Structure-activity relationships of omega-conotoxins MVIIA, MVIIC and 14 loop splice hybrids at N and P/Q-type calcium channels

The omega-conotoxins are a set of structurally related, four-loop, six cysteine containing peptides, that have a range of selectivities for different subtypes of the voltage-sensitive calcium channel (VSCC). To investigate the basis of the selectivity displayed by these peptides, we have studied the binding affinities of two naturally occurring omega-conotoxins, MVIIA and MVIIC and a series of 14 MVIIA/MVIIC loop hybrids using radioligand binding assays for N and P/Q-type Ca2+ channels in rat brain tissue. A selectivity profile was developed from the ratio of relative potencies at N-type VSCCs (using [I-125]GVIA radioligand binding assays) and P/Q-type VSCCs (using [I-125]MVIIC radioligand binding assays). in these peptides, loops 2 and 4 make the greatest contribution to VSCC subtype selectivity, while the effects of loops 1 and 3 are negligible. Peptides with homogenous combinations of loop 2 and 4 display clear selectivity preferences, while those with heterogeneous combinations of loops 2 and 4 are less discriminatory. H-1 NMR spectroscopy revealed that the global folds of MVIIA, MVIIC and the 14 loop hybrid peptides were similar; however, several differences in local structure were identified. Based on the binding data and the 3D structures of MVIIA, GVIA and MVIIC, we have developed a preliminary pharmacophore based on the omega-conotoxin residues most Likely to interact with the N-type VSCC. (C) 1999 Academic Press.

Carbon structure and porosity of carbonaceous adsorbents in relation to their adsorption properties

A number of carbonaceous adsorbents were prepared by carbonisation at 600 degrees C following acidic oxidation under various conditions. Effects of the chemical nature of the precursor, such as the ratio of aromatic to aliphatic carbons and oxygen content, on the chemical and structural characteristics of the resultant chars were investigated using C-13 NMR and Raman spectroscopy, respectively. The C-13 NMR spectral parameters of the coal samples show that as the severity of oxidation conditions increased, the ratio of aromatic to aliphatic carbons increased. Furthermore, it was also found that the amount of disorganised carbon affects both the pore structure and the adsorption properties of carbonaceous adsorbents. It is demonstrated that higher amount of the disorganised carbon indicates smaller micropore size. (C) 1999 Elsevier Science Ltd. All rights reserved.

Chemical synthesis and folding pathways of large cyclic polypeptide: Studies of the cystine knot polypeptide kalata B1

Kalata B1 is a member of a new family of polypeptides, isolated from. plants, which have a cystine knot structure embedded within an amide-cyclized backbone. This family of molecules are the largest known cyclic peptides, and thus, the mechanism of synthesis and folding is of great interest. To provide information about both these phenomena, we have synthesized kalata B1 using two distinct strategies. In the first, oxidation of the cysteine residues of a linear precursor peptide to form the correct disulfide bonds results in folding of the three-dimensional structure and preorganization of the termini in close proximity for subsequent cyclization. The second approach involved cyclization prior to oxidation. In the first method, the correctly folded peptide was produced only in the presence of partially hydrophobic solvent conditions. These conditions are presumably required to stabilize the surface-exposed hydrophobic residues. However,; in the synthesis,involving cyclization prior to oxidation, the cyclic reduced peptide folded to a significant degree in the absence of hydrophobic solvents and even more efficiently in the presence of hydrophobic solvents. Cyclization clearly has a major effect on the folding pathway and facilitates formation of the correctly disulfide-bonded form in aqueous solution; In addition to facilitating folding to a compact stable structure cyclization has an important effect on biological activity as assessed by hemolytic activity.

Pyruvated carrageenans from Solieria robusta and its adelphoparasite Tikvahiella candida

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.

Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif

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.

Conformations of cyclic octapeptides and the influence of heterocyclic ring constraints upon calcium binding

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).

Chemical synthesis, characterization and activity of RK-1, a novel alpha-defensin-related peptide

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.

Acyclic permutants of naturally occurring cyclic proteins - Characterization of cystine knot and beta-sheet formation in the macrocyclic polypeptide kalata B1

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.

The relationship between radiation-induced chemical processes and transverse relaxation times in polymer gel dosimeters

The effects of ionizing radiation in different compositions of polymer gel dosimeters are investigated using FT-Raman spectroscopy and NMR T-2 relaxation times. The dosimeters are manufactured from different concentrations of comonomers (acrylamide and N,N'-methylene-bis-acrylamide) dispersed in different concentrations of an aqueous gelatin matrix. Results are analysed using a model of fast exchange of magnetization between three proton pools. The fraction of protons in each pool is determined using the known chemical composition of the dosimeter and FT-Raman spectroscopy. Based on these results, the physical and chemical processes in interplay in the dosimeters are examined in view of their effect on the changes in T-2 The precipitation of growing macroradicals and the scavenging of free radicals by gelatin are used to explain the rate of polymerization. The model describes the changes in T-2 as a function of the absorbed dose up to 50 Gy for the different compositions. This is expected to aid the theoretical design of new, more efficient dosimeters, since it was demonstrated that the optimum dosimeter (i.e, with the lowest dose resolution) must have a range of relaxation times which match the range of T-2 values which can be determined with the lowest uncertainty using an MRI scanner.

Cyclic octapeptides containing thiazole. Effect of stereochemistry and degree of flexibility on calcium binding properties

Solution conformation and calcium binding properties have been investigated for the two cyclic octapeptides cyclo(-D-Thr-D-Val(Thz)-Ile-)(2) (4) and cyclo(-Thr-Gly(Thz)-Ile-Ser-Gly(Thz)-Ile-)(5) and the results are compared to those for the cyclic octapeptides previously studied; ascidiacyclamide (1), patellamide D (2), cyclo(-Thr-D-Val(Thz)-Ile-)(2) (3), and cyclo(-Thr-D-Val-alphaAbu-Ile-)2 (6). Both 4 and 5 contain two heterocyclic thiazole ring constraints but the latter has a larger degree of flexibility as a consequence of the glycine residues within the cyclic framework. The solution conformation of 4 and 5 was determined from H-1 NMR spectra and found to be a twisted figure of eight similar to that for 2. Complexation studies using H-1 NMR and CD spectroscopy yielded 1 : 1 calcium-peptide binding constants (logK) for the two peptides (2.3 (4) and 5.7 (5)). For 5 the magnitude of the binding constant was verified by a competition titration using CD. The different calcium-binding affinities of 3 (logK = 4.0) and 4 is attributed to the stereochemistry of the threonine residue. The magnitude of the binding constant for 5 compared to 3 and 4 (all peptides containing two thiazole ring constrains) demonstrates that the increase in flexibility of the cyclic peptide has a dramatic effect on the Ca2+ binding ability. The affinity for Ca2+ thus decreases in the order (6 similar to 5 > 3 > 2 similar to 1 > 4). The number of carbonyl donors available on each peptide has only a limited effect on calcium binding. The most important factor is the flexibility, which allows for a conformation of the peptide capable of binding calcium efficiently.

Conformations of platypus venom C-type natriuretic peptide in aqueous solution and sodium dodecyl sulfate micelles

Nuclear magnetic resonance spectroscopy was used to investigate the conformations of the platypus venom C-type natriuretic peptide A (OvCNPa) in aqueous solutions and in solutions containing sodium dodecyl sulfate (SDS) micelles. The chemically synthesized OvCNPa showed a substantial decrease in flexibility in aqueous solution at 10 degreesC, allowing the observation of medium- and long-range nuclear Overhauser enhancement (NOE) connectivities. Three-dimensional structures calculated using these data showed flexible and reasonably well-defined regions, the locations of which were similar in the two solvents. In aqueous solution, the linear part that spans residues 3-14 was basically an extended conformation while the cyclic portion, defined by residues 23-39, contained a series of beta-turns. The overall shape of the cyclic portion was similar to that observed for an atrial natriuretic peptide (ANP) variant in aqueous solution. OvCNPa adopted a different conformation in SDS micelles wherein the N-terminal region, defined by residues 2-10, was more compact, characterised by turns and a helix, while the cyclic region had turns and an overall shape that was fundamentally different from those structures observed in aqueous solution. The hydrophobic cluster, situated at the centre of the ring of the structure in aqueous solution, was absent in the structure in the presence of SDS micelles. Thus, OvCNPa interacts with SDS micelles and can possibly form ion-channels in cell membranes. (C) 2002 Elsevier Science Ltd. All rights reserved.

Luminescent Langmuir-Blodgett film of a new amphiphilic Eu(3+) beta-diketonate

This work reports on the synthesis and characterization of the ligand 3-hexadecylpentane-2,4-drone (Hhdacac) and its Eu(3+) complexes Eu(hdacac)(6) center dot 2H(2)O, Eu(hdacac)(6) center dot phen and Eu(hdacac)(6) center dot tta, where phen and tta denote 1,10-phenanthroline and thenoyltrifluoroacetone, respectively. These new compounds present long carbon chains and their expected miscibility into non-polar ambients is confirmed by the emission spectra of Eu(hdacac)6 center dot tta in hexane. Moreover, the amphiphilic properties of Eu(hdacac)6 complexes allow the obtainment of thin luminescent films by the Langmuir-Blodgett technique. In both cases (solids and films), the typical antenna effect of beta-diketonates is observed. The alluring characteristics of these compounds raise great interest in many fields of Materials Science, like photo- and electro-luminescent materials (mainly thin ""organic"" films), metal catalysts or probes in non-polar solutions, and Langmuir-Blodgett films of several compositions. For the characterization of these products, nuclear magnetic resonance spectroscopy ((1)H NMR), thermogravimetric analysis, elementary analyses (C, H), scanning electron microscopy (energy dispersive X-ray spectroscopy), absorption (UV-vis/FT-IR) and photoluminescence spectroscopies were used. (c) 2008 Elsevier B.V. All rights reserved.

H-1 NMR spectroscopic studies of the stability of an oxazolidine condensation product

Condensation of (-)-norephedrine with excess formaldehyde under mild conditions leads to formation of the 2:1 condensation product N,N'-methylenebis(4-methyl-5-phenyl)oxazolidine compared with the reaction with 1 mol of formaldehyde, which leads to 4-methyl-5-phenyloxazolidine. H-1 and C-13 NMR spectroscopy was used to monitor the stability of this compound and its decomposition products. The 2:1 condensation product is found to be stable in CDC1(3) but breaks down rapidly in CD3OD to yield a 50:50 mixture of 4-methyl-5-phenyloxazolidine and 3-hydroxymethyl-4-methyl-5-phenyloxazolidine. Upon addition of D2O to this equimolar mixture, the latter compound decomposes to norephedrine and formaldehyde, whereas the former compound is stable. (C) 1997 by John Wiley & Sons, Ltd.

Molecular motion in miscible polymer blends .1. Motion in blends of PEO and PVPh studied by solid-state C-13 T-1 rho measurements

Changes in molecular motion in blends of PEO-PVPh have been studied using measurements of C-13 T-1 rho relaxation times. C-13 T-1 rho relaxation has been confirmed as arising from spin-lattice interactions by observation of the variation in T-1 rho with rf field strength and temperature. In the pure homopolymers a minimum in T-1 rho is observed at ca. 50 K above the glass transition temperatures detected by DSC. After blending, the temperature of the minimum in T-1 rho for PEO increased, while that for PVPh decreased, however, the minima, which correspond to the temperatures where the average correlation times for reorientation are close to 3.1 mu s, are separated by 45 K (in a 45% PEO-PVPh blend). These phenomena are explained in terms of the local nature of T-1 rho measurements. The motions of the individual homopolymer chains are only partially coupled in the blend. A short T-1 rho has been observed for protonated aromatic carbons, and assigned to phenyl rings undergoing large-angle oscillatory motion, The effects of blending, and temperature, on the proportion of rings undergoing oscillatory motion are analyzed.