Protein and Peptide Letters - Volume 16, Issue 9, 2009

Polyribonucleotide phosphorilase from the psychrophilic Antarctic eubacterium Pseudoalteromonas haloplanktis (PhPNPase) has been purified. This enzyme catalyzes both the RNA polymerisation and degradation reaction, showing the highest activity at temperatures below 40°C. PhPNPase is quite sensitive to heat treatment and it is endowed with remarkable halotolerance.

We have synthesised nine analogues of the antibacterial peptide anoplin with a peptoid residue at position 5 (HGLLKXIKTLL- NH2). The most active compounds showed MIC-values of 12.5 and 25 μM against E.coli and S.aureus. These MIC-values are comparable with anoplin which showed 23 μM and 11 μM against E.coli and S.aureus. However, the selectivity was reversed. Our results indicate that peptoid analogues of anoplin are promising lead structures for developing new antibacterial agents.

Peptidylarginine deiminase IV (PAD4) catalyzes the conversion of an Arg residue to a citrulline residue in various proteins. In particular, citrullination of histone subunits, such as H2A and H3, by PAD4 is thought to be related to rheumatoid arthritis. However, the details of the citrullination mechanism of histone H2A and H3 are not yet well known. Moreover, the effects of N-terminal acetylation on histone subunits with respect to PAD4 recognition have not yet been studied. To further study the mechanism of PAD4 recognition of histone H2A and H3 subunits, a series of the N-terminal peptides was chemically synthesized and the citrullination sites were identified using MALDI-TOF/MS. N-terminal acetylation of histone H2A was not significant with respect to PAD4 recognition in vitro, but the acetylation of H3 peptide had a significant effect on PAD4 recognition in vitro, resulting in predominant citrullination at the Arg2 residue.

Since there are not much experimental data available about different structural properties of Isfahan virus (ISFV), in the present investigation, computational study of G protein of ISFV was performed and the results were compared with G proteins of Chandipura virus (CHPV) and Piry virus (PV). Calculation of amino acid compositions of G proteins of viruses was done by PseAAC server. Predictions of localization, sequence of signal peptides, C, N and O glycosylation sites, transmembrane helices, cysteine bond positions and B cell epitopes of G proteins were performed by Virus- PLoc, Signal-CF, EnsembleGly, MemBrain, DiANNA and BCPREDS servers respectively. Similarities and differences between these glycoproteins were predicted and discussed.

New analogues of galanthamine containing peptide fragments either at 6 or 11 position, were synthesized by reaction between galanthamine molecule and dipeptides and tripeptide, derivatives of N-(3,4-dichlorophenyl)-D,L-Ala- OH. The best results according to yields, easily purification of the target products, and simplicity of the scheme realisation was achieved by using of cyanomethyl ester of Boc-Gly-OH as activated compound.

We report a one pot synthesis of Fmoc amino acid derived 4-amino-thiazole derivatives and thiazole linked Northogonally protected dipeptidomimetics by the condensation of Nα-Fmoc α-halomethylketones with thiourea and Boc/Z-α-amino acid thioamides via modified Hantzch protocol. Side chain modified Fmoc amino acids containing 4- amino thiazole moiety have also been synthesized following the similar protocol.

A novel oligomeric decaprenyl pyrophosphate synthase was identified. Circular dichroism measurements indicated that it is alpha-helical, and stable against pH changes and denaturants. Three peptides corresponding to the conserved regions were synthesized and metal affinities were investigated. Crystallographic analyses of this oligomeric DDS are currently in progress.

To study the effect of probenazole on the induced systemic resistance mechanism of rice-bacterial interaction, a proteomic approach was applied. Oryza sativa cv. Java 14 seedlings were treated with probenazole, followed by inoculation with compatible (Xo7435) and incompatible (T7174) races of Xanthomonas oryzae pv. oryzae. Cytosolic proteins were fractionated from leaf blades, separated by two-dimensional polyacrylamide gel electrophoresis. Pathogenesisrelated protein 5 (PR5) was significantly induced with probenazole treatment followed by inoculation with T7174 or Xo7435. The sense PR5 transgenic rice plants were more highly resistant than the susceptible vector control against Xo7435. These results indicate that probenazole strongly induces PR5 in the interaction between rice and X. oryzae pv. oryzae, and might be involved in the resistance mechanism of rice against bacterial blight.

G-protein βγ dimers are prime regulators transmitting extracellular signals to wide-ranging cellular effectors including phosphoinositide 3-kinase (PI3K) isoforms β and γ. Recombinant Gβ?? purified from Sf9 cells via metal-affinity and anion exchange chromatography exhibited a wortmannin-insensitive phospholipid kinase activity that copurified from the insect cells. To exclude false-positive results of Gβγ-dependent lipid kinase activity, the elimination of insect phospholipid kinase from Gβγ protein samples is necessary to avoid interference with the intrinsic lipid kinase activity of PI3K isoforms in reconstitution experiments. Here we describe an improved procedure of Gβ1γ2 purification from cell membranes that separates the contaminating phospholipid kinase.

To study the intrinsic structural properties of three human prion helices and analyse their stability, we conducted molecular dynamics simulations, applied helix propensity predictions and evaluated the energetic contribution of the helical regions to the PrP protein stability. Our results suggest that three helices present different stability and the helix 2 results the least stable.

Single crystal X-ray diffraction studies and solvent dependent 1H NMR titrations reveal that a set of four tetrapeptides with general formula Boc-Xx(1)-Aib(2)-Yy(3)-Zz(4)-OMe, where Xx, Yy and Zz are coded L-amino acids, adopt equivalent conformations that can be described as overlapping double turn conformations stabilized by two 4→1 intramolecular hydrogen bonds between Yy(3)-NH and Boc C=O and Zz(4)-NH and Xx(1)C=O. In the crystalline state, the double turn structures are packed in head-to-tail fashion through intermolecular hydrogen bonds to create supramolecular helical structures. Field emission scanning electron microscopic (FE-SEM) images of the tetrapeptides in the solid state reveal that they can form flat tape-like structures. The results establish that synthetic Aib containing supramolecular helices can form highly ordered self-aggregated amyloid plaque like human amylin.

In neonatal rat metatarsal organ culture, a bell-shaped dose-related curve in length of mineralized area, increases in the height of proliferative and hypertrophic zones, in the number of hypertrophic chondrocyte and in the amount of Runx2 mRNA, were revealed after treatment with OGP(10-14). We conclude that OGP(10-14) accelerates bone growth.

Single site-directed mutations at each of the four EF-hand loops of potassium channel-interacting protein 2.2 (KChIP2.2) were carried out to explore the functional roles of EF-hands in KChIP2.2. In contrast to those on EF-hands 1 and 2, mutations on EF-hands 3 or 4 distorted the high affinity Ca2+-binding site of KChIP2.2. However, the Mg2+-binding ability of KChIP2.2 was marginally affected by the mutations. The gross conformation of mutated KChIP2.2 was indistinguishable from wild-type KChIP2.2 as revealed by CD spectra. The results of size exclusion chromatography showed that, with exception of EF-hand 4 mutant, mutations on EF-hands 1, 2 or 3 caused KChIP2.2 to form oligomer. Pull-down assay revealed that, unlike wild-type KChIP2.2, the interaction between mutated KChIP2.2 and Kv4.2 was not notably enhanced by Ca2+ and Mg2+. Coexpression of Kv4.2 and KChIP2.2 in HeLa cells revealed that mutations on EF-hands did not alter the intracellular co-localization of KChIP2.2 and Kv4.2. Together with previous findings that EF-hand mutants of KChIP proteins are unable to regulate the kinetics of Kv4.2, our data show that the intact EF-hands should be crucial for the formation of active conformation of KChIP2.2 when the protein is loaded with Ca2+ and Mg2+.

Diocleinae lectins administered per oral route in mice inhibited the abdominal constrictions induced by acetic acid. The percentage of the lectins antinociception varied from 61% for Canavalia grandiflora (ConGf) to 20% for Dioclea violacea. ConGf inhibited contortions at all doses tested but not in a dose-dependent manner, involving carbohydrate recognition.

Protein inactivation frequently occurs through partially unfolded states under native conditions, and temperature is an important parameter that affects the susceptibility of proteins to inactivation. While the effect of temperature on global unfolding is well documented, however, experimental characterizations of the temperature effect on partial unfolding are rare. Proteolysis offers a valuable chance to investigate the temperature effect on partial unfolding. By investigating proteolysis kinetics, the energetics of the partially unfolded state responsible for proteolysis (the cleavable state) can be studied. E. coli ribonuclease H (RNase H) has been shown to be cleaved by thermolysin at the amide bond between Thr92 and Ala93 through partial unfolding. Using this cleavage as a model system, we evaluated quantitatively the temperature effect on conformational equilibrium between the native state and a cleavable state. The analysis shows that decrease in temperature from 37°C to 4°C decreases the population in the cleavable state and reduces proteolytic susceptibility of the substrate protein. The conformational change leading to the cleavable state has a temperature-independent positive ΔH° with negligible ΔCp°. This thermodynamic characteristic of partial unfolding for proteolysis is quite distinct from that of global unfolding of RNase H that has a considerable ΔCp° and a negative ΔH° at low temperature. The distinct thermodynamic characteristics of partial unfolding from global unfolding mainly result from the difference in the changes of solvent-accessible surface area, which confirms that the temperature effect on partial unfolding is strongly scaledependent.

Here we report for the first time heat of ionization of invertase (E.C.3.2.1.26) active site residues from hyperproducer strain of Aspergillus niger (34.1 U ml-1), along with its physiochemical properties, kinetics and thermodynamics of stability-function. The Invertase showed great potential for industry as being highly efficient (kcat = 24167 s-1 at 65 °C, pH 5.0) and stable (half life= 12 h at 56°C).

An insulin-binding protein was isolated from Canavalia ensiformis seed coat, by using an insulin-Sepharose 4B affinity chromatography, and the protein was identified as canavalin (Canavalia 7S globulin) by mass spectrometry analysis. The major novelty of these data is the acidic nature of this globulin insulin-binding, in contrast to the basic Bg-like insulin- binding proteins so far reported in plants.

Based on the primary sequence, by selecting the pseudo amino acid composition, position weight matrix score, the predicted secondary structure and the second neighbor dipeptide composition as characteristic parameters, an approach of diversity increment for predicting 27-class protein folds is proposed. Overall recognition accuracy reaches 61.10% in the independent testing.

From 1 kg of defatted Pterocarpus angolensis (mukwa tree) seed meal, 21.6 grams of an α,Dmannose/ glucose-specific lectin can be purified on mannose-Sepharose. Relative affinities for several (oligo)saccharides and glycoproteins were studied by haemagglutination-inhibition. Gel filtration shows that the lectin exists as a dimer above pH 5 and as a monomer below pH 3.5. This is confirmed by studies on the release of lectin subunits that were adsorbed from solution to lectin monomers immobilized onto Eupergit-c. From the gel filtration patterns it is calculated that a residue with pKa of about 4.4 is involved in dimer dissociation. Titration of glutamic acids (E60, E209) is postulated to be involved. CD spectroscopy shows that the secondary structure of the lectin is unchanged between pH 1 and 12.5, and that the tertiary structure remains unchanged between pH 5 and 12. In the acid pH region, reversible spectral changes occur that may be due to the titration of one or more amino acids with a pKa value of 3.9-4.2, probably aspartic acid. These residues are implicated in sugar-binding but not in dimerization of the lectin. Only at pH 12.5, irreversible denaturation occurs. Mukwa lectin displays full carbohydrate-binding capacity between pH 4 and 12, as is concluded from ELLA (Enzyme Linked Lectin Assay) using ovalbumin and fetuin, and from binding of the same glycoproteins to immobilized lectin monomers. The lectin is rapidly and fully reversibly demetallized at pH 2.5 with 5 mM EDTA. The demetallized lectin is completely devoid of sugar-binding activity. Mukwa lectin is a very thermostable molecule (at least till 85°C). However, addition of non-ionic detergents substantially lowers its thermostability.