p-cresol as a reversible acylium ion scavenger in solid-phase peptide synthesis

In this work we have defined the nature of the p-cresol and p-thiocresol adducts generated from acylium ions during HF cleavage, following contemporary Boc/benzyl solid-phase peptide synthesis. Contrary to the results in previous reports, we found that both p-cresol and p-thiocresol predominantly form. aryl esters under typical cleavage conditions. Initially we investigated a number of small peptides containing either a single glutamate residue or a C-terminal long-chain amino acid which allowed us to unambiguously characterize the scavenged side products. Whereas, the p-cresol esters are stable at 0 degrees C they rearrange irreversibly at higher temperatures (5-20 degrees C) to form aryl ketones. By contrast, p-thiocresol esters do not undergo a Fries rearrangement but readily undergo further additions of p-thiocresol to form ketenebisthioacetals and trithio ortho esters, even at low temperatures. Importantly, we found by LC/MS and FT-ICR MS analysis that peptides containing p-cresol esters at glutamyl side chains are susceptible to amidation and fragmentation reactions at these sites during standard mild base workup procedures. The significance of these side reactions was further demonstrated in the synthesis of neutrophil immobilization factor, a 26-residue peptide, containing four glutamic acid residues. The side reactions were largely avoided by mild hydrogen peroxide-catalyzed hydrolysis which converted the p-cresol adducts to the free carboxylic acids in near quantitative yield. The choice of p-cresol as a reversible acylium ion scavenger when coupled with the simple workup conditions described is broadly applicable to Boc/benzyl peptide synthesis and will significantly enhance the quality of peptides produced.

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.

Synthesis of [Cu-2(Me-2[9]aneN(2)S)(2)(OH)(2)] (PF6)(2), a hydroxo-bridged copper(II) complex of N,N '-dimethyl-1-thia-4,7-diazacyclononane (Me-2[9]aneN(2)S). X-ray structural analysis, magnetic susceptibility, EPR and visible spectroscopy

The bis(mu-hydroxo) complex [Cu-2(Me-2[9]aneN(2)S)(2)(OH)(2)](PF6)(2) (Me-2[9]aneN(2)S = N,N'-dimethyl-1-thia-4,7-diazacyclononane) results after reaction of [Cu(Me-2[9]aneN(2)S)(MeCN)] (PF6) with dioxygen at -78 degrees C in acetonitrile. The complex has been characterized by X-ray crystallography: orthorhombic, space group Pnma, with a 18.710(3), b 16.758(2), c 9.593(2) Angstrom, and Z = 4. The structure refined to a final R value of 0.051. The complex contains two copper(II) ions bridged by two hydroxo groups with Cu ... Cu 2.866(1) Angstrom. The solid-state magnetic susceptibility study reveals ferromagnetic coupling, the fitting parameters being J = +46+/-5 cm(-1), g = 2.01+/-0.01 and theta = -0.58+/-0.03 K. The frozen-solution e.p.r. spectrum in dimethyl sulfoxide is characteristic of a monomeric copper(II) ion (g(parallel to) 2.300, g(perpendicular to) 2.063; A(parallel to) 156.2 x 10(-4) cm(-1), A(perpendicular to) 9.0 x 10(-4) cm(-1)) with an N2O2 donor set. Thioether coordination to the copper(II) in solution is supported by the presence of an intense absorption assigned to a sigma(S)-->Cu-II LMCT transition at c. 34000 cm(-1). The single-crystal spectrum of [Cu-2(Me-2[9]aneN(2)S)(2)(OH)(2)] (PF6)(2) (273 K) reveals d-->d transitions at 14500 and 18300 cm(-1) and a weak pi(S)-->Cu-II charge-transfer band at approximately 25000 cm(-1).

Chemical synthesis, antibacterial activity and conformation of diptericin, an 82-mer peptide originally isolated from insects

The small amounts of antibacterial peptides that can be isolated from insects do not allow detailed studies of their range of activity, side-chain sugar requirements, or their conformation, factors that frequently play roles in the mode of action. In this paper, we report the solid-phase step-by-step synthesis of diptericin, an 82-mer peptide, originally isolated from Phormia terranovae. The unglycosylated peptide was purified to homogeneity by conventional reversed-phase high performance liquid chromatography, and its activity spectrum was compared to that Of synthetic unglycosylated drosocin, which shares strong sequence homology with diptericin's N-terminal domain. Diptericin appeared to have antibacterial activity:for only a limited number of Gram-negative bacteria. Diptericin's submicromolar potency against Escherichia coli strains indicated that, in a manner similar to drosocin, the presence of the carbohydrate side chain is not,necessary to kill bacteria. Neither the N-terminal, drosocin-analog fragment, nor the C-terminal, glycine-rich attacin-analog region was active against any of the bacterial strains studied, regardless of whether the Gal-GalNAc disaccharide units were attached. This suggested that the active site of diptericin fell outside the drosocin or attacin homology domains. In addition, the conformation of diptericin did not seem to play a role in the antibacterial activity, as was demonstrated by the complete lack of ordered structure by two-dimensional nuclear magnetic resonance spectroscopy and circular dichroism. Diptericin completely killed bacteria within I h, considerably faster than drosocin and the attacins; unlike some other, fast-acting antibacterial peptides, diptericin did not lyse normal mammalian cells. Taken together, these data suggest diptericin does not belong to any known class of antibacterial peptides.

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.

Evolutionary History of Arabidopsis thaliana Aminoacyl-tRNA Synthetase Dual-Targeted Proteins

Aminoacyl-transfer RNA (tRNA) synthetases (aaRS) are key players in translation and act early in protein synthesis by mediating the attachment of amino acids to their cognate tRNA molecules. In plants, protein synthesis may occur in three subcellular compartments (cytosol, mitochondria, and chloroplasts), which requires multiple versions of the protein to be correctly delivered to its proper destination. The organellar aaRS are nuclear encoded and equipped with targeting information at the N-terminal sequence, which enables them to be specifically translocated to their final location. Most of the aaRS families present organellar proteins that are dual targeted to mitochondria and chloroplasts. Here, we examine the dual targeting behavior of aaRS from an evolutionary perspective. Our results show that Arabidopsis thaliana aaRS sequences are a result of a horizontal gene transfer event from bacteria. However, there is no evident bias indicating one single ancestor (Cyanobacteria or Proteobacteria). The dual-targeted aaRS phylogenetic relationship was characterized into two different categories (paralogs and homologs) depending on the state recovered for both dual-targeted and cytosolic proteins. Taken together, our results suggest that the dual-targeted condition is a gain-of-function derived from gene duplication. Selection may have maintained the original function in at least one of the copies as the additional copies diverged.

Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins

An Escherichia coli cell-free transcription/translation system was used to explore the high-level incorporation Of L-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI-MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean N-15-HSQC NMR spectra. A redox-staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell-free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and/or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome-mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High-yield cell-free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains.

Total chemical synthesis and chemotactic activity of human S100A12 (EN-RAGE)

Human S100A12 (extracellular newly identified RAGE (receptor for advanced glycosylation end products)binding protein), a new member of the S100 family of EF-hand calcium-binding proteins, was chemically synthesised using highly optimised 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/tert-butoxycarbonyl in situ neutralisation solid-phase chemistry. Circular dichroism studies indicated that CaCl2 decreased the helical content by 27% whereas helicity was marginally increased by ZnCl2. The propensity of S100A12 to dimerise was examined by electrospray ionisation time-of-flight mass spectrometry which clearly demonstrated the prevalence of the non-covalent homodimer (20 890 Da). Importantly, synthetic human S100A12 in the nanomolar range was chemotactic for neutrophils and macrophages in vitro. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis

Several members of the Rubiaceae and Violaceae families produce a series of cycloticles or macrocyclic peptides of 29-31 amino acids with an embedded cystine knot. We aim to understand the mechanism of synthesis of cyclic peptides in plants and have isolated a cDNA clone that encodes the cyclotide kalata Ell as well as three other clones for related cycloticles from the African plant Olden-landia affinis. The cDNA clones encode prepropeptides with a 20-aa signal sequence, an N-terminal prosequence of 46-68 amino acids and one, two, or three cyclotide domains separated by regions of about 25 aa. The corresponding cycloticles have been isolated from plant material, indicating that the cyclotide domains are excised and cyclized from all four predicted precursor proteins. The exact processing site is likely to lie on the N-terminal side of the strongly conserved GlyLeuPro or SerLeuPro sequence that flanks both sides of the cyclotide domain. Cyclotides have previously been assigned an antimicrobial function; here we describe a potent inhibitory effect on the growth and development of larvae from the Lepidopteran species Helicoverpa punctigera.

Synthesis of an analog of the thyroid hormone-binding protein transthyretin via regioselective chemical ligation

Transthyretin is an essential protein responsible for the transport of thyroid hormones and retinol in human serum and is also implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases, Here we report the solid phase synthesis of the monomeric unit of a transthyretin analog (equivalent to 127 amino acids) using t-Boc chemistry and peptide ligation and its folding to form a functional 54-kDa tetramer, The monomeric unit of the protein was chemically synthesized in three parts (positions 1-51, 54-99, and 102-127) and ligated using a chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of transthyretin's native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, transthyretin antibody recognition, and thyroid hormone binding. Other folding products included a high molecular weight aggregate as well as a transient dimeric species. This represents one of the largest macromolecules chemically synthesized to date and demonstrates the potential of protein chemical synthesis for investigations of protein-ligand interactions.

Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins

Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. Contractile state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (alpha -SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, beta -NM actin was localised to the cell periphery and basal cortex. The dense body protein alpha -actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In synthetic state SMC (passages 2-3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (beta -non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic chan-es in their distribution. The distinct compartmentalisation of structural proteins observed in contractile state SMC was no longer obvious, with proteins more evenly distributed throughout die cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation. (C) 2001 Wiley-Liss, Inc.

Regioselective synthesis of antiparallel loops on a macrocyclic scaffold constrained by oxazoles and thiazoles

[GRAPHICS] The regioselective syntheses and structures are reported for two tris-macrocylic compounds, each possessing two antiparallel loops on a macrocyclic scaffold constrained by two oxazoles and two thiazoles. NMR solution structures show the loops projecting from the same face of the macrocycle. Such molecules are shown to be prototypes for mimicking multiple loops of proteins.

Circular proteins - no end in sight

Circular proteins are a recently discovered phenomenon. They presumably evolved to confer advantages over ancestral linear proteins while maintaining the intrinsic biological functions of those proteins. In general, these advantages include a reduced sensitivity to proteolytic cleavage and enhanced stability. In one remarkable family of circular proteins, the cyclotides, the cyclic backbone is additionally braced by a knotted arrangement of disulfide bonds that confers additional stability and topological complexity upon the family. This article describes the discovery, structure, function and biosynthesis of the currently known circular proteins. The discovery of naturally occurring circular proteins in the past few years has been complemented by new chemical and biochemical methods to make synthetic circular proteins; these are also briefly described.

Influence of ageing, heat shock treatment and in vivo total antioxidant status on gene-expression profile and protein synthesis in human peripheral lymphocytes

Ageing results in a progressive, intrinsic and generalised imbalance of the control of regulatory systems. A key manifestation of this complex biological process includes the attenuation of the universal stress response. Here we provide the first global assessment of the ageing process as it affects the heat shock response, utilising human peripheral lymphocytes and cDNA microarray analysis. The genomic approach employed in our preliminary study was supplemented with a proteomic approach. In addition, the current study correlates the in vivo total antioxidant status with the age-related differential gene expression as well as the translational kinetics of heat shock proteins (hsps). Most of the genes encoding stress response proteins on the 4224 element microarray used in this study were significantly elevated after heat shock treatment of lymphocytes obtained from both young and old individuals albeit to a greater extent in the young. Cell signaling and signal transduction genes as well as some oxidoreductases showed varied response. Results from translational kinetics of induction of major hsps, from 0 to 24 It recovery period were broadly consistent with the differential expression of HSC 70 and HSP 40 genes. Total antioxidant levels in plasma from old individuals were found to be significantly lower by comparison with young, in agreement with the widely acknowledged role of oxidant homeostasis in the ageing process. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.