Protein and Peptide Letters - Volume 20, Issue 11, 2013

In this study, the influence of isoleucine and arginine on the biological activity and peptide-membrane interactions of linear avian β-defensin-4 (RL38) analogs was investigated. Results of biological activities showed that the antimicrobial activities of AvBD-4 analogs were closely related to hydrophobicity and amphipathicity. The peptide GLI19 with high hydrophobicity value and amphipathicity displayed broad spectrum antimicrobial activity against both gram-negative and gram-positive, whereas GLR19 with increasing multiple charges only exhibited activity against gram-negative. The interaction between peptides and the liposome membrane demonstrated that the peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which supported the antimicrobial activity data. The outer membranes assay further demonstrated that GLI19 had a greater capacity than the other tested peptides to penetrate the cell membrane at a low concentration. Collectively, the peptides derived from the bactericidal domain of linear β- defensins by truncation and hydrophobic amino acid substitution may be effective high-potential antibacterial agents.

The interaction of non-phosphorylated L-type pyruvate kinase (L-PK) with fructose 1,6-bisphosphate (FBP), which is an allosteric activator of the phosphorylated enzyme, and peptides that mimic the phosphorylatable N-terminal regulatory domain of the enzyme, was studied. It was found that the catalytic activity of the enzyme was not enhanced in the presence of FBP, and this ligand acted as a relatively weak reversible inhibitor of the enzyme activity in the micromolar concentration range. The phosphorylation site analogue peptides RRASVA and RRAAVA had no effect on the activity of the enzyme, while the phosphorylated peptide RRAS(Pi)VA reversibly inhibited the enzyme and this process was characterised by the Ki value 47 µM. As the phosphorylated form of L-PK is a subject of significant allosteric regulation by FBP, it was concluded that phosphorylation should function as a molecular switch of the allosteric properties of this enzyme.

VML is a lectin from Vatairea macrocarpa seeds that has various biological activities. Here, we describe three new lectin isoforms from V. macrocarpa identified through genomic DNA analysis. One of these isoforms has high similarity to VML, while another that has noteworthy differences. We have denoted the new isoforms as VML-2, VML-3 and VML-4. Recombinant VML (rVML) and VML-2 (rVML-2) were expressed in Escherichia coli and were anticipated to have similar biological activity compared to native VML. Recombinant lectins were produced using a synthetic gene strategy to improve the expression levels. We obtained two active recombinant lectin isoforms from V. macrocarpa, and there was no significant difference between their biological activities. The conservation between carbohydrate-binding sites of recombinant and native proteins was demonstrated by specific inhibition of hemagglutin activity by D-galactose and lactose. However, no inhibition was observed in the presence of glucose and mannose. Our data show that the recombinant lectins VML and VML-2 are active and capable of recognising D-galactose and lactose. Moreover, the absence of glycosylation does not interfere with their biological activity.

Human ryanodine receptor 2 (hRyR2) is a calcium ion channel present in the membrane of the sarcoplasmic reticulum of cardiac myocytes that mediates release of calcium ions from the sarcoplasmic reticulum stores during excitation- contraction coupling. Disease-causing mutations of hRyR2 are clustered into N-terminal (amino acids 1-600), central (amino acids 2100-2500) and C-terminal (amino acids 3900-5000) regions. These regions are believed to be involved in regulation of channel gating. The N-terminal region of hRyR2 has been implicated in regulating basal channel activity by interaction with the central hRyR2 region. This paper reports preparation, crystallization and preliminary X-ray analysis of recombinant hRyR21-606 N-terminal fragment. Soluble hRyR21-606 was expressed in Escherichia coli. Purification conditions were optimized using thermal shift assay. The quality and stability of the sample was probed by dynamic light scattering. A monomeric protein showing over 95% purity was obtained. The protein was crystallized by the hanging drop vapor-diffusion method. Diffraction data with resolution 2.39 Å were collected and processed.

Amphibian skin secretions contain a plethora of pharmacologically-active substances and represent established sources of bioactive peptides, including tachykinins. Tachykinins are one of the most widely-studied peptide families in animals and are found in neuroendocrine tissues from the lowest vertebrates to mammals. They are characterized by the presence of a highly-conserved C-terminal pentapeptide amide sequence motif (-FXGLM-amide) that also constitutes the bioactive core of the peptide. Amidation of the C-terminal methioninyl residue appears to be mandatory in the expression of biological activity. Here, we describe the isolation, characterization and molecular cloning of a novel tachykinin named ranachensinin, from the skin secretion of the Chinese brown frog, Rana chensinensis. This peptide, DDTSDRSN QFIGLM-amide, contains the classical C-terminal pentapeptide amide motif in its primary structure and an Ile (I) residue in the variable X position. A synthetic replicate of ranachensinin, synthesized by solid-phase Fmoc chemistry, was found to contract the smooth muscle of rat urinary bladder with an EC50 of 20.46 nM. However, in contrast, it was found to be of low potency in contraction of rat ileum smooth muscle with an EC50 of 2.98 µM. These data illustrate that amphibian skin secretions continue to provide novel bioactive peptides with selective effects on functional targets in mammalian tissues.

Present study dealt with the production, purification and characterization of xylanase from Bacillus subtilis- BS05 grown in submerged fermentation at 37°C for 48h using sugarcane bagasse as a substrate. Xylanase enzyme was purified to homogeneity by ammonium sulphate fractionation followed by sephadex G-100 chromatography. The molecular mass of xylanase enzyme was found to be 23kDa by SDS-PAGE. The optimum pH and temperature of xylanase enzyme was 5 and 50°C with stability range of 5-6 and 30°C -5°C respectively. The enzyme had half life of 1386-1155 minutes at 30°C -50°C respectively. Metal profile of the enzyme showed that Mn2+ (127.4 %) and Fe2+ (115%) were the activators while Hg2+ (14%) and EDTA (19%) were the inhibitors. Kinetic parameters revealed that the enzyme is specific to birchwood xylan with Km, Kcat, Vmax and Kcat/Km value of 1.15 mg/ml, 850 s-1, 117.64 U/mg and 739.13 s-1 mg-1.mL respectively.

Hypobromous acid (HOBr) is a powerful oxidant produced by stimulated neutrophils and eosinophils. Taurine, a non-protein amino acid present in high amounts in the leukocytes, reacts instantaneously with HOBr leading to their haloamine derivative taurine dibromamine (Tau-NBr2). Lysozyme is a bactericidal enzyme also present in leukocytes and in secretory fluids. The inhibition of lysozyme is a pathway for bacterial proliferation in inflammatory sites. Here, we investigated the inhibition of the enzymatic activity of lysozyme when it was submitted to oxidation by Tau-NBr2. We found that the oxidation of lysozyme by Tau-NBr2 decreased its enzymatic activity in 80percnt;, which was significant higher compared to the effect of its precursor HOBr (30%). The study and comparison of Tau-NBr2& and HOBr regarding the alterations provoked in the intrinsic fluorescence, synchronous fluorescence, resonance light scattering and near and far-UV circular dichroism spectra of lysozyme and oxidized lysozyme revealed that tryptophan residues in the active site of the protein were the main target for Tau-NBr2 and could explain its efficacy as inhibitor of lysozyme enzymatic activity. This property of Tau-NBr2 may have pathological significance, since it can be easily produced in the inflammatory sites.

The crystal complex propofol/GLIC (the bacterial homologue from Gloeobacter violaceus) recently determined also showed sensitivity to general anesthetics, belonging to the pLGIC (pentameric ligand-gated ion channels) family as a homopentameric member, which are potentiated and inhibited by general anesthetics and mimic changes elicited by anesthetics in physiologic targets as ion channels, providing the useful information for general anesthetics and allosteric modulators design. Thirteen active compounds selected from the previous data sets are used as reference and seed structures for developing novel general anesthetics via the scoring function of lead optimization using pharmacophoric & shape similarity integrated in Muse™ molecular design workflow. Autodock4.0 is validated its accuracy by two computational procedures and used for docking calculations in this study. Finally, the top ten invented propofol analogs reported here exhibit more favorable binding energies geometric matching than propofol. Especially, the comp#1 fits the binding site well in geometric shape with the lowest binding energy than any other compounds, and makes the hydrophobic interaction with the binding site formed by M4 helixes and lipids of receptor as well. More importantly, the interactions between ligand and lipids caused by general-anesthetic binding would be specially considered in novel general anesthetic design. accordingly, the findings reported here may provide useful insights for discovering more effective general anesthetics.

Aurora-A, B and C are non-receptor serine/threonine kinases in Homo sapiens. In spite of high similarity in their sequences, they possess distinct binding partners. These kinases play an important role in cell division and overexpressed in certain cancers. It has been demonstrated that Gly198 in Aurora-A kinase is responsible for its basal kinase activity, the mutation G198N transforms Aurora-A to Aurora-B like function and localization by binding to Inner centromere protein (INCENP). The molecular mechanisms, structural determinants and the binding energetics of the Aurora-A – INCENP complex owing to a single amino acid G198N mutation are not studied. Therefore, we have docked INCENP into human Aurora-A kinase, mutated Gly198 to Asn, Leu and Ala. The wild type and mutant Aurora-A – INCENP complexes were subjected to 40 ns molecular dynamics (MD) simulations. The Asn198 is located in the amphipathic cavity comprising Leu869IN, Glu868IN, Thr872IN, Tyr197AurA and Tyr199AurA and the interactions mediated via hydrogen bonds are important to stabilize the Aurora-AG198N – INCENP complex. The fluctuations in the secondary structural elements and the solvent accessible surface area of all the four complexes during the MD simulations were studied. We calculated the binding free energy upon mutation in the three mutant complexes. The Aurora-AG198N – INCENP complex with hydrophilic amino acid mutation has the negative free energy of solvation indicating favorable interactions with INCENP. Our results provide the structural basis and energetics of the human Aurora-AG198N – INCENP complex.

Ribosome-inactivating proteins (RIPs) function to inhibit protein synthesis through the removal of specific adenine residues from eukaryotic ribosomal RNA and rending the 60S subunit unable to bind elongation factor 2. They have received much attention in biological and biomedical research due to their unique activities toward tumor cells, as well as the important roles in plant defense. Alpha-momorcharin (α-MC), a member of the type I family of RIPs, is rich in the seeds of Momordica charantia L. Previous studies demonstrated that α-MC is an effective antifungal and antibacterial protein. In this study, a detailed analysis of the DNase-like activity of α-MC was conducted. Results showed that the DNase-like activity toward plasmid DNA was time-dependent, temperature-related, and pH-stable. Moreover, a requirement for divalent metal ions in the catalytic domain of α-MC was confirmed. Additionally, Tyr93 was found to be a critical residue for the DNase-like activity, while Tyr134, Glu183, Arg186, and Trp215 were activity-related residues. This study on the chemico-physical properties and mechanism of action of α-MC will improve its utilization in scientific research, as well as its potential industrial uses. These results may also assist in the characterization and elucidation of the DNase-like enzymatic properties of other RIPs.

We modified amylin chemically by conjugating methoxyl polyethyleneglycol succinimidyl carbonate (mPEGSC) of varying molecular weights (1 kDa, 2 kDa and 5 kDa). The reaction occurred within a few minutes, resulting in at least four distinct PEGylated products. The reaction products were separated by reversed-phase chromatography and identified by mass-spectrometry. The monoPEGylated and diPEGylated amylin products were generated rapidly through conjugation to the two amino groups of the N-terminal lysine residue. Both PEGylated amylin products bound to the receptor activity-modifying protein 1 (RAMP1). Pharmacological evaluation by subcutaneous administration in mice of monoPEGylated and diPEGylated amylin obtained with mPEG-SC 5 kDa revealed that both compounds modulated glycemia for longer times than unmodified amylin. Collectively, these data demonstrate the potential of bioconjugation with mPEG for the design of amylin therapeutics with sustained action.

Human N-formyl peptide receptors (FPRs) belong to the G protein-coupled receptors (GPCRs) superfamily, the most frequently addressed drug targets in the pharmaceutical industry, and are considered to play important roles in innate immunity and host defense mechanisms. Although still a highly challenging task, the availability of soluble and functional GPCRs including FPRs in milligram quantities is essential to spur the advancement of protein-based structural and functional studies for drug discovery. In this report, the applicability of E. coli extracts-based cell-free expression system to producing soluble and active human FPRs and hence to FPRs protein-based research was evaluated, during which human FPR3 was selected as our prototype receptor. To better solubilize the freshly expressed human FPR3, a panel of different detergents (mostly nonionic detergents) were selected and evaluated in the cell-free system devoid of natural membrane. After one-step immunoaffinity purification, the secondary structure and biological function of purified FPR3 were characterized. A final yield of 0.6 mg functional human FPR3 per ml reaction volume was obtained. The demonstrated proper folding and functionality of the cell-free produced human FPR3 opens a new avenue for the fast and efficient generation of human FPRs (and even other GPCRs) for structural and functional analysis.

This research is focussed on predicting through Naive Bayes learning, the possible p53 rescue mutants from amino-acid substitutions at the second, third and fourth site recombination that could reinstate normal p53 activity. The Naive Bayes probability values of the amino-acid substitutions at the respective site-wise recombination were utilized to formulate the proposed Genetic Mutant Marker Extraction (GMME) technique that could unearth the hot spot cancer, strong rescue and weak rescue mutants. The p53 mutation records depicting the amino-acid substitutions obtained by yeast assays comprising of nearly 16,700 records, available at the University of California, Machine Learning Repository, were utilized as the training dataset for the GMME technique. The proposed GMME technique revealed the hot spot cancer mutants, strong rescue and weak rescue mutants leading to the detection of probable genetic markers for Cancer prediction from the surface regions 96-289 constituting the second, third and fourth site recombinations. Thus far, computational approaches have been able to predict rescue markers at region-specific mutations (96-105, 114-123, 130-156 and 223- 232) with respect to the second site recombination for three hot spot cancer mutants only viz, P152L, R158L and G245S. The GMME technique aimed at predicting possible rescue markers for p53 mutants at the second, third and fourth site recombinations revealing novel rescue markers for fourteen hot spot cancer mutants. Moreover, the GMME technique can be extended effectively to increasing number of recombinant sites that can be efficiently utilized to predict novel rescue markers.