910 resultados para MAS NMR


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Solieria, the type genus of the commercially important red algal family Solieriaceae (Gigartinales), contains seven or eight species, three of which are represented in Australia. The cell-wall galactans of the most common Australian Solieria species, S. robusta (Greville) Kylin, were analysed by a combination of compositional assays, linkage analysis, and Fourier transform infrared (FTIR) and C-13 nuclear magnetic resonance (NMR) spectroscopy. They are shown to be composed predominantly of carrabiose 2,4'-disulphate units (the repeating unit of iota-carrageenan) and a significant proportion of 4',6'-pyruvated carrabiose 2-sulphate units. The constituent sugars, pyruvate content, FTIR spectrum, and linkage and substitution patterns of the galactans from Tikvahiella candida Kraft et Gabrielson, an adelphoparasite of Solieria robusta, closely resemble those of its host and furnish evidence in support of a close phylogenetic relationship between the two species.

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Polymer hydrogels based upon methacrylates are used extensively in the pharmaceutical industry, particularly as controlled release drug delivery systems. These materials are generally prepared by chemically initiated polymerization, but this can lead to the presence of unwanted initiator fragments in the polymer matrix. In the present work, initiation of polymerization by gamma-irradiation of hydroxyethyl methacrylate, with and without added crosslinkers, has been investigated, and the diffusion coefficients for water in the resulting polymers have been measured through mass uptake by the polymers. The diffusion of water in poly(hydroxyethyl methacrylate) at 310 K was found to be Fickian, with a diffusion coefficient of 1.96 +/- 0.1 x 10(11) m(2) s(-1) and an equilibrium water content of 58%, NMR imaging analyses confirmed the adherance to a Fickian model of the diffusion of water into polymer cylinders. The incorporation of small amounts (0.2-0.5 wt%) of added ethyleneglycol-dimethacrylate-based crosslinkers was found to have only a small effect on the diffusion coefficient and the equilibrium water content for the copolymers. (C) 1999 Society of Chemical Industry.

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MiAMP1 is a recently discovered 76 amino acid residue, highly basic protein from the nut kernel of:Macadamia integrifolia which possesses no sequence homology to any known protein and inhibits the growth of several microbial plant pathogens in vitro while having no effect on mammalian or plant cells. It is considered to be a potentially useful tool for the genetic engineering of disease resistance in transgenic crop plants and for the design of new fungicides. The three-dimensional structure of MiAMP1 was determined through homonuclear and heteronuclear (N-15) 2D NMR spectroscopy and subsequent simulated annealing calculations with the ultimate aim of understanding the structure-activity relationships of the protein. MiAMP1 is made up of eight beta-strands which are arranged in two Greek key motifs. These Greek key motifs associate to form a Greek key beta-barrel. This structure is unique amongst plant antimicrobial proteins and forms a new class which we term the beta-barrelins. Interestingly, the structure of MiAMP1 bears remarkable similarity to a yeast killer toxin from Williopsis mrakii. This toxin acts by inhibiting beta-glucan synthesis and thereby cell wall construction in sensitive strains of yeast. The structural similarity of MiAMP1 and WmKT, which originate from plant and fungal phyla respectively, may reflect a similar mode of action. (C) 1999 Academic Press.

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The bulk free radical copolymerizations of 2-hydroxyethyl methacrylate (HEMA) with n-butyl methacrylate (BMA) or cyclohexyl methacrylate (CHMA) were studied over the composition mole fraction interval of 0-1 for HEMA in the monomer feed. The C-13 NMR (125 MHz) spectra of the copolymers were analysed to determine the copolymer composition and the stereochemical configuration of the copolymers. The terminal model reactivity ratios of HEMA and BMA were found to be r(HEMA) = 1.73 and r(BMA) = 0.65 and for HEMA and CHMA, r(HEMA) = 1.26 and r(CHMA) = 0.31. The BMA and CHMA homopolymers were found to be predominantly syndiotactic with isotacticity parameters of theta(BB) = 0.18 and theta(CC) = 0.19, respectively. The copolymers were also found to be predominantly syndiotactic, indicating a strong tendency for racemic additions of the monomers in the formation of the copolymers. The diffusion of water into cylinders of poly(HEMA-co-BMA) and poly(HEMA-co-CHMA) was studied over a range of copolymer compositions and was found to be Fickian. The diffusion coefficients of water at 37 degrees C were determined from swelling measurements and were found to vary from 1.72 x 10(-11) m(2) s(-1) for polyHEMA to 0.97 x 10(-11) m(2) s(-1) for poly(HEMA-co-BMA) having a mole fraction F-HEMA = 0.80 and to 0.91 x 10(-11) m(2) s(-1) for a poly(HEMA-co-CHMA) also having F-HEMA = 0.80. The mass of water absorbed at equilibrium relative to the mass of dry polymer varied from 58.8 for polyHEMA to 27.2% for poly(HEMA-co-BMA) having F-HEMA = 0.85 and to 21.3% for poly(HEMA-co-CHMA) having F-HEMA = 0.80. (C) 1999 Elsevier Science Ltd. All rights reserved.

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This paper describes a hybrid numerical method for the design of asymmetric magnetic resonance imaging magnet systems. The problem is formulated as a field synthesis and the desired current density on the surface of a cylinder is first calculated by solving a Fredholm equation of the first kind. Nonlinear optimization methods are then invoked to fit practical magnet coils to the desired current density. The field calculations are performed using a semi-analytical method. A new type of asymmetric magnet is proposed in this work. The asymmetric MRI magnet allows the diameter spherical imaging volume to be positioned close to one end of the magnet. The main advantages of making the magnet asymmetric include the potential to reduce the perception of claustrophobia for the patient, better access to the patient by attending physicians, and the potential for reduced peripheral nerve stimulation due to the gradient coil configuration. The results highlight that the method can be used to obtain an asymmetric MRI magnet structure and a very homogeneous magnetic field over the central imaging volume in clinical systems of approximately 1.2 m in length. Unshielded designs are the focus of this work. This method is flexible and may be applied to magnets of other geometries. (C) 1999 Academic Press.

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The alpha-conotoxins, a class of nicotinic acetylcholine receptor (nAChR) antagonists, are emerging as important probes of the role played by different nAChR subtypes in cell function and communication, In this study, the native alpha-conotoxins PnIA and PnIB were found to cause concentration-dependent inhibition of the ACh-induced current in all rat parasympathetic neurons examined, with IC50 values of 14 and 33 nM, and a maximal reduction in current amplitude of 87% and 71%, respectively. The modified alpha-conotoxin [N11S]PnIA reduced the ACh-induced current with an IC50 value of 375 nM and a maximally effective concentration caused 91% block, [A10L]PnIA was the most potent inhibitor, reducing the ACh-induced current in similar to 80% of neurons, with an IC50 value of 1.4 nM and 46% maximal block of the total current, The residual current was not inhibited further by alpha-bungarotoxin, but was further reduced by the cu-conotoxins PnIA or PnIB, and by mecamylamine. H-1 NMR studies indicate that PnIA, PnIB, and the analogues, [A10L]PnIA and [N11S]PnIA, have identical backbone structures. We propose that positions 10 and II of PnIA and PnIB influence potency and determine selectivity among alpha 7 and other nAChR subtypes, including alpha 3 beta 2 and alpha 3 beta 4, Four distinct components of the nicotinic ACh-induced current in mammalian parasympathetic neurons have been dissected with these conopeptides.

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The 12-membered macrocyclic ligand 1-thia-4,7, 10-triazacyclododecane ([12]aneN(3)S) has been synthesised, although upon crystallization from acetonitrile a product in which carbon dioxide had added to one secondary amine in the macrocyclic ring (H[12]aneN(3)SCO(2). H2O) was isolated and subsequently characterised by X-ray crystallography. The protonation constants for [12]aneN(3)S and stability constants with Zn(II), Pb(II), Cd(II) and Cu(II) have been determined either potentiometrically or spectrophotometrically in aqueous solution, and compared with those measured or reported for the ligands 1-oxa-4,7,10-triazacyclododecane ([12]aneN(3)O) and 1,4,7,10-tetraazacyclododecane ([12]aneN(4)). The magnitudes of the stability constants are consistent with trends observed previously for macrocyclic ligands as secondary amine donors are replaced with oxygen and thioether donors although the stability constant for the [Hg([12]aneN(4))](2+) complex has been estimated from an NMR experiment to be at least three orders of magnitude larger than reported previously. Zinc(II), mercury(II), lead(II), copper(II) and nickel(II) complexes of [12]aneN(3)S have been isolated and characterised by X-ray crystallography. In the case of copper(II), two complexes [Cu([12]aneN(3)S)(H2O)](ClO4)(2) and [Cu-2([12]aneN(3)S)(2)(OH)(2)](ClO4)(2) were isolated, depending on the conditions employed. Molecular mechanics calculations have been employed to investigate the relative metal ion size preferences of the [3333], asym-[2424] and sym-[2424] conformation isomers. The calculations predict that the asym-[2424] conformer is most stable for M-N bond lengths in the range 2.00-2.25 Angstrom whilst for the larger metal ions the [3333] conformer is dominant. The disorder seen in the structure of the [Zn([12]aneN(3)S)(NO3)](+) complex is also explained by the calculations. (C) 1999 Elsevier Science Ltd. All rights reserved.

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Several macrocyclic peptides (similar to 30 amino acids), with diverse biological activities, have been isolated from the Rubiaceae and Violaceae plant families over recent years. We have significantly expanded the range of known macrocyclic peptides with the discovery of 16 novel peptides from extracts of Viola hederaceae, Viola odorata and Oldenlandia affinis. The Viola plants had not previously been examined for these peptides and thus represent novel species in which these unusual macrocyclic peptides are produced. Further, we have determined the three-dimensional struc ture of one of these novel peptides, cycloviolacin O1, using H-1 NMR spectroscopy. The structure consists of a distorted triple-stranded beta-sheet and a cystine-knot arrangement of the disulfide bonds. This structure is similar to kalata B1 and circulin A, the only two macrocyclic peptides for which a structure was available, suggesting that despite the sequence variation throughout the peptides they form a family in which the overall fold is conserved. We refer to these peptides as the cyclotide family and their embedded topology as the cyclic cystine knot (CCK) motif. The unique cyclic and knotted nature of these molecules makes them a fascinating example of topologically complex proteins. Examination of the sequences reveals they can be separated into two subfamilies, one of which tends to contain a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-Pro peptide bond and may conceptually be regarded as a molecular Moebius strip. Here we define the structural features of the two apparent subfamilies of the CCK peptides which may be significant for the likely defense related role of these peptides within plants. (C) 1999 Academic Press.

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A comparison is made between the structures and calcium binding properties of four cyclic octapeptides that differ in the number of heterocyclic thiazole and oxazoline ring constraints. The conformations of the naturally occurring cyclic octapeptides ascidiacyclamide 1 and patellamide D 2, which each contain two oxazoline and two thiazole rings, are compared by H-1 NMR spectroscopy with the analogues cyclo(Thr-D-Val(Thz)-Ile)(2) 3 with just two thiazoles, and cyclo(Thr-D-Val-alpha Abu-Ile)(2) 4, with no 5-membered rings. The conformations observed in the solid state for ascidiacyclamide (saddle) and patellamide D (twisted figure of eight) were retained in solution, whilst peptide 3 was found to have a chair shape and peptide 4 displayed a range of conformations. The solid state structure of 4 revealed that the peptide takes a relatively planar conformation with a number of transannular hydrogen bonds, which are apparently retained in solution. Complexation studies utilising H-1 NMR and CD spectroscopy yielded 1∶1 calcium-peptide binding constants (log K) for the four peptides (2.9 (1), 2.8 (2), 4.0 (3) and 5.5 (4)) as well as a 1 : 2 metal-peptide binding constant for 3 (log K = 4.5). The affinity for Ca2+ thus decreases with increasing number of 5-membered ring constraints in the macrocycle (4 > 3 > 2 approximate to 1).

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The 32-residue peptide, RK-1, a novel kidney-derived three disulfide-bonded member of the antimicrobial alpha-defensin family, was synthesized by the continuous now Fmoc-solid phase method. The crude, cleaved and S-reduced Linear peptide was both efficiently folded and oxidized in an acidic solution of aqueous dimethyl sulfoxide. Following purification of the resulting product, it was shown by a variety of analytical techniques, including matrix assisted laser desorption time of flight mass spectrometry, to possess a very high degree of purity. The disulfide bond pairing of the synthetic peptide was determined by H-1-NMR spectroscopy and confirmed to be a Cys(1)-Cys(6), Cys(2)-Cys(4), Cys(3)-Cys(5) arrangement similar to other mammalian alpha-defensin peptides. The synthetic RK-1 was also shown to inhibit the growth of Escherichia coli type strain NCTC 10418, Copyright (C) 2000 European Peptide Society and John Wiley & Sons, Ltd.

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A method is presented for including path propagation effects into models of radiofrequency resonators for use in magnetic resonance imaging. The method is based on the use of Helmholtz retarded potentials and extends our previous work on current density models of resonators based on novel inverse finite Hilbert transform solutions to the requisite integral equations. Radiofrequency phase retardation effects are most pronounced at high field strengths (frequencies) as are static field perturbations due to the magnetic materials in the resonators themselves. Both of these effects are investigated and a novel resonator structure presented for use in magnetic resonance microscopy.

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Inhibitors of proteolytic enzymes (proteases) are emerging as prospective treatments for diseases such as AIDS and viral infections, cancers, inflammatory disorders, and Alzheimer's disease. Generic approaches to the design of protease inhibitors are limited by the unpredictability of interactions between, and structural changes to, inhibitor and protease during binding. A computer analysis of superimposed crystal structures for 266 small molecule inhibitors bound to 48 proteases (16 aspartic, 17 serine, 8 cysteine, and 7 metallo) provides the first conclusive proof that inhibitors, including substrate analogues, commonly bind in an extended beta-strand conformation at the active sites of all these proteases. Representative superimposed structures are shown for (a) multiple inhibitors bound to a protease of each class, (b) single inhibitors each bound to multiple proteases, and (c) conformationally constrained inhibitors bound to proteases. Thus inhibitor/substrate conformation, rather than sequence/composition alone, influences protease recognition, and this has profound implications for inhibitor design. This conclusion is supported by NMR, CD, and binding studies for HIV-1 protease inhibitors/ substrates which, when preorganized in an extended conformation, have significantly higher protease affinity. Recognition is dependent upon conformational equilibria since helical and turn peptide conformations are not processed by proteases. Conformational selection explains the resistance of folded/structured regions of proteins to proteolytic degradation, the susceptibility of denatured proteins to processing, and the higher affinity of conformationally constrained 'extended' inhibitors/substrates for proteases. Other approaches to extended inhibitor conformations should similarly lead to high-affinity binding to a protease.

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Kalata B1 is a prototypic member of the unique cyclotide family of macrocyclic polypeptides in which the major structural features are a circular peptide backbone, a triple stranded beta-sheet, and a cystine knot arrangement of three disulfide bonds. The cyclotides are the only naturally occurring family of circular proteins and have prompted us to explore the concept of acyclic permutation, i.e. opening the backbone of a cross-linked circular protein in topologically permuted ways. We have synthesized the complete suite of acyclic permutants of kalata B1 and examined the effect of acyclic permutation on structure and activity. Only two of six topologically distinct backbone loops are critical for folding into the native conformation, and these involve disruption of the embedded ring in the cystine knot. Surprisingly, it is possible to disrupt regions of the p-sheet and still allow folding into native-like structure, provided the cystine knot is intact. Kalata B1 has mild hemolytic activity, but despite the overall structure of the native peptide being retained in all but two cases, none of the acyclic permutants displayed hemolytic activity. This loss of activity is not localized to one particular region and suggests that cyclization is critical for hemolytic activity.

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We have shown that 44 amino acid residues N-terminal segment of kappa-casein exhibits considerable a-helical structure. This prompted us to investigate the structures of the remaining segments of kappa-casein. Thus, in this study the chemical synthesis and structure elucidation of the peptide 45-87 amino acid residues of kappa-casein is reported. The peptide was assembled using solid phase peptide synthesis methodology on pam resin, cleaved via HF, freeze dried and, after purification, characterised by mass spectrometry (observed m/z 4929; calculated mit 4929.83). The amino acid sequence of the peptide is: CKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKA Its structure elucidation has been carried out using circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques. CD spectrum of the peptide shows it to be a random structure in water but in 30% trifluoroethanol the peptide exhibits considerable structure. The 1D and 2D NMR spectra corroborated the results of CD. The structure elucidation of the peptide using TOCSY and NOESY NMR techniques will be discussed.

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The three-dimensional solution structure of conotoxin TVIIA, a 30-residue polypeptide from the venom of the piscivorous cone snail Conus tulipa, has been determined using 2D H-1 NMR spectroscopy. TVIIA contains six cysteine residues which form a 'four-loop' structural framework common to many peptides from Conus venoms including the omega-, delta-, kappa-, and mu O-conotoxins. However, TVIIA does not belong to these well-characterized pharmacological classes of conotoxins, but displays high sequence identity with conotoxin GS, a muscle sodium channel blocker from Conus geographus. Structure calculations were based on 562 interproton distance restraints inferred from NOE data, together with 18 backbone and nine side-chain torsion angle restraints derived from spin-spin coupling constants. The final family of 20 structures had mean pairwise rms differences over residues 2-27 of 0.18 +/- 0.05 Angstrom for the backbone atoms and 1.39 +/- 0.33 Angstrom for all heavy atoms. The structure consists of a triple-stranded, antiparallel beta sheet with +2x, -1 topology (residues 7-9, 16-20 and 23-27) and several beta turns. The core of the molecule is formed by three disulfide bonds which form a cystine knot motif common to many toxic and inhibitory polypeptides. The global fold, molecular shape and distribution of amino-acid sidechains in TVIIA is similar to that previously reported for conotoxin GS, and comparison with other four-loop conotoxin structures provides further indication that TVIIA and GS represent a new and distinct subgroup of this structural family. The structure of TVIIA determined in this study provides the basis for determining a structure-activity relationship for these molecules and their interaction with target receptors.