A change on blending in the H-1 T-1rho, and the H-1 NMR line width of the signal from the protons of PVAc was consistent with an increase in the amplitude and frequency of motion of this component. The PVAc chains reside within the inter-lamellar space, as confirmed by spin diffusion measurements after H-1 T-1rho preparation. (C) 2003 Society of Chemical Industry.

Sulfated galactans from Australian specimens of the red alga Phacelocarpus peperocarpos (Gigartinales, Rhodophyta)

Polysaccharides from the red alga Phacelocarpos peperocarpos were extracted with hot water, clarified, and precipitated with 2-propanol. The native preparation was highly sulfated (36.2% w/w). Alkali modification decreased the sulfate content by 2.0% w/w. The alkali-modified polysaccharide is composed mostly of galactose (Gal, 51 mol%) and 3,6-anhydrogalactose (AnGal, 41 mol%), with minor amounts of a mono-O-methylgalactose (MeGal, 1 mol%), xylose (Xyl, 6 mol%), and glucose (Glc, 1 mol%). The FTIR spectrum of the alkali-modified polysaccharide resembled K-carrageenan with absorption at 930 cm(-1) (indicative of AnGal) and 850 cm(-1) (Gal ii-sulfate). However, an additional, major band of absorption occurred at 820 cm(-1) indicating the presence of equatorial sulfate ester substitution at O-6 of Gal residues, A combination of linkage and C-13 NMR spectroscopic analyses showed that the polysaccharide was composed predominantly of a novel repeating-unit, O-beta-D-galactopyranosyl 4,6-disulfate)-(1 --> 4)-3,6-anhydro-alpha-D-galactopyranose. Minor structural variations also occurred, including alternative patterns of sulfation and the presence of terminal Xylp, The location of the terminal Xylp residues was not certain but evidence supported their attachment at O-3 of some 4-linked Galp residues. The cell-wall galactans remain unchanged during the life cycle of the alga. (C) 1996 Elsevier Science Ltd.

Bipolar disorder comorbid with alcoholism: A (1)H magnetic resonance spectroscopy study

Alcoholism is highly prevalent among bipolar disorder (BD) patients, and its presence is associated with a worse outcome and refractoriness to treatment of the mood disorder. The neurobiological underpinnings that characterize this comorbidity are unknown. We sought to investigate the neurochemical profile of the dorsolateral prefrontal cortex (DLPFC) of BD patients with comorbid alcoholism. A short-TE, single-voxel (1)H spectroscopy acquisition at 1.5T from the left DLFPC of 22 alcoholic BD patients, 26 non-alcoholic BD patients and 54 healthy comparison subjects (HC) were obtained. Absolute levels of N-acetyl aspartate, phosphocreatine plus creatine, choline-containing compounds, myo-inositol, glutamate plus glutamine (Glu + Gln) and glutamate were obtained using the water signal as an internal reference. Analysis of co-variance was used to compare metabolite levels among the three groups. In the primary comparison, non-alcoholic BD patients had higher glutamate concentrations compared to alcoholic BD patients. In secondary comparisons integrating interactions between gender and alcoholism, non-alcoholic BD patients presented significantly higher glutamate plus glutamine (Glu + Gln) than alcoholic BD patients and HC. These results appeared to be driven by differences in male subjects. Alcoholic BD patients with additional drug use disorders presented significantly lower myo-inositol than BD patients with alcoholism alone. The co-occurrence of BD and alcoholism may be characterized by neurochemical abnormalities related to the glutamatergic system and to the inositol second messenger system and/or in glial pathology. These abnormalities may be the neurochemical correlate of an increased risk to develop alcoholism in BD, or of a persistently worse clinical and functional status in BD patients in remission from alcoholism, supporting the clinical recommendation that efforts should be made to prevent or early diagnose and treat alcoholism in BD patients. (C) 2009 Elsevier Ltd. All rights reserved.

Antileishmanial and antitrypanosomal activity of bufadienolides isolated from the toad Rhinella jimi parotoid macrogland secretion

Amphibian skin secretions are considered a rich source of biologically active compounds and are known to be rich in peptides, bufadienolides and alkaloids. Bufadienolides are cardioactive steroids from animals and plants that have also been reported to possess antimicrobial activities. Leishmaniasis and American Trypanosomiasis are parasitic diseases found in tropical and subtropical regions. The efforts toward the discovery of new treatments for these diseases have been largely neglected, despite the fact that the only available treatments are highly toxic drugs. In this work, we have isolated, through bioguided assays, the major antileishmanial compounds of the toad Rhinella jimi parotoid macrogland secretion. Mass spectrometry and (1)H and (13)C NMR spectroscopic analyses were able to demonstrate that the active molecules are telocinobufagin and hellebrigenin. Both steroids demonstrated activity against Leishmania (L.) chagasi promastigotes, but only hellebrigenin was active against Trypanosoma cruzi trypomastigotes. These steroids were active against the intracellular amastigotes of Leishmania, with no activation of nitric oxide production by macrophages. Neither cytotoxicity against mouse macrophages nor hemolytic activities were observed. The ultrastructural studies with promastigotes revealed the induction of mitochondrial damage and plasma membrane disturbances by telocinobufagin, resulting in cellular death. This novel biological effect of R. jimi steroids could be used as a template for the design of new therapeutics against Leishmaniasis and American Trypanosomiasis. (C) 2008 Elsevier Ltd. All rights reserved.

Three-dimensional structure of RTD-1, a cyclic antimicrobial defensin from rhesus macaque leukocytes

Most mammalian defensins are cationic peptides of 29-42 amino acids long, stabilized by three disulfide bonds. However, recently Tang et al. (1999, Science 286, 498-502) reported the isolation of a new defensin type found in the leukocytes of rhesus macaques. In contrast to all the other defensins found so far, rhesus theta defensin-1 (RTD-1) is composed of just 18 amino acids with the backbone cyclized through peptide bonds. Antibacterial activities of both the native cyclic peptide and a linear form were examined, showing that the cyclic form was 3-fold more active than the open chain analogue [Tang et al. (1999) Science 286, 498-502]. To elucidate the three-dimensional structure of RTD-1 and its open chain analogue, both peptides were synthesized using solid-phase peptide synthesis and tert-butyloxycarbonyl chemistry. The structures of both peptides in aqueous solution were determined from two-dimensional H-1 NMR data recorded at 500 and 750 MHz. Structural constraints consisting of interproton distances and dihedral angles were used as input for simulated-annealing calculations and water refinement with the program CNS. RTD-1 and its open chain analogue oRTD-1 adopt very similar structures in water. Both comprise an extended beta -hairpin structure with turns at one or both ends. The turns are well defined within themselves and seem to be flexible with respect to the extended regions of the molecules. Although the two strands of the beta -sheet are connected by three disulfide bonds, this region displays a degree of flexibility. The structural similarity of RTD-1 and its open chain analogue oRTD-1, as well as their comparable degree of flexibility, support the theory that the additional charges at the termini of the open chain analogue rather than overall differences in structure or flexibility are the cause for oRTD-1's lower antimicrobial activity. In contrast to numerous other antimicrobial peptides, RTD-1 does not display any amphiphilic character, even though surface models of RTD-1 exhibit a certain clustering of positive charges. Some amide protons of RTD-1 that should be solvent-exposed in monomeric beta -sheet structures show low-temperature coefficients, suggesting the possible presence of weak intermolecular hydrogen bonds.

NMR analysis of covalent intermediates in thiamin diphosphate enzymes

Enzymic catalysis proceeds via intermediates formed in the course of substrate conversion. Here, we directly detect key intermediates in thiamin diphosphate (ThDP)-dependent enzymes during catalysis using H-1 NMR spectroscopy. The quantitative analysis of the relative intermediate concentrations allows the determination of the microscopic rate constants of individual catalytic steps. As demonstrated for pyruvate decarboxylase (PDC), this method, in combination with site-directed mutagenesis, enables the assignment of individual side chains to single steps in catalysis. In PDC, two independent proton relay systems and the stereochemical control of the enzymic environment account for proficient catalysis proceeding via intermediates at carbon 2 of the enzyme-bound cofactor. The application of this method to other ThDP-dependent enzymes provides insight into their specific chemical pathways.

Cobalt and Zinc Compounds Bearing 1,10-Phenanthroline-5,6-dione or 1,3,5-Triaza-7-phosphaadamantane Derivatives - Synthesis, Characterization, Cytotoxicity, and Cell Selectivity Studies

The compounds [mPTA][CoCl4] (1, mPTA = N-methyl-1,3,5-triaza-7-phosphaadamantane cation), [CoCl(H2O)(DION)(2)][BF4] (2, DION = 1,10-phenanthroline-5,6-dione), [Zn(DION)(2)]Cl-2 (3) and [ZnCl(O-PTA=O)(DION)][BF4] (4) were synthesized by reaction of CoCl2 with [mPTA]I or DION and ZnCl2 with DION or 1,3,5-triaza-7-phosphaadamantane-7-oxide (PTA=O) and DION, respectively. All complexes are water soluble and have been characterized by IR, far-IR, H-1, C-13 and P-31{H-1} NMR spectroscopy, ESI-MS, elemental analyses and single-crystal X-ray diffraction structural analysis (for 1). They were screened against the human tumour cell lines HCT116, HepG2 and MCF7. Complexes 2 and 3 exhibit the highest in vitro cytotoxicity and show lower cytotoxic activities in normal human fibroblast cell line than in HCT116 tumour cell line, which demonstrates their slight specificity for this type of tumour cell.

Silver coordination polymers with tri- and hexacyanoethyl-functionalized macrocyclic ligands

Tri-and hexa-cyanoethyl functionalized 17-(L-1) and 42-membered (L-2) macrocyclic compounds were obtained by [1 + 1] (for L-1) or [2 + 2] (for L-2) cyclocondensation of the corresponding dialdehyde and diethylenetriamine, followed by hydrogenation by KBH4 and subsequent cyano-functionalization with acrylonitrile. They react with silver nitrate, leading to the formation of [AgL1](NO3) (1) and of the metalorganic coordination polymers [Ag-2(NO3)(2)L-1](n) (2) and {[Ag2L2](NO3)(2)}(n) (3). The complexes were characterized by elemental analysis, H-1 NMR, C-13 NMR, IR spectroscopies, and ESI-MS; moreover, L-2, 1, 2 and 3 were also characterized by single crystal X-ray diffraction. The metal cation in 1 is pentacoordinated with a N3O2 coordination environment; in 2, the metal cations display N4O2 octahedral and N2O3 square-pyramid coordination and in 3 they are in square-planar N-4 sites. In 1, the ligand acts as a pentadentate chelator, and in the other two cases, the ligands behave as octadentate chelators in a 1 kappa N-3:kappa O-2,2 kappa N,3 kappa N,4 kappa N (in 2) or 1 kappa N-3,2 kappa N-3,3 kappa N,4 kappa N fashion (in 3). The cyanoethyl strands of the ligands are directly involved in the formation of the 2D frameworks of 2 and 3, which in the former polymer can be viewed as a net composed of hexametallic 36-membered macrocyclic rings and in the latter generates extra hexametallic 58-membered cyclic sets that form zig-zag layers. The thermal analytical and electrochemical properties of these silver complexes were also studied.

Quantification of in vivo short echo-time proton magnetic resonance spectra at 14.1 T using two different approaches of modelling the macromolecule spectrum

Reliable quantification of the macromolecule signals in short echo-time H-1 MRS spectra is particularly important at high magnetic fields for an accurate quantification of metabolite concentrations (the neurochemical profile) due to effectively increased spectral resolution of the macromolecule components. The purpose of the present study was to assess two approaches of quantification, which take the contribution of macromolecules into account in the quantification step. H-1 spectra were acquired on a 14.1 T/26 cm horizontal scanner on five rats using the ultra-short echo-time SPECIAL (spin echo full intensity acquired localization) spectroscopy sequence. Metabolite concentrations were estimated using LCModel, combined with a simulated basis set of metabolites using published spectral parameters and either the spectrum of macromolecules measured in vivo, using an inversion recovery technique, or baseline simulated by the built-in spline function. The fitted spline function resulted in a smooth approximation of the in vivo macromolecules, but in accordance with previous studies using Subtract-QUEST could not reproduce completely all features of the in vivo spectrum of macromolecules at 14.1 T. As a consequence, the measured macromolecular 'baseline' led to a more accurate and reliable quantification at higher field strengths.

Selective resonance suppression 1H-[13C] NMR spectroscopy with asymmetric adiabatic RF pulses.

Despite obvious improvements in spectral resolution at high magnetic field, the detection of 13C labeling by 1H-[13C] NMR spectroscopy remains hampered by spectral overlap, such as in the spectral region of 1H resonances bound to C3 of glutamate (Glu) and glutamine (Gln), and C6 of N-acetylaspartate (NAA). The aim of this study was to develop, implement, and apply a novel 1H-[13C] NMR spectroscopic editing scheme, dubbed "selective Resonance suppression by Adiabatic Carbon Editing and Decoupling single-voxel STimulated Echo Acquisition Mode" (RACED-STEAM). The sequence is based on the application of two asymmetric narrow-transition-band adiabatic RF inversion pulses at the resonance frequency of the 13C coupled to the protons that need to be suppressed during the mixing time (TM) period, alternating the inversion band downfield and upfield from the 13C resonance on odd and even scans, respectively, thus suppressing the detection of 1H resonances bound to 13C within the transition band of the inversion pulse. The results demonstrate the efficient suppression of 1H resonances bound to C3 of Glu and Gln, and C4 of Glu, which allows the 1H resonances bound to C6 of NAA and C4 of Gln to be revealed. The measured time course of the resolved labeling into NAA C6 with the new scheme was consistent with the slow turnover of NAA.

Triterpenóides pentacíclicos de Mentha villosa: identificação estrutural e atribuição dos deslocamentos químicos dos átomos de hidrogênio e carbono

The structures of seven oleanene and ursene triterpenoids (1-7) isolated from aerial parts of Mentha villosa were identified. In addition, the complete ¹H and 13C resonance assignments of these triterpenoids were accomplished using 1D and 2D NMR spectroscopic experiments.

Essential factors for control of the equilibrium in the reversible rearrangement of M3N@Ih-C80 fulleropyrrolidines: Exohedral functional groups versus endohedral metal clusters

The effects of exohedral moieties and endohedral metal clusters on the isomerization of M3N@Ih-C80 products from the Prato reaction through [1,5]-sigmatropic rearrangement were systematically investigated by using three types of fulleropyrrolidine derivatives and four different endohedral metal clusters. As a result, all types of derivatives provided the same ratios of the isomers for a given trimetallic nitride template (TNT) as the thermodynamic products, thus indicating that the size of the endohedral metal clusters inside C80 was the single essential factor in determining the equilibrium between the [6,6]-isomer (kinetic product) and the [5,6]-isomer. In all the derivatives, the [6,6]- and [5,6]-Prato adducts with larger metal clusters, such as Y3N and Gd3N, were equally stable, which is in good agreement with DFT calculations. The reaction rate of the rearrangement was dependent on both the substituent of exohedral functional groups and the endohedral metal-cluster size. Further DFT calculations and 13C NMR spectroscopic studies were employed to rationalize the equilibrium in the rearrangement between the [6,6]- and [5,6]-fulleropyrrolidines