Protein and Peptide Letters - Volume 15, Issue 4, 2008

Peroxidases (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase) are part of a large group of enzymes. In this study, peroxidase, a primer antioxidant enzyme, was purified with 19.3 fold and 0.2% efficiency from cauliflower (Brassica oleracea L.) by ammonium sulphate precipitation, dialysis, CM-Sephadex ion-exchange chromatography and Sephadex G-25 purification steps. The substrate specificity of peroxidase was investigated using 2,2'-azino-bis(3- ethylbenz-thiazoline-6-sulphonic acid) (ABTS), 2-methoxyphenol (guaiacol), 1,2-dihydroxybenzene (catechol), 1,2,3- trihyidroxybenzene (pyrogallol) and 4-methylcatechol. Also, optimum pH, optimum temperature, optimum ionic strength, stable pH, stable temperature, thermal inactivation conditions were determined for guaiacol/H2O2, pyrogallol/H2O2, ABTS/H2O2, catechol/H2O2 and 4-methyl catechol/H2O2 substrate patterns. The molecular weight (Mw) of this enzyme was found to be 44 kDa by gel filtration chromatography method. Native polyacrylamide gel electrophoresis (PAGE) was performed for isoenzyme determination and a single band was observed. Km and Vmax values were calculated from Lineweaver-Burk graph for each substrate patterns.

A precursor of ConBr, a glucose/mannose-binding plant lectin, was expressed in the yeast Pichia pastoris. Western blot analysis of transformed cells detected an intracellularly recombinant protein band with ca. 34.5 kDa. The recombinant protein was apparently active as suggested by its strong interaction with the mannose-rich yeast cell debris.

E. coli acetyl esterase (Aes) and β-cysthationase (MalY) interact, probably with the same mechanism, with the N-terminus of transcriptional activator of maltose regulon (MalT). In order to investigate the basic mechanism of this interaction, we used both a proteomic and a bioinformatic approach. Affinity-based mass spectrometry experiments with purified Aes protein as bait allowed to fish twenty-three, apparently specific, interactors from crude extracts of E. coli cells grown in different conditions. The group of interactors appeared quite heterogeneous, comprising Aes itself, some molecular chaperons, metabolic enzymes, and several proteins of unknown function. Among the identified proteins, two are in some way related to the maltose metabolism and two are related to the lipopolysaccharide metabolism. By superposing the structures of the Alicyclobacillus acidocaldarius EST2, an Aes homolog, and MalY, a region of structural similarity was discovered that allowed detecting a short stretch of nine residues with sequence similarity among EST2, AES and MalY. Degenerated sequence consensuses derived from the alignment were used to analyse the E. coli proteome in the Swiss Prot database and permitted to retrieve sequences of Aes interactors already known or detected in this study. Most of these interactors (14 out of 25) contain the expected consensus. A site-directed mutant of Aes R179A made in the consensus sequence resulted in complete loss of interaction. Based on the analysis of the available three-dimensional structures and mutagenic and structural data inferred from literature, we predict a role of this motif in protein-protein interaction.

244 point mutations have been recorded in human hemoglobin β-chain, of which some change the oxygen affinity of human hemoglobin β-chain. We use the amino-acid distribution probability to quantify these mutations, and use the cross-impact analysis with Bayes' law to determine the probability that changes the oxygen affinity of human hemoglobin β-chain under mutations.

We have previously characterized an acid-unfolded (UA) state of Mucor miehei lipase at pH 2. The effect of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) resulted in characterization of molten-globule (MG) like states with β-sheet secondary structure at 15% (v/v) TFE and 6% (v/v) HFIP. α-Helical states accumulate at 80% (v/v) TFE and 30% (v/v) HFIP.

The sheep skins unhairing process with preliminary alkaline treatment of the wool leads to two unnatural dipeptide mimetics lysinoalanine (Lys* - Ala) and ornithinoalanine (Orn*- Ala) obtaining. They are result from the keratin hydrolysis process. The changes of wool keratin make it resistant to sulphide degradation. We synthesized and characterized these unnatural dipeptides under the experimental conditions. The structures and mechanism of Lys* - Ala and Orn*- Ala obtaining were elucidated. The using of newly synthesized products as markers for control of wool's keratin changes during skin unhairing process was demonstrated. The developments have also been the result of economic and environmental pressures to meet environmental regulations.

A series of N-phosphoryl dipeptide derivatives with trimethoxyaniline moiety were synthesized, in which compound 2a DIPP-Val-Phe-Ar exhibited the best inhibitory activity against K562 cells with the IC50 at 9.7 μM. Its antitumor effects are due to the induction of apoptosis which was further confirmed by morphological study and flow cytometry analysis.

The interaction between cholesterol and Human Serum Albumin (HSA) was studied by fluorescence technique. Addition of cholesterol causes decreasing of the fluorescence intensity of HSA and the mechanism can be attributed to static quenching. Both negative enthalpy and entropy change indicate this binding was an “enthalpy-driven” reaction. The number of binding site and distance between residues and ligands were also calculated: n=0.98, r=3.84nm. UV-vis spectra showed HSA molecules unfolded to some extent and the hydrophobicity was decreased in the presence of cholesterol.

Triose phosphate isomerase (TIM) was prepared and purified from chicken breast muscle. The equilibrium unfolding of TIM by urea was investigated by following the changes of intrinsic fluorescence and circular dichroism spectroscopy, and the equilibrium thermal unfolding by differential scanning calorimetry (DSC). Results show that the unfolding of TIM in urea is highly cooperative and no folding intermediate was detected in the experimental conditions used. The thermodynamic parameters of TIM during its urea induced unfolding were calculated as ΔG =3.54 kcal•mol-1, and mG = 0.67 kcal•mol-1•M-1, which just reflect the unfolding of dissociated folded monomer to fully unfolded monomer transition, while the dissociation energy of folded dimer to folded monomer is probe silence. DSC results indicate that TIM unfolding follows an irreversible two-state step with a slow aggregation process. The cooperative unfolding ratio, ΔHcal/ΔHvH, was measured close to 2, indicating that the two subunits of chicken muscle TIM unfold independently. The van't Hoff enthalpy, ΔHvH, was estimated as about 200 kcal•mol-1. These results support the unfolding mechanism with a folded monomer formation before its tertiary structure and secondary structure unfolding.

Peptides with more than one biological activity are many a times multifunctional peptides. Two peptides with multifunctional properties from αS2-casein were stabilized in presence of cosolvents for their biological activities like ACE inhibition activity and antioxidant activity. These bioactive peptides in cosolvents were also thermostable. Infra red spectra of peptides in cosolvents reveal no change in the secondary structure in presence of cosolvents. Correlation between sequence, structure and composition of peptides on biological activities were studied.

Peroxidase from bitter gourd was purified by three step purification scheme; ammonium sulphate fractionation, gel filtration and affinity chromatography. The enzyme was purified 42 fold with the retention of 67% of the initial activity. The enzyme exhibited its maximum activity at pH 5.6 and 40 °C. The enzyme retained half of its activity even after 1 h incubation at 60 °C. Molecular weight of the purified glycosylated bitter gourd peroxidase determined by Sephacryl S- 100 and SDS-PAGE was 43 kDa. The stokes radius, diffusion coefficient and sedimentation coefficient of the purified peroxidase were 27.3 Å, 8.17 x 10-7 cm2/sec and 3.74 S, respectively. Km for o-dianisidine and ABTS were 1.3 and 4.9 mM, respectively. The activity of the enzyme was inhibited by sulfide, azide and L-cysteine. The carbohydrate content and sulfydryl groups of the enzyme were 25% (w/w) mass of the protein and 16 mmoles/mole of the protein, respectively.

Advanced glycation end products (AGEs) are formed from the non-enzymatic reaction between reducing sugars and amine residues on proteins, lipoproteins or nucleic acids. AGEs are found on long-lived proteins and their tissue accumulation is associated with normal ageing. The formation of AGEs can be accelerated in certain pathological conditions such as diabetes where hyperglycaemia is present. AGE modification of proteins can lead to alterations of normal function by binding to intracellular or extracellular cell components, or through receptor binding. This consequently can initiate a cascade of events, which includes the activation of signal transduction pathways, which activate inflammatory responses causing tissue damage. Such tissue injury contributes to the development of microvascular complications and is of particular relevance in diabetes where interventions to reduce the accumulation of AGEs is desirable.

The function of protein is closely correlated with it subcellular location. Prediction of subcellular location of apoptosis proteins is an important research area in post-genetic era because the knowledge of apoptosis proteins is useful to understand the mechanism of programmed cell death. Compared with the conventional amino acid composition (AAC), the Pseudo Amino Acid composition (PseAA) as originally introduced by Chou can incorporate much more information of a protein sequence so as to remarkably enhance the power of using a discrete model to predict various attributes of a protein. In this study, a novel approach is presented to predict apoptosis protein solely from sequence based on the concept of Chou's PseAA composition. The concept of approximate entropy (ApEn), which is a parameter denoting complexity of time series, is used to construct PseAA composition as additional features. Fuzzy K-nearest neighbor (FKNN) classifier is selected as prediction engine. Particle swarm optimization (PSO) algorithm is adopted for optimizing the weight factors which are important in PseAA composition. Two datasets are used to validate the performance of the proposed approach, which incorporate six subcellular location and four subcellular locations, respectively. The results obtained by jackknife test are quite encouraging. It indicates that the ApEn of protein sequence could represent effectively the information of apoptosis proteins subcellular locations. It can at least play a complimentary role to many of the existing methods, and might become potentially useful tool for protein function prediction. The software in Matlab is available freely by contacting the corresponding author.

The repetitive [RTRG]6 peptide was fortuitously identified as a potent nuclear localization signal when linked to the green fluorescent reporter protein. Replacing the arginines by lysines, or the threonines by glycines, both resulted in a decreased nuclear targeting ability of the peptide within this context. By contrast, the sequence [RT]12 proved able to drive nuclear accumulation of the linked protein as efficiently as the starting peptide. Remarkably, [RTRG]n peptides where n=2 to 6 showed a gradual, copy-number dependent, increase in their ability to target the green fluorescent protein to the cell nucleus. As a consequence, the nuclear to cytoplasmic concentration ratio of the linked protein within the cell could be adjusted to different values depending on the number of repeats used in the fusion. Our observation may open the way to the use of [RTRG]n repeats of given lengths (n=2 to 6) for fixing the nuclear-cytoplasmic partition of shuttling protein domains in the course of their functional study.

Bacillus strain CTP-09 yielded maximum productivity (1120 IU/L.h) of extracellular endoglucanase (CMCase) on 0.5% cellobiose after 10 h fermentation at 55° C. The purified enzyme is mono-meric in nature and exhibits stability up to 80° C and over a pH range (6.0-9.0). Activation energy, enthalpy and entropy of catalysis, and inactivation indicated that this CMCase is highly thermos-table. Purified enzyme possessed high power of defibrillation of textile and was minutely inhibited by anionic detergent and oxidizing agent comparable with inhibition by commercial enzyme. This polypeptide could be exploited for mass production and application in local industries.

A novel antiretroviral protein Polygonatum cyrtonema lectin (PCL) belonging to the monocot mannose-binding lectin (MMBL) superfamily has been crystallized using hanging-drop vapor-diffusion method. The crystals diffract to 2.0 Å resolution and belong to space group P21, with unit-cell parameters of a=39.308 Å, b=48.317 Å, c=112.221 Å, and β=90.12° . Preliminary analysis indicates that the asymmetric unit contains four PCL molecules with a solvent content of about 45%. A set of X-ray data has been collected for the crystal structure determination.

The Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) produces a cold-active iron superoxide dismutase (SOD). PhSOD is a homodimeric enzyme, that displays a high catalytic activity even at low temperature. Using hanging-drop vapour-diffusion technique, PhSOD has been successfully crystallized in two different crystal forms. Both crystal forms are monoclinic with space group P21 and diffract to 2.1 Å resolution. Form I has unit-cell parameters a=45.49Å b=103.63Å c=50.37Å β=108.2° and contains a homodimer in the asymmetric unit. Form II has unit-cell parameters a=50.48Å b=103.78Å c=90.25Å β=103.8° and an asymmetric unit containing two PhSOD homodimers. Structure determination has been achieved using molecular replacement. The crystallographic study of this cold-adapted enzyme could contribute to the understanding of the molecular mechanisms of cold-adaptation and of the high catalytic efficiency at low temperature.

Galectin LEC-1 isolated from the nematode Caenorhabditis elegans was the first galectin found in invertebrates and also the first tandem-repeat-type galectin identified, containing two homologous carbohydrate-binding sites. This galectin is localized most abundantly in the adult cuticle and possibly plays a role in the formation of epidermal layers. We succeeded in crystallizing LEC-1 composed of 279 amino acids with a calculated molecular weight of 31,809 Da under two independent sets of conditions as a result of extensive screening. The crystals grown under one set of conditions belong to the triclinic space group P1, with unit-cell parameters a = 48.44, b = 52.13, c = 64.24 Å, α = 108.73, β = 91.39, and γ = 98.45 and two protein molecules per unit cell. The crystals grown under the other set of conditions which included lactose belong to the monoclinic space group P21, with unit-cell parameters a = 52.90, b = 47.01, c = 66.16 Å, and β = 113.30° and one protein molecule per asymmetric unit.

Cytochrome P450 monooxygenases are a superfamily of heme-thiolate proteins involved in the metabolism of a wide variety of endogenous and xenobiotic compounds. The P450 enzyme CYP195A2 from Rhodopseudomonas palustris CGA009, a metabolically versatile bacterium, was overproduced in E. coli and purified. Two distinct crystal forms were obtained under separately optimized conditions by the hanging-drop vapor-diffusion method. Native data sets extending to resolutions of 2.3 Å and 2.8 Å have been collected and processed in space groups P222 and C2221 respectively.