Protein and Peptide Letters - Volume 20, Issue 1, 2013

The synthesis and analysis of nanostructures in the cavities of protein molecules is a promising research field in the industry of nanosystems. In this study, atomic force microscopy (AFM) has been used to evaluate the properties of CdS quantum dots synthesized in the tunnel cavities of R-phycoerythrin, a 290 kDa water-soluble pigment protein responsible for light harvesting in red algae. It has been shown that R-phycoerythrin dissolved in deionized water to a concentration of 50 μg/ml is prone to self-organization into regular spatial structures upon adsorption on the surface of mica, but no such structuring takes place in films prepared from R-phycoerythrin solutions diluted tenfold. In the latter case, protein molecules are deformed, as judged from the analysis of the surface profile. R-phycoerythrin with CdS quantum dots in protein cavities (the concentration of the preparation was (48 μg/ml) loses the self-organization ability and is not deformed upon adsorption on the mica surface. Analysis of AFM images by flicker-noise spectroscopy has shown that incorporation of CdS quantum dots into R-phycoerythrin molecules provides for “smoothing” of the protein surface, with various irregularities being leveled off. Conversely, the irregularity of the protein surface increases when R-phycoerythrin molecules are arranged into three-dimensional branching structures. It is concluded that CdS quantum dots interfere with protein–protein interactions and restrain the conformational mobility of the protein. The anomalously rigid structure of Rphycoerythrin in the presence of CdS is due to its conformational rearrangements during the synthesis of quantum dot.

Neurospora crassa has been widely used as a model organism and contributed to the development of biochemistry and molecular biology by allowing the identification of many metabolic pathways and mechanisms responsible for gene regulation. Nuclear proteins are synthesized in the cytoplasm and need to be translocated to the nucleus to exert their functions which the importin-α receptor has a key role for the classical nuclear import pathway. In an attempt to get structural information of the nuclear transport process in N. crassa, we present herein the cloning, expression, purification and structural studies with N-terminally truncated IMPα from N. crassa (IMPα-Nc). Circular dichroism analysis revealed that the IMPα-Nc obtained is correctly folded and presents a high structural conservation compared to other importins-α. Dynamic light scattering, analytical size-exclusion chromatography experiments and molecular dynamics simulations indicated that the IMPα-Nc unbound to any ligand may present low stability in solution. The IMPα-Nc theoretical model displayed high similarity of its inner concave surface, which binds the cargo proteins containing the nuclear localization sequences, among IMPα from different species. However, the presence of non-conserved amino acids relatively close to the NLS binding region may influence the binding specificity of IMPα-Nc to cargo proteins.

The current study was aimed at analyzing putative protein sequences of the protamine-like proteins of 12 Drosophila species based on the reference sequences of two protamine-like proteins (Mst35Ba and Mst35Bb) found in Drosophila melanogaster sperm nuclei. Protamine-like proteins belong to a larger group of proteins that are involved in DNAbinding known as sperm nuclear basic proteins (SNBPs). SNBPs play a role in chromatin condensation during the postmeiotic stage of spermatogenesis, termed spermiogenesis. During spermiogenesis, nuclear transformation occurs where histones are exchanged for SNBPs, the chromatin condenses, and the nucleus transforms into a needle-like shape in Drosophila. Our goal was to search the 12 sequenced Drosophila genomes for protamine-like proteins based on the known sequences for D. melanogaster. Searches were performed on genomic DNA, mRNA transcripts and amino acid sequences using NCBI basic local alignment search tool (BLAST). Sequence alignments and analysis of amino acid content indicate that homologs for Mst35Ba and Mst35Bb are present in all 12 species of flies analyzed in this study. Functional analyses of a conserved region found within the proteins indicate the presence of a DNA-binding domain, possibly a high mobility group DNA- binding box. This study represents the first large-scale, single-genus dataset for protamine-like proteins and provides the basis for a fine-grained analysis of their evolution.

Recently, the cloning of the ORF Dr0930 from Deinococcus radiodurans displaying, as primary activity, a lactonase activity and a promiscuous phosphotriesterase activity was reported. The crystal structure of the resulting recombinant enzyme has been solved, and many mutants have been generated in order to increase the phosphotriesterase activity, with the aim to reach the level of activity of the related pPTE from Pseudomonas diminuta. In this paper we report an additional biochemical characterization of DrPLL and show that this enzyme has an optimal temperature for catalysis of 85 °C and possesses promiscuous carboxylesterase, phophodiesterase and thioesterase activities which were not previously described. A metal analysis was performed on the purified protein by inductively coupled plasma mass spectrometry (ICP-MS ELAN DRC-e), which confirmed the presence of Ni2+ as a main metal in the recombinant protein. Surprisingly, the specificity constants (s=kcat/KM) for the pNP-decanoate and pNP-dodecanoate esters were only one order of magnitude lower than that for the lactone substrate thio-buthyl-γ-butyric-lactone (TBBL), and the KM value for TBBL was more than ten-fold higher than those for the esters. We named this enzyme DrPLL, based on its structural and biochemical features it belongs to the Phosphotriesterase Like Lactonase group, a small protein family belonging to the amidohydrolase superfamily.

PepX is a X-prolyl dipeptidyl aminopeptidase of S15 family that cleaves dipeptides from the N-terminus of polypeptides having a proline or alanine residue at the second position. Involved in bacterial nutrition and in peptide maturation, this serine exopeptidase, counterpart of the mammalian DDP-4, has been proposed to play a role in pathogenicity for Streptococci and to be a promising target against trypanosomes. Searching for specific inhibitors, we undertook docking simulations on the whole surface of PepX from Lactococcus lactis, type example of the S15 family, which revealed a new putative binding site in connection with the active site and involving the C-terminal domain. Accordingly to the results of the computations, we synthesized two peptidomimetics of low molecular weight: the valinephenylpiperazine and the valine-isopropylpiperazine that can accommodate to this putative binding site. Experiments revealed that the valine-phenylpiperazine was an uncompetitive inhibitor whereas the valine-isopropylpiperazine showed to be an activator of the enzyme activity. The valine-phenylpiperazine is interacting with ASN 379, GLU 383, GLU 474, residues in connection with the specificity and active sites, and with the residues from the C-terminal domain LEU 693, GLU 710 and GLN 712. These results point out a role of the C-terminal domain in controlling access to the active site of enzymes of the S15 family, like PepX, the cocaine esterase or the alpha-amino acid ester hydrolase, and could have applications in human health giving new perspectives to struggle against streptococci or trypanosomes by designing inhibitors specific to the S15 family of enzymes.

OG2 is a modified antimicrobial peptide of Palustrin-OG1 (OG1), which is derived from Odorrana grahami frog. OG2 has shown much higher selective antimicrobial activity and lower hemolytic activity than OG1, indicating OG2 may be a promising antimicrobial agent. In this study, we investigated three fusion partners, including thioredoxin, Mxe GyrA intein, and small ubiquitin-like modifier (SUMO), each fused with OG2, and examined their effects on the expression level and solubility of OG2 in Escherichia coli. The codon-optimized OG2 gene was cloned into pET32a (+) and pTWIN1 for fusion with thioredoxin and Mxe GyrA intein, respectively. In addition, the SUMO-OG2 gene was amplified by splice overlap extension PCR method and was cloned into pET30a (+). All recombinant plasmids were then transformed into E. coli BL21(DE3)pLysS, and the expressed fusion proteins were verified. Upon isopropyl β-D-1- thiogalactopyranoside (IPTG) induction, OG2 fused with thioredoxin (Trx-OG2) showed the highest yield as a soluble fusion protein (50 mg/L), followed by Mxe GyrA intein (44 mg/L) and SUMO (11 mg/L). The thioredoxin-fused protein (Trx-OG2) was then purified by nickel-nitrilotriacetic acid chromatography and desalted by Sephadex G25. The OG2 released by both tobacco etch virus protease and enterokinase from Trx-OG2 showed strong antimicrobial activity against Staphylococcus aureus ATCC25923.

Kidney cells of animals including human and marine invertebrates contain high amount of the protein denaturant, urea. Methylamine osmolytes are generally believed to offset the harmful effects of urea on proteins in vitro and in vivo. In this study we have investigated the possibility of glycine to counteract the effects of urea on three proteins by measuring thermodynamic stability, ΔGD o and functional activity parameters (Km and kcat). We discovered that glycine does not counteract the effects of urea in terms of both protein stability and functional activity. We also observed that the glycine alone is compatible with enzymes function and increases protein stability in terms of Tm (midpoint of thermal denaturation) to a great extent. Our study indicates that a most probable reason for the absence of a stabilizing osmolyte, glycine in the urea-rich cells is due to the fact that this osmolyte is non-protective to macromolecules against the hostile effects of urea, and hence is not chosen by evolutionary selection pressure.

Protein methylation is an important and reversible post-translational modification which regulates diverse protein properties. Many methylation sites on arginine and lysine have been identification through experiments. However, experimental identification without prior knowledge is laborious and costly. Hence, there is interest in the development of computational methods for reliable prediction of methylation sites. Prediction of methylation sites may provide researches with useful information for further productivity in methylation candidate sites discovery. This work proposes Methcrf, a computational predictor based on conditional random field (CRF) for predicting protein methylation sites limit to lysine and arginine residues due to the absence of enough experimentally verified data for other residues. The approach is developed to consider combining protein sequence features with structural information such as solvent accessibility of amino acids that surround the methylation sites. In 10-fold cross validation Methcrf can achieve the area under receiver operating characteristic curve (AUC) of 0.85 and 0.80 for arginine and lysine, respectively. The proposed method has comparable performance with previous methods for accurately predicting methylation sites.

Copepods are the most abundant multicellular animal group and are often most important component of zooplankton and indicators of local and global climate change. Among this broad group of animals, Calanus sinicus holds an important role in the ecosystems of The East China Sea, The Korea Strait, and The East Sea. By comparing the response of their proteomes to ecologically viable environment changes, this study tried to identify molecular responses accountable for compensation of a change in metabolic rates. Using two-dimensional gel electrophoresis and mass spectrometry on C. sinicus sampled at its native environment, 45 distinct proteins were identified that changed abundance as a function of environment changes i.e., temperature elevation and/or oxygen decrease, and 14 that are only present in proteome adapted to higher temperature/lower oxygen. Although the study failed to find heat shock proteins, the abundance of disulfide-isomerase A3 precursor was higher in species thriving at higher temperatures/lower oxygen. The abundance of proteins responsible for redox homeostasis, DNA maintenance, and chromatin rearrangement was also higher at elevated temperature. Also, the molecular machinery responsible for xenobiotic metabolism is mobilized at higher temperatures/ lower oxygen levels. These data indicate fine adjustment of molecular apparatus in response to changes in living environment.

Obtaining soluble proteins in sufficient concentrations helps increase the overall success rate in various experimental studies. Protein solubility is an individual trait ultimately determined by its primary protein sequence. Exploring the interconnection between the protein solubility and the compositions of protein sequence is instrumental for setting priorities on targets in large scale proteomics projects. In this paper, amino acid composition (20 dimensions) and the dipeptide composition (400 dimensions) were extracted to form the total candidate feature pool (420 dimensions), and each feature was selected into the feature vectors one by one, which were sorted by the absolute value of the correlation coefficient. Finally, we evaluated and recorded the 420 results of Support Vector Machine (SVM) as the prediction engine. According to the results of SVM, the first 208 features were chosen from the 420 dimensions, which were considered as the efficient ones. By analyzing the composition of the former 208 features, we found that the protein solubility was significantly influenced by the occurrence frequencies of the acidic amino acids, basic amino acids, non-polar hydrophobic amino acids and the two polar neutral amino acids(C, Q) in the protein sequences. Additionally, we detected that the dipeptides composed by the acidic amino acids (D, E) and basic amino acids (K, R and H), especially the dipeptide composed by the acidic amino acids (D, E), had strong interconnection with the protein solubility.

Human transferrin receptor 1 (TfR1) is expressed on malignant cells at levels several fold higher than those on normal cells and its expression can be correlated with tumor stage or cancer progression. It is a potentially rational target for drug delivery. To identify novel ligands which can recognize the TfR1 specifically, a random phage displayed 12-mer peptide library was screened and two consensus motifs of the peptides which are displayed by the positive phages RXR and RXXXR (x is any amino acid) were yield. The phage displaying peptide SPRPRHTLRLSL (designated as B18) exhibited the highest affinity to TfR1 in phage ELISA and B18 could bind to TfR1 specifically in a dose dependent manner. The flow cytometry assay and immunocellularchemistry assay showed that the B18 could bind to TfR1 positive carcinoma cells with specificity. In addition, the biodistribution assay indicated that B18 could home to TfR1 positive tumor tissue specifically. Our study suggests that the peptide exhibited by B18 is a potentially promising targeting peptide for tumor diagnosis and treatment.

The propensity to catalysis of transglycosylation of the β-glucosidase Tmβgly is higher than for Sfβgly. Moreover the propensity to catalysis of transglycosylation is directly proportional to the substrate concentration for Tmβgly, whereas for Sfβgly it is constant. For instance, 60% of a Tmβgly sample catalyzes transglycosylation reactions at 40 mM p-nitrophenyl β-glucoside, whereas only 40% is engaged in hydrolysis of this substrate. For Sfβgly the fraction involved in transglycosylation is only 30 %. In addition, 48 % of a Tmβgly sample catalyzes transglycosylation reactions at 8 mM methylumbelliferyl β-glucoside, whereas Sfβgly does not catalyze transglycosylation using this substrate. Interestingly, these Tmβgly properties were grafted into Sfβgly by a single replacement of a residue forming a channel involved in supplying the catalytic water molecules for attack on the covalent intermediate present in the reaction catalyzed by β-glucosidases. Hence a single residue determines the ratio of hydrolysis to transglycosylation reactions catalyzed by these β-glucosidases.

Sequence alignments and homology modeling of Pyrococcus furiosus thermostable glycosidase (PFTG) showed that the residue 150 is conserved as tryptophan in β-glycosidase and in other related enzymes such as β- mannosidase and β-galactosidase. To elucidate the relationship between the substrate size and geometric shape of the catalytic site of thermophilic β-glycosidase and category of PFTG, the Q77, Q150 and D206 located at the interface of the dimer were replaced with Trp and Asn. Also, to confirm the role of active sites of PFTG, the Q77R/Q150W double mutant was created through subcloning. Temperature and pH optima of both mutants and native enzyme were same at 100°C and pH 5.0 in sodium citrate buffer, respectively. The catalytic efficiencies (kcat/Km) of the mutants on synthetic and natural substrates by Isothermal Titration Calorimetry were slightly changed, but indicated the characteristics of β-glycosidase activity. Kinetic parameters of the mutant enzymes indicated that they possess characteristics of both β- galactosidase and β-mannosidase activities. Although the mutant enzymes showed similar substrate specificities compared to the recombinant enzyme, they had more affinity (Km) to substrates with low turnover number (kcat).