Protein and Peptide Letters - Volume 19, Issue 11, 2012

Protein structure information is very useful for the confirmation of protein function. The protein structural class can provide information for protein 3D structure analysis, causing the conformation of the protein overall folding type plays a significant part in molecular biology. In this paper, we focus on the prediction of protein structural class which was based on new feature representation. We extract features from the Chou-Fasman parameter, amino acid compositions, amino acids hydrophobicity features, polarity information and pair-coupled amino acid composition. The prediction result by the Support vector machine (SVM) classifier shows that our method is better than some others.

Yeast tRNA-thiouridine modification protein 1 (Tum1) plays essential role in the sulfur transfer process of Urm1 system, which in turn is involved in many important cellular processes. In the rhodanese-like domain (RLD), conserved cysteine residue is proved to be the centre of active site of sulfurtransferases and crucial for the substrate recognition. In this report, we describe the crystal structure of Tum1 protein at 1.90 A resolution which, despite consisting of two RLDs, has only one conserved cysteine residue in the C-terminal RLD. An unaccounted electron density is found near the active site, which might point to the new cofactor in the sulfur transfer mechanism.

A novel α-helical antimicrobial peptide G6 rich in Val/Arg residues has been screened previously. In this study, we further evaluated the biochemical stability, interaction with whole bacteria, and in vivo therapeutic or prophylactic role of the peptide in Salmonella typhimurium-infected mice. The results showed that G6 exhibited strong resistance to pH, heat, and salts. But G6 lost the antimicrobial activity when treated with proteolytic enzymes. G6 had no toxicity on mammalian cell. An intraperitoneal model of sepsis caused by Salmonella typhimurium was established in mice. G6 was administered intraperitoneally 1 h before or after mice were infected with Salmonella typhimurium. For the mice given peptide post-bacterial infection, the mortality of the mice and the peritoneal bacterial counts were significantly lower in the groups that were administered 2.5 mg/kg BW and 5.0 mg/kg BW of G6 (P < 0.05) compared to the PBS-treated group. Similar trend was obtained when G6 was given 1 h prior to Salmonella typhimurium infection. Peptide-membrane experiments showed that G6 was effective in permeabilizing the outer and inner membrane in a dose dependent manner, indicating that the peptide targets the cell membrane. Taken together, the results revealed that the peptide G6 may provide a useful alternative to antibiotic agents to treat or prevent bacterial infections.

Rheumatoid arthritis (RA) is a chronic disease with an autoimmunological background. RA is mostly characterized by systemic inflammation and injuries of synovial joints. There is a hypothesis that bacterial infections may be connected with development of the disease. It has been suggested that molecular mimicry between bacterial and human antigens may be one of possible mechanisms of RA development. One of potential antigens involved in this mechanism is urease - enzyme with high structural conservatism, occurring in pathogenic and commensal bacteria. We found that the level of antibodies against peptide mimicking urease “flap” region is significantly higher in sera from patients with rheumatoid arthritis in comparison with volunteer blood donor sera. We also observed that antibodies present in RA sera may bind not only specific peptide antigens but also peptides with a slightly different structure.

Bacterial lipoproteins play critical roles in various physiological processes including the maintenance of pathogenicity and numbers of them are being considered as potential candidates for generating novel vaccines. In this work, we put forth an algorithm to identify and predict ligand-binding sites in bacterial lipoproteins. The method uses three types of pocket descriptors, namely fpocket descriptors, 3D Zernike descriptors and shell descriptors, and combines them with Support Vector Machine (SVM) method for the classification. The three types of descriptors represent shape-based properties of the pocket as well as its local physio-chemical features. All three types of descriptors, along with their hybrid combinations are evaluated with SVM and to improve classification performance, WEKA-InfoGain feature selection is applied. Results obtained in the study show that the classifier successfully differentiates between ligand-binding and non-binding pockets. For the combination of three types of descriptors, 10 fold cross-validation accuracy of 86.83% is obtained for training while the selected model achieved test Matthews Correlation Coefficient (MCC) of 0.534. Individually or in combination with new and existing methods, our model can be a very useful tool for the prediction of potential ligand-binding sites in bacterial lipoproteins.

Machine learning is a kind of reliable technology for automated subcellular localization of viral proteins within a host cell or virus-infected cell. One challenge is that the viral protein samples are not only with multiple location sites, but also class-imbalanced. The imbalanced dataset often decreases the prediction performance. In order to accomplish this challenge, this paper proposes a novel approach named imbalance-weighted multi-label K-nearest neighbor to predict viral protein subcellular location with multiple sites. The experimental results by jackknife test indicate that the presented algorithm achieves a better performance than the existing methods and has great potentials in protein science.

Transcription factors (TF) are proteins that control the first step of gene expression, the transcription of DNA into RNA sequences. The mechanism of transcriptional regulatory can be much better understood if the category of transcription factors is known. We developed a new method for predicting the classification of transcription factors by incorporating their binding site properties into a novel mode of Chou's pseudo amino acid composition. The properties include the length of TFBSs for a TF, a new_PWM value, the proportion of not conservative TFBSs, the proportion of nonucleosome of TFBSs, the proportion of conserved-nucleosome of TFBSs, and the GC content of TFBSs. We construct a vector with these properties to represent a TF. Then the vectors which stand for TFs were classified with SVMs. The high accuracy obtained shows that these properties are of great significance for a TF.

β-N-acetyl-D-glucosaminidase (NAGase) is a major member in chitinolytic enzymes system, which plays an important role in the hatching and molting processes of marine organism. The effects of guanidinium chloride (GuHCl) on the activity of NAGase from green crab (Scylla serrata) were investigated in this study. In results, GuHCl causes reversible inactivation of the enzyme at below 0.8 M concentrations, and the IC50 is estimated to be 0.15 M. The relationship between the enzyme activity and conformation was charaterized by monitoring the change of protein fluorescence spectra. With increasing GuHCl concentration, the fluorescence intensity of the enzyme distinctly decreases , and the maximal emission peaks appear red-shifted (from 338 nm to 343 nm). The enzyme inactivation precedes conformational changes, indicating that the enzyme active site is more flexible than the whole enzyme molecule. The result of the kinetics of inactivation shows that the value of k+0 is larger than that of k+0 '. It suggests that the substrate could protect the enzyme to a certain extent during guanidine denaturation. Our results provide important new insights in marine organism culture, especially in crustacean growth.

The NAD+-requiring enzymes of the aldehyde dehydrogenase (ALDH) family contain a glycine motif, GX1– 2GXXG, which is reminiscent of the fingerprint region of the Rossman fold, a conserved structural motif of the classical nicotinamide nucleotide-binding proteins. In this research, the role of three glycine residues situated within the putative NAD+-binding motif (211-GPGSSAG) together with Gly233 and Gly238 of Bacillus licheniformis ALDH (BlALDH) were probed by site-directed mutatgenesis. Fifteen mutant BlALDHs were obtained by substitution of the indicated glycine residues with alanine, glutamate and arginine. Except for the Ala replacement at positions 211, 213, 217 and 238, the remaining mutant enzymes lost the dehydrogenase activity completely. Tryptophan fluorescence and far-UV circular dichroism spectra allowed us to discriminate BlALDH and the inactive mutant enzymes, and unfolding analyses further revealed that they had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlALDH and the functional variants had a comparable Tm value, but the value was reduced by more than 5.1°C in the rest of mutant enzymes. Acrylamide quenching analysis showed that the inactive mutant enzymes had a dynamic quenching constant greater than that of BlALDH. Native BlALDH started to unfold beyond ∼ 0.21 M GdnHCl and reached an unfolded intermediate, [GdnHCl]0.5, N-U, at 0.92 M equivalent to free energy change ( N-U H2O Δ G ) of 12.34 kcal/mol for the N → U process, whereas the denaturation midpoints for mutant enzymes were 0.45–1.61 M equivalent to N-U H2O Δ G of 0.31–4.35 kcal/mol. Taken together, these results strongly suggest that the explored glycines are indeed important for the catalytic activity and structural stability of BlALDH.

The gap between known protein sequences and structures is increasing rapidly and experimental methods alone will not be able to fill in this gap. Therefore it is necessary to use computational methods to predict protein structures. Template based modeling methods could be used for sequences, which have detectable relationship with sequences of one or more experimentally determined protein structures. For predicting the structure of proteins, which does not share a detectable sequence relationship with experimental structures, ab initio protein structure prediction techniques must be used. The methods under ab initio protein structure prediction category aim to predict the structure of a protein from the sequence information alone, without any explicit use of previously known structures. These methods use thermodynamic principles and try to identify the native structure of a protein as the global minimum of a potential energy landscape. However, such methods are computationally complex and are extraordinarily challenging. There has been significant progress in the development of ab inito protein structure prediction methods over the past few years. This review describes the basic principles, the complexity, challenges and recent progresses of ab initio protein structure prediction.

Pectin methylesterase (PME) (3.1.1.11) is the pectin degrading enzyme which catalyses the hydrolysis of pectin methylester group, resulting in de-esterification. PME is widely distributed in plants, fungi, yeast and bacteria. In the present study, PME was extracted from tomato by using 8.8% NaCl (4°C). The crude enzyme precipitated with 60% ammonium sulphate resulted in 1.02 fold purification of the enzyme. The purification was done by ion exchange chromatography using DEAE-Cellulose column. This resulted in 1.82 fold purification of the enzyme. The molecular weight of purified enzyme was determined by SDS-PAGE which was found to be 34.0 kDa. During characterization of the purified enzyme, the maximum activity was found at temperature 50°C, pH 6.5, reaction time 45 min. Citrus pectin was the best substrate for maximum enzyme activity. The enzyme did not require any metal ion to express its activity, enzyme was found to be very stable at 4°C and at 50°C the enzyme was stable upto 2 h as it retained 70% of its activity. The Km and Vmax values of the enzyme were found to be 0.115 mg/ml and 1.03 μmol/ml/min. PME enhanced the pectin degradation process in apple juice clarification in combination with polygalacturonase and increased %T650 from 1.7% to 5.6%.

Short antimicrobial peptides were designed and synthesized by C-terminal truncation and residue substitution of avian β-defensin-4. The biological activity of these peptides was examined to elucidate the quantitative structureactivity relationships and find a lead peptide for the development of a novel antimicrobial peptide. The results showed that the truncation of the avian β-defensin-4 eliminated the hemolysis of the peptide. The GLI13 derivative, developed by substituting the Cys of the truncated peptide with Ile, led to increased antimicrobial activity. These results suggest that the peptides with antimicrobial activity can be derived by truncating the avian β-defensin-4. We further developed the GLI13 derivative with an increased net charge by residue substitution. The results showed that the GLI13-5 with 5 net charges had the highest cell selectivty. An increase in the net charge from 6 to 8 did not result in the improvement of antimicrobial potency. Membrane-simulating experiments showed that the peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which led to greater cell selectivity. A membrane depolarization assay showed that GLI13-5 killed bacteria by targeting the cytoplasmic membrane. These results suggest that the short peptide developed by truncation of linear β-defensin may be a promising candidate for future antibacterial agents.

Nucleotide phosphorylation is a key step towards DNA replication and during viral infections the maintenance of the nucleotide triphosphates pool is required. Deoxythymidine triphosphate (dTTP) is the unique nucleotide that is produced either by de novo or salvage pathways. Thymidine monophosphate kinase (TMK) is the enzyme that phosphorylates deoxythymidine monophosphate (dTMP) using adenosine triphosphate (ATP) as a phosphate group donor in presence of Mg2+ yielding deoxythymidine diphosphate (dTDP) and adenosine diphosphate. The TMK region of the WSSV TK-TMK chimeric protein was overexpressed and purified. This recombinant protein had TMK activity, this is that dTMP was phosphorylated to dTDP and we found that the dimeric state of the protein was the functional and a theoretical structural model was built as such. Future work will focus towards a structural characterization as an antiviral target.

ThyX, a flavin-dependent thymidylate synthase that is involved in the synthesis of dTMP from dUMP, is a promising target for the development of novel antibacterial drugs that aimed at blocking the biosynthesis of dTMP, one of the building blocks of DNA. This enzyme has been recently identified in some dsDNA viruses and pathogenic bacteria, including the gastric pathogen Helicobacter pylori. It shares neither sequence nor structural homology with the classical ThyA in humans and other organisms. Further more, ThyX and ThyA are the only source of dTMP in these organisms and other pathways cannot substitute for their function. Thus, ThyX-specific inhibitors could be effective antibacterial reagents while having no impact on human cells. Here we report the crystal structure of ThyX from Helicobacter pylori strain 26695 in complex with co-factor FAD and substrate dUMP at 2.5 A resolution, which consists of a 1.5 tetramer of ThyX with a total of 1248 residues, six FAD and six dUMP molecules in an asymmetric unit. The structure revealed the key residues that are involved in co-factor FAD and substrate dUMP binding, site-directed mutagenesis were performed to analysis the importance of these residues on ThyX activity by genetic complementation and FAD binding assay.

A microwave assisted peptide synthesis in water using nanosized Fmoc-amino acids was developed. 5, 7, and 10 mer peptides (Leu-enkephalinamide, dermorphinamide, and a typical difficult sequence, ACP (65-74) peptide) were successfully synthesized in water according to Fmoc chemistry using water-dispersible nanoparticles with microwave irradiation.