Protein and Peptide Letters - Volume 16, Issue 2, 2009

The synthesis of Bsmoc-N-methyl amino acids is presented. The first step involves p-toluenesulfonic acid (TsOH) catalysed condensation of a Bsmoc-amino acid with paraformaldehyde to furnish N-Bsmoc-5-oxazolidinone under MW irradiation. This intermediate is reduced to the corresponding N-methyl amino acid using triethylsilane (Et3SiH) and trifluoroacetic acid (TFA) at r.t. The N-methyl amino acids are converted into corresponding acid fluorides using diethylaminosulfur trifluoride (DAST) and employed as coupling agents in the synthesis of dipeptides. The peptide coupling was mediated by KOAt in CH2Cl2.

Spectroscopic and structural elucidation of the peptides L-Valyl-L-Prolyl-L-Proline (1) and L-Isoleucyl-L-Prolyl- L-Proline (2) are reported on the basis of experimental linear-polarized IR-spectroscopy in solid-state, 1H-NMR data and DFT. Curiously, the experimental data shown that both peptides stabilized in solution and in solid-state neutral H2N-RCOOH form. Conformational analysis made, shown two strong intramolecular NH2…O=C-N(Amide) and O=C-OH…NH2 hydrogen bonds with lengths of 2.979 Å and 2.475 Å in (1) and 2.599 Å and 2.507 Å in (2) respectively. The presence of the Pro-Pro fold resulted to strong steric effect leading to the stabilization of free COOH and NH2 groups. The Erel values of zwitterion form are significant higher than the neutral forms with a difference of 1.2 and 0.9 kJ/mol. The manner of interaction of the peptides with angiotensin-I converting enzyme is proposed.

Currently, no pharmaceuticals for the etiological treatment of neurodegenerative protein-misfolding diseases (e.g., ALS, Alzheimer's or prion diseases) are available. In this brief communication the development of chaperone-based medications from medicinal plants (e.g., Ginkgo biloba) are reviewed as referred to specific protein-protein interactions of plant metallochaperones and human enzymes. It is indicated that bioactive copper chaperones for superoxide dismutase isolated from medicinal plants may be lead molecules for drug development in several diseases concerning metal ion metabolisms of man and animals.

Evidence for a key role of beta-amyloid (Aβ) in Alzheimer's disease has led to considerable interest in potential therapeutic strategies targeting enzymes involved in processing the amyloid precursor protein (APP). Beta-site APP Cleaving Enzyme (BACE or β-secretase) is a membrane bound aspartyl protease that has been shown to be directly involved in Aβ production and, therefore, is at the forefront of therapeutic targets in the treatment of Alzheimer's disease. BACE-2, an enzyme closely related to BACE, regulates Aβ production in a manner antagonistic to BACE, suggesting that non-selective inhibition of BACE-2 by BACE inhibitors might impair the lowering of Aβ. The design of BACE inhibitors that do not inhibit BACE-2 would be enhanced by structural and kinetic studies, efforts that typically demand considerable amounts of both enzymes. A BACE-2 construct containing 19 residues of the BACE prosegment followed by the BACE-2 catalytic domain sequence, Asp36…..Trp447, was produced in E. coli inclusion bodies (IB) at 110-140 mg/L cell culture. Exploration of a variety of refolding conditions resulted in an efficient method for refolding the resulting pro- BACE-2 construct, and this protein undergoes facile autocatalytic cleavage, optimal at pH 4, at the Leu40-↓-Ala41 bond. Refolded BACE-2 was purified by anion exchange, molecular sieve, and affinity chromatographies, yielding 105 mg of homogeneous enzyme (kcat/ Km = 1.2 x 104 x M-1 x sec-1) from 8 liters of E. coli cell culture.

A novel neuroprotective peptide, Humanin (HN), has a strong tendency to aggregate in phosphate-buffered saline. Here we attempted to reduce aggregation employing an aqueous phosphate solution, without NaCl, at pH 6.0 and low peptide concentrations wherever possible. Such a condition, though not fully physiological, allowed us to determine the secondary structure and molecular weight of the peptides. Comparison of a parent HN peptide, an inactive analog (S7AHN) and a 1000-fold more active analog (S14G-HN) showed no apparent differences in the secondary structure. These peptides were all disordered over the wide range of peptide concentration. Sedimentation analysis was done only for HN and S7A-HN and showed aggregation into soluble oligomers in 20 mM phosphate at pH 6.0. Aggregation was greatly suppressed in 5 mM phosphate at the same pH in terms of aggregate size, with the formation of smaller oligomers. Sedimentation velocity experiments at 60,000 rpm in 5 mM phosphate at pH 6.0 showed that both HN and S7A-HN distributed into soluble aggregates that sedimented to the bottom of the cell and low molecular weight species that approached sedimentation equilibrium. The mass of this low molecular weight species was determined by sedimentation equilibrium to be close to monomers for both peptides. Thus, these results clearly demonstrate that the active HN and inactive S7AHN are identical in structure and hence there is no apparent correlation between solution structure and biological activity.

We attempted to develop a stable radiolabeled transferrin (Tf) useful in experimental studies related to Tf receptor. 67Ga and 111In were used as labeling radioisotopes. The results from gel chromatography, dialysis, and electrophoresis showed that 111In-DTPA-Tf was the most stable among the radiolabeled Tfs examined in the present study. 111In- DTPA-Tf was also the most stable radiolabeled transferrin in the blood.

In this paper, HESH, which was a new set of amino acid descriptors including Hydrophobic, Electronic, Steric and Hydrogen bond contribution properties, were derived from multi-dimensional properties of 20 coded amino acids. The quantitative structure-activity relationship (QSAR) of 101 synthetic cationic antimicrobial polypeptides (CAMELs) was then characterized with HESH scales and studied by genetic algorithm-partial least square (GA-PLS) method. It was found that the robust QSAR model constructed with electronic and hydrophobic properties parameters of HESH descriptors was a better one. Through further analysis, electronic and hydrophobic properties of the 3rd, 6th, 7th, 11th and 12th residue of CAMELs sequence made high contribution to antimicrobial potencies. Based on this PLS model, a series of cationic antimicrobial peptides (AMPs), with relatively high antimicrobial activity was designed. Meanwhile, a robust QSAR model with favorable predictive capability for 34 antimicrobial peptides was constructed with HESH descriptors. The results showed that HESH descriptors had many obvious advantages, for it contains abundant information and its physico-chemical characteristics are clear and easily explained. The developed descriptors can be further expanded for the larger sets of biologically activities peptides and can serve as a useful quantitative tool for the rational design and discovery of antibiotics.

Molecular recognition of a series of oxadiazole-substituted α-isopropoxy phenylpropanoic acids by PPARα and PPARγ was investigated by using molecular modeling and 3D-QSAR analyses. The binding models of these compounds were determined by hydrophobic property analyses and molecular docking procedure FlexX. It was found that the hydrophilic heads of these compounds form four specific conserved hydrogen bonds with the ligand binding pockets of PPARα and PPARγ, which results in fixed head conformations. On the contrary, their hydrophobic tails adopt different configurations to make contacts with hydrophobic region. The oxadiazole-ring-related hydrogen bond interactions well elucidate the structural features governing the different binding behavior of these agonists against PPARα and PPARγ. Based on these active conformations, highly predictive comparative molecular similarity indices analysis (CoMSIA) models were derived, which not only is consistent with the experimental results but also could be mapped back to the receptor topology and the ligand-receptor interaction models. The simulation results reveal the structure-activity relationship of these compounds at the molecular level and provide new insights for the design of novel potent PPARα and PPARγ dual agonists.

The focuses of this work are: to propose a novel method for building an ensemble of classifiers for peptide classification based on substitution matrices; to show the importance to select a proper set of the parameters of the classifiers that build the ensemble of learning systems. The HIV-1 protease cleavage site prediction problem is here studied. The results obtained by a blind testing protocol are reported, the comparison with other state-of-the-art approaches, based on ensemble of classifiers, allows to quantify the performance improvement obtained by the systems proposed in this paper. The simulation based on experimentally determined protease cleavage data has demonstrated the success of these new ensemble algorithms. Particularly interesting it is to note that also if the HIV-1 protease cleavage site prediction problem is considered linearly separable we obtain the best performance using an ensemble of non-linear classifiers.

Calcineurin (CN) is a heterodimer of a catalytic subunit, calcineurin A (CNA), and a regulatory subunit (CNB). Here, we find that the mechanism by which CNB regulates CNA depends on the substrate involved. The regulation mechanism involving tau and its truncation segments is distinct from that involving RII peptide, and the efficiencies of CNA to dephosphorylate tau are constant regardless of whether CNB was present or not. The findings shed some light on the role of CNB in controlling phosphorylation of tau in vivo and the pathogenesis of tauopathies such as Alzheimer's Disease.

Till date various plants extract have been studied to reduce the incidence of urolithiasis but the identification of naturally occurring calcium oxalate (CaOx) inhibitory biomolecules from plants was hampered in past by limitation in identification method. The present study is aimed at examining the efficacy of Trachyspermum ammi on CaOx crystallization in vitro and further by combining conventional biochemical methods with recent advances in mass spectrometry, a novel calcium oxalate (CaOx) crystal growth inhibitor was purified from the seeds of Trachyspermum ammi. An anticalcifying protein from the seeds of Trachyspermum ammi was purified by three step purification scheme; ammonium sulphate fractionation, anion exchange chromatography and molecular sieve chromatography based on its ability to inhibit calcium oxalate crystallization in vitro. An anticalcifying protein having molecular weight 107 kDa and isolectric point 6.2 was isolated. Amino acid analysis of Trachyspermum ammi anticalcifying protein (TAP) showed abundant presence of acidic amino acids (Asp and Glu). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry of TAP showed similarities with an unnamed protein product of Vitis vinifera (CAO23876) after matching peptide mass fingerprints in MASCOT search engine. Two EF hand domains were identified in unnamed protein product of Vitis vinifera (CAO23876) by SMART normal module. Due to a significant similarity of TAP with unnamed protein product of Vitis vinifera, presence of two EF hand domains in TAP was anticipated, signifying its calcium binding properties which is a feature of most kidney stone inhibitory proteins.

We examined the effects of air-water and water-sevoflurane interfaces on conformational properties of amyloid- β peptide (ABP). Fractions were extracted from sub-interfacial (air-water) and supra-interfacial (water-sevoflurane) layers and compared with aqueous bulk layers using fluorescence properties of ABP provided by a single tyrosine. The observations suggest that interfacial ABP may be more disordered than bulk ABP.

Synbindin is one component of Transport protein particle (TRAPP) complexes. In the hippocampal neurons, synbindin binds syndecan-2 by its atypical PDZ domain (APD) and may regulate the formation of dendritic spines. To investigate the interaction of synbindin and syndecan-2, we determined the solution structure of the synbindin APD by NMR. The structure of APD is different from the classical canonical PDZ domains by lacking the typical αA helix and the signature sequence Gly-Ψ-Gly-Ψ. These differences indicate that APD may not bind syndecan-2 with the typical binding mode of other PDZ domain proteins. In NMR titration experiments, APD do not bind with the C-terminal TKEFYA peptide of syndecan-2, but can interact with the 32-residue cytoplasmic domain of syndecan-2 very weakly.

The most promising targets for Leishmania-specific drug design are two key enzymes involved in the unique thiol-based metabolism, common to all parasites of the Trypanosomatidae family: trypanothione synthetase (TryS) and trypanothione reductase (TR). Recently, new inhibitors of TR have been identified such as polyamines and tricyclic compounds. The knowledge of the three-dimensional structure of Leishmania TR will shed light on the mechanism of interaction of these inhibitors with TR and will be the starting point to design novel lead candidates to facilitate the development of new effective and affordable drugs. Trypanothione reductase from Leishmania infantum has been cloned, expressed in E. coli and purified. Crystals were obtained at 294 K by the hanging drop vapour diffusion method using ammonium sulfate as precipitant agent and diffract to better than 2.95 Å resolution using a synchrotron radiation source. The crystals exhibit an unusually high solvent content of 74 %, belong to the tetragonal space group P41 with units cell parameters a=b=103.45 Å, c=192.62 Å and two molecules in the asymmetric unit. The protein X-ray structure has been solved by Molecular Replacement and the model is under construction.

We have successfully expressed an active human brain serine racemase (hSR) with His-tag using moderate halophile. The purified His-hSR showed high elimination and racemization activities on L-serine: the elimination activity was 2.6-fold higher than racemization activity. Both enzyme activities showed an optimum reaction pH at around 9.0 and were stimulated 5- to 7-fold by such divalent cations as Mg++, Mn++ and Ca++.

Chick-pea (Cicer arietinum L.) cotyledons are unique source of aminopeptidase - 8-9 U/g cotyledons was observed using L-leucine-p-nitroanilide as substrate. The aminopeptidase was purified (65 kDa, pI 4.8 ) reaching a specific activity of 220 U/mg at pH 7.0-7.2 and 35-40°C. The determined constant of specificity kcat/Km during hydrolysis of Nunsubstituted amino acid-p-nitroanilides showed a decrease order: Phe>Leu>Pro>Ile>Val>Ala. The enzyme was strongly inhibited by p-chloromercuribenzoic acid as well as in a competitive rate by the antihypertensive peptides Ile-Pro-Pro and Val-Pro-Pro.

Hemoglobin is a tetrameric, iron-containing metalloprotein, which plays a vital role in the transportation of oxygen from lungs to tissues and carbon dioxide back to lungs. Though good amount of work has already been done on hemoglobins, the scarcity of data on three dimensional structures pertaining to low oxygen affinity hemoglobins from mammalian species, motivated our group to work on this problem specifically. Herein, we report the preliminary crystallographic analysis of buffalo hemoglobin, which belongs to low oxygen affinity species. The buffalo blood was collected, purified by anion exchange chromatography and crystallized with PEG 3350 using 50mM phosphate buffer at pH 6.7 as a precipitant by hanging drop vapor diffusion method. Data collection was carried out using mar345dtb image plate detector system. Buffalo hemoglobin crystallizes in orthorhombic space group P212121 with one whole biological molecule (α2β2) in the asymmetric unit with cell dimensions a=63.064Å, b=74.677Å, c=110.224Å.