Identification of a new ligand binding domain in the al subunit of the inhibitory glycine receptor

Four discontinuous extracellular sequence domains have been proposed to form the ligand binding sites of the ligand-gated ion channel receptor superfamily. In this study, we investigated the role of 12 contiguous residues of the inhibitory glycine receptor that define the proposed loop A ligand binding domain; Using the techniques of site-directed mutagenesis and patch-clamp electrophysiology, four of the 12 residues were shown to have impaired ligand binding. Three mutants, I93A, A101H, and N102A, resulted in significant (17-44-fold) increases in the agonist EC50 values as compared with the wild-type glycine receptor, whereas Hill coefficients, I-max values, and antagonist affinity remained largely unaffected. Consideration of receptor efficacy values indicates that these residues are involved in ligand binding rather than channel activation. A fourth mutant, W94A, failed to give rise to any glycine-activated currents, although cell-surface expression was observed, suggesting that this residue may also be involved in agonist binding. These data provide the most extensive characterization of the loop A ligand binding domain available to date and define two new residue locations, Ile(93) and Asn(102), as contributing to the four-loop model of ligand binding.

High level of aspartic acid-bond isomerization during the synthesis of an N-linked tau glycopeptide

An increased degree of utilization of the potential N-glycosylation site In the fourth repeat unit of the human tau protein may be involved in the inability of tau to bind to the corresponding tubulin sequence(s) and in the subsequent development of the paired helical filaments of Alzheimer's disease. To model these processes, we synthesized the octadecapeptide spanning this region without sugar, and with the addition of an N-acetyl-glucosamine moiety. The carbohydrate-protected, glycosylated asparagine was incorporated as a building block during conventional Fmoc-solid phase peptide synthesis. While the crude non-glycosylated analog was obtained as a single peptide, two peptides with, the identical, expected masses, in approximately equal amounts, were detected after the cleavage of the peracetylated glycopeptide. Surprisingly, the two glycopeptides switched positions on the reversed-phase high performance liquid chromatogram after removal of the sugar-protecting acetyl groups. Nuclear magnetic resonance spectroscopy and peptide sequencing identified the more hydrophobic deprotected peak as the target peptide, and the more hydrophilic deprotected peak as a peptide analog in which the aspartic acid-bond just preceding the glycosylated asparagine residue was isomerized resulting in the formation of a beta-peptide. The anomalous chromatographic behavior of the acetylated beta-isomer could be explained on the basis of the generation of an extended hydrophobic surface which is not present in any of the other three glycopeptide variants. Repetition of the syntheses, with altered conditions and reagents, revealed reproducibly high levels of aspartic acid-bond isomerization of the glycopeptide as well as lack of isomerization for the non-glycosylated parent analog. If similar increased aspartic acid-bond isomerization occurs in vivo, a protein modification well known to take place for both the amyloid deposits and the neurofibrillary tangles in Alzheimer's disease, this process may explain the aggregation of glycosylated tau into the paired helical filaments in the affected brains. Copyright (C) 1999 European Peptide Society and John Wiley & Sons, Ltd.

MiAMP1, a novel protein from Macadamia integrifolia adopts a Greek key beta-barrel fold unique amongst plant antimicrobial proteins

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.

Development and characterization of novel potent and stable inhibitors of endopeptidase EC 3.4.24.15

Solid-phase synthesis was used to prepare a series of modifications to the selective and potent inhibitor of endopeptidase EC 3.4.24.15 (EP24.15), N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP), which is degraded at the Ala-Tyr bond, thus severely limiting its utility in vivo. Reducing the amide bond between the Ala and Tyr decreased the potency of the inhibitor to 1/1000. However, the replacement of the second alanine residue immediately adjacent to the tyrosine with alpha-aminoisobutyric acid gave a compound (JA-2) that was equipotent with cFP, with a K-i of 23 nM. Like cFP, JA-2 inhibited the closely related endopeptidase EC 3.4.24.16 1/20 to 1/30 as potently as it did EP24.15, and did not inhibit the other thermolysin-like endopeptidases angiotensin-converting enzyme, endothelin-converting enzyme and neutral endopeptidase. The biological stability of JA-2 was investigated by incubation with a number of membrane and soluble sheep tissue extracts. In contrast with cFP, JA-2 remained intact after 48 h of incubation with all tissues examined. Further modifications to the JA-2 compound failed to improve the potency of this inhibitor. Hence JA-2 is a potent, EP24.15-preferential and biologically stable inhibitor, therefore providing a valuable tool for further assessing the biological functions of EP24.15.

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.

Leu(10) of alpha-conotoxin PnIB confers potency for neuronal nicotinic responses in bovine chromaffin cells

Two alpha-conotoxins PnIA and PnIB (previously reported as being mollusc specific) which differ in only two amino acid residues (AN versus LS at residues 10 and 11, respectively), show markedly different inhibition of the neuronal nicotinic acetylcholine receptor response in bovine chromaffin cells, a mammalian preparation. Whereas alpha-conotoxin PnIB completely inhibits the nicotine-evoked catecholamine release at 10 mu M, with IC50 = 0.7 mu M, alpha-conotoxin PnIA is some 30-40 times less potent. Two peptide analogues, [A10L]PnIA and [N11S]PnIA were synthesized to investigate the extent to which each residue contributes to activity. [A10L]PnIA (IC50 = 2.0 mu M) completely inhibits catecholamine release at 10 mu M whereas [N11S]PnIA shows Little inhibition. In contrast, none of the peptides inhibit muscle-type nicotinic responses in the rat hemi-diaphragm preparation. We conclude that the enhanced potency of alpha-conotoxin PnIB over alpha-conotoxin PnIA in the neuronal-type nicotinic response is principally determined by the larger, more hydrophobic leucine residue at position 10 in alpha-conotoxin PnIB. (C) 2000 Elsevier Science B.V. All rights reserved.

Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa - 2. Three-dimensional solution structure

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.

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.

Functional analysis, using in vitro mutagenesis, of amino acids located in the phenylalanine hydroxylase active site

The 3-dimensionaI structure determination of rat phenylalanine hydroxylase (PAH) has identified potentially important amino acids lining the active site cleft with the majority of these having hydrophobic side-chains including several with aromatic side chains. Here we have analyzed the effect on rat PAH enzyme kinetics of in vitro mutagenesis of a number of these amino acids lining the PAH active site. Mutation of F299, Y324, F331, and Y343 caused a significant decrease in enzyme activity but no change in the K-m for substrate or cofactor. me conclude that these aromatic residues are essential for activity but are not significantly involved in binding of the substrate or cofactor. in contrast the PAH mutant, S349T, showed an 18-fold increase in K-m for phenylalanine, showing the first functional evidence that this residue was binding at or near the phenylalanine binding site. This confirms the recently published model for the binding of phenylalanine to the PAH active site that postulated S349 interacts with the amino group on the main chain of the phenylalanine molecule. This result differs with that found for the equivalent mutation (S395T), in the closely related tyrosine hydroxylase, which had no effect on substrate K-m, showing that while the architecture of the two active sites are very similar the amino acids that bind to the respective substrates are different. (C) 2000 Academic Press.

Residues of zeta-cypermethrin in bovine tissues and milk following pour-on and spray application

The depletion of zeta-cypermethrin residues in bovine tissues and milk was studied. Beef cattle were treated three times at 3-week intervals with 1 ml 10 kg(-1) body weight of a 25 g litre(-1) or 50 g litre(-1) pour-on formulation (2.5 and 5.0 mg zeta-cypermethrin kg(-1) body weight) or 100 mg kg(-1) spray to simulate a likely worst-case treatment regime. Friesian and Jersey dairy cows were treated once with 2.5 mg zeta-cypermethrin kg(-1) in a pour-on formulation. Muscle, liver and kidney residue concentrations were generally less than the limit of detection (LOD = 0.01 mg kg(-1)). Residues in renal-fat and back-fat samples from animals treated with 2.5 mg kg(-1) all exceeded the limit of quantitation (LOQ = 0.05 mg kg(-1)), peaking at 10 days after treatment. Only two of five kidney fat samples were above the LOQ after 34 days, but none of the back-fat samples exceeded the LOQ at 28 days after treatment. Following spray treatments, fat residues were detectable in some animals but were below the LOQ at all sampling intervals. Zeta-cypermethrin was quantifiable (LOQ = 0.01 mg kg(-1)) in only one whole-milk sample from the Friesian cows (0.015 mg kg(-1), 2 days after treatment). In whole milk from Jersey cows, the mean concentration of zeta-cypermethrin peaked 1 day after treatment, at 0.015 mg kg(-1), and the highest individual sample concentration was 0.025 mg kg(-1) at 3 days after treatment. Residues in milk were not quantifiable beginning 4 days after treatment. The mean concentrations of zeta-cypermethrin in milk fat from Friesian and Jersey cows peaked two days after treatment at 0.197 mg kg(-1) and 0.377 mg kg(-1), respectively, and the highest individual sample concentrations were 2 days after treatment at 0.47 mg kg(-1) and 0.98 mg kg(-1), respectively. (C) 2001 Society of Chemical Industry.

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.

The p35 relative, p49, inhibits mammalian and Drosophila caspases including DRONC and protects against apoptosis

This study characterized the ability of a new member of the p35 family, p49, to inhibit a number of mammalian and insect caspases. p49 blocked apoptosis triggered by treatment with Fas ligand (FasL), Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or ultraviolet (UV) radiation but provided negligible protection against apoptosis induced by the chemotherapeutic drug cisplatin. The caspase cleavage site in p49 was determined, and mutation of the 131 residue of this site abolished the ability of p49 to inhibit caspases, implying that p49 inhibits caspases through an analogous suicide-substrate mechanism to p35. Unlike p35, p49 inhibited the upstream insect caspase DRONC.

Effects of restricted cochlear lesions in adult cats on the frequency organization of the inferior colliculus

Restricted cochlear lesions in adult animals result in plastic changes in the representation of the lesioned cochlea, and thus in the frequency map, in the contralateral auditory cortex and thalamus. To examine the contribution of subthalamic changes to this reorganization, the effects of unilateral mechanical cochlear lesions on the frequency organization of the central nucleus of the inferior colliculus (ICC) were examined in adult cats. Lesions typically resulted in a broad high-frequency hearing loss extending from a frequency in the range 15-22 kHz. After recovery periods of 2.5-18 months, the frequency organization of ICC contralateral to the lesioned cochlea was determined separately for the onset and late components of multiunit responses to tone-burst stimuli. For the late response component in all but one penetration through the ICC, and for the onset response component in more than half of the penetrations, changes in frequency organization in the lesion projection zone were explicable as the residue of prelesion responses. In half of the penetrations exhibiting nonresidue type changes in onset-response frequency organization, the changes appeared to reflect the unmasking of normally inhibited inputs. In the other half it was unclear whether the changes reflected unmasking or a dynamic process of reorganization. Thus, most of the observed changes were explicable as passive consequences of the lesion, and there was limited evidence for plasticity in the ICC. The implications of the data with respect to the primary locus of the changes and to the manner in which they contribute to thalamocortical reorganization are considered. (C) 2003 Wiley-Liss, Inc.

A comparison of the self-association behavior of the plant cyclotides kalata B1 and kalata B2 via analytical ultracentrifugation

The recently discovered cyclotides kalata B1 and kalata B2 are miniproteins containing a head-to-tail cyclized backbone and a cystine knot motif, in which disulfide bonds and the connecting backbone segments form a ring that is penetrated by the third disulfide bond. This arrangement renders the cyclotides extremely stable against thermal and enzymatic decay, making them a possible template onto which functionalities can be grafted.We have compared the hydrodynamic properties of two prototypic cyclotides, kalata B1 and kalata B2, using analytical ultracentrifugation techniques. Direct evidence for oligomerization of kalata B2 was shown by sedimentation velocity experiments in which a method for determining size distribution of polydisperse molecules in solution was employed. The shape of the oligomers appears to be spherical. Both sedimentation velocity and equilibrium experiments indicate that in phosphate buffer kalata B1 exists mainly as a monomer, even at millimolar concentrations. In contrast, at 1.6 mM, kalata B2 exists as an equilibrium mixture of monomer (30%), tetramer (42%), octamer (25%), and possibly a small proportion of higher oligomers. The results from the sedimentation equilibrium experiments show that this self-association is concentration dependent and reversible. We link our findings to the three-dimensional structures of both cyclotides, and propose two putative interaction interfaces on opposite sides of the kalata B2 molecule, one involving a hydrophobic interaction with the Phe(6), and the second involving a charge-charge interaction with the Asp(25) residue. An understanding of the factors affecting solution aggregation is of vital importance for future pharmaceutical application of these molecules.

Production and properties of xylanases from Aspergillus terricola Marchal and Aspergillus ochraceus and their use in cellulose pulp bleaching

Aspergillus terricola and Aspergillus ochraceus, isolated from Brazilian soil, were cultivated in Vogel and Adams media supplemented with 20 different carbon sources, at 30 A degrees C, under static conditions, for 120 and 144 h, respectively. High levels of cellulase-free xylanase were produced in birchwood or oat spelt xylan-media. Wheat bran was the most favorable agricultural residue for xylanase production. Maximum activity was obtained at 60 A degrees C and pH 6.5 for A. terricola, and 65 A degrees C and pH 5.0 for A. ochraceus. A. terricola xylanase was stable for 1 h at 60 A degrees C and retained 50% activity after 80 min, while A. ochraceus xylanase presented a t (50) of 10 min. The xylanases were stable in an alkali pH range. Biobleaching of 10 U/g dry cellulose pulp resulted in 14.3% delignification (A. terricola) and 36.4% (A. ochraceus). The brightness was 2.4-3.4% ISO higher than the control. Analysis in SEM showed defibrillation of the microfibrils. Arabinase traces and beta-xylosidase were detected which might act synergistically with xylanase.