Protein and Peptide Letters - Volume 18, Issue 6, 2011

The prolactin (PRL) permeation through the pericardium depending on the species of origin (porcine, bovine and ovine) was studied, and the parameters of its bioavailability were calculated. An in vitro model using pericardium as a natural membrane and Frantz cell method was applied. Significant differences in permeation were observed depending on the species of origin. Within 5 h, 17.5% of bovine PRL, 27.2% of porcine PRL and 90.3% of ovine PRL permeated the pericardium. The amount of permeated ovine PRL was 3.3-fold higher than porcine PRL and 5.2-fold higher than bovine PRL. The maximum concentration of permeated PRL was reached in the thirtieth minute of the experiment and was the highest for ovine PRL (Cmax = 677.21 μg/cm2) and the lowest for bovine PRL (Cmax=259.97 μg/cm2). Bioavailability of PRL through the pericardium is 3.3-fold greater for ovine PRL in comparison to porcine or bovine PRL. The relative extent of bioavailability for bovine and ovine prolactin versus the porcine PRL standard was 85.6% and 229.3%, respectively.

Aqualysin I, is a subtilisin-like serine proteinase, from the thermophilic bacterium Thermus aquaticus. It is predicted that the enzyme contains a salt bridge, D17-R259, connecting the N- and C-terminal regions of the enzyme. Previously we reported on the stabilizing effect of the incorporation of a salt bridge at a corresponding site in VPR, a related cold adapted enzyme from a marine Vibrio sp. Here we describe the effect of the reverse change, i.e. the elimination of the salt bridge on the thermal stability and kinetic properties of aqualysin I. Deletion of the putative salt bridge in the D17N mutant of the enzyme destabilized the enzyme by 8-9 °C in terms of T50%, determined by thermal inactivation and over 4°C in Tm, as measured from melting curves of the inhibited enzyme. The mutation, however, had no significant effect on the kinetic parameters of the enzyme under standard assay conditions.

Proteases are essential to most biological processes though they themselves remain intact during the processes. In this research, a computational approach was developed for predicting the families of proteases based on their sequences. According to the concept of pseudo amino acid composition, in order to catch the essential patterns for the sequences of proteases, the sample of a protein was formulated by a series of its biological features. There were a total of 132 biological features, which were sourced from various biochemical and physicochemical properties of the constituent amino acids. The importance of these features to the prediction is rated by Maximum Relevance Minimum Redundancy algorithm and then the Incremental Feature Selection was applied to select an optimal feature set, which was used to construct a predictor through the nearest neighbor algorithm. As a demonstration, the overall success rate by the jackknife test in identifying proteases among their seven families was 92.74%. It was revealed by further analysis on the optimal feature set that the secondary structure and amino acid composition play the key roles for the classification, which is quite consistent with some previous findings. The promising results imply that the predictor as presented in this paper may become a useful tool for studying proteases.

The aim of this study was prediction of epitopes and medically important structural properties of protein E of Alkhurma hemorrhagic fever virus (AHFV) and comparing these features with two closely relates viruses, i.e. Kyasanur Forest disease virus (KFDV) and Tick-borne encephalitis virus (TBEV) by bioinformatics tools. Prediction of evolutionary distance, localization, sequence of signal peptides, C, N O glycosylation sites, transmembrane helices (TMHs), cysteine bond positions and B cell and T cell epitopes of E proteins were performed. 2D-MH, Virus-PLoc, Signal-CF, EnsembleGly, MemBrain, DiANNA, BCPREDS and MHCPred servers were applied for the prediction. According to the results, the evolutionary distance of E protein of AHFV and two other viruses was almost equal. In all three proteins of study, residues 1-35 were predicted as signal sequences and one asparagine was predicted to be glycosylated. Results of prediction of transmembrane helices showed one TMH at position 444-467 and the other one at position 476-490. Twelve cysteines were potentially involved to form six disulfide bridges in the proteins. Four parts were predicted as B cell epitopes in E protein of AHFV. One epitope was conserved between three proteins of study. The only conserved major histocompatibility complex (MHC) binding epitope between three viruses was for DRB0401 allele. As there are not much experimental data available about AHFV, computer-aided study and comparison of E protein of this virus with two closely related flaviviruses can help in better understanding of medical properties of the virus.

Heterologous protein expression levels can not be evaluated in real time by experimental procedures commonly used for most expression systems during host cell culture. Rb. sphaeroides has provided an ideal system for studying both photosynthesis and membrane development and exhibited potential as a novel expression system. We constructed the puc1BA and puc2BA mutant strain Rb. sphaeroides CQU68 and used it as a novel expression system to heterologously express proteins fused to β-subunit of light-harvesting 2 complexes (LH2). The presence of LH2 with β-subunit fusion proteins was spectrally detected by the LH2 typical absorption at ∼800 nm and ∼850 nm, and the formation of these complexes were further confirmed by SDS-PAGE and Western blot analysis. The expression levels of heterologous protein measured by SDS-PAGE and Western blot turned out to be higher as the typical spectral peak heights increase. These findings suggested that the production of the heterologous protein could be rapidly detected through the LH2 absorption at ∼800 nm and ∼850 nm. Moreover, the typical absorption could be used as a monitor for rapid and real-time evaluation of heterologous protein expression levels.

Protein S-nitrosylation plays a key and specific role in many cellular processes. Detecting possible Snitrosylated substrates and their corresponding exact sites is crucial for studying the mechanisms of these biological processes. Comparing with the expensive and time-consuming biochemical experiments, the computational methods are attracting considerable attention due to their convenience and fast speed. Although some computational models have been developed to predict S-nitrosylation sites, their accuracy is still low. In this work,we incorporate support vector machine to predict protein S-nitrosylation sites. After a careful evaluation of six encoding schemes, we propose a new efficient predictor, CPR-SNO, using the coupling patterns based encoding scheme. The performance of our CPR-SNO is measured with the area under the ROC curve (AUC) of 0.8289 in 10-fold cross validation experiments, which is significantly better than the existing best method GPS-SNO 1.0's 0.685 performance. In further annotating large-scale potential S-nitrosylated substrates, CPR-SNO also presents an encouraging predictive performance. These results indicate that CPR-SNO can be used as a competitive protein S-nitrosylation sites predictor to the biological community. Our CPR-SNO has been implemented as a web server and is available at http://math.cau.edu.cn/CPR -SNO/CPR-SNO.html.

The use of small carbohydrates that stabilize proteins from misfolding is important from pharmaceutical point of view. We have investigated the role of small isomeric amino sugars on the in vitro aggregation of insulin amyloid. Using mass spectrometry, we screened 6 isomeric aminosugars for their role on inhibition of insulin amyloid formation and the results were compared with transmission electron microscopy imaging. We found that three N-acetylamino sugars promote insulin fibril formation. Among three isomeric aminosugars studied, only galactosamine showed few fibrils whereas other two isomers showed enhanced fibrils. The results demonstrated here may contribute to future designing of small amine derivatised galactose sugars as amyloid inhibitors and understanding their action.

A 7.3-kDa antifungal peptide was purified from dried red kidney beans. The purification procedure entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, followed by fast protein liquid chromatography-gel filtration on Superdex 75. The peptide was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and CM-cellulose. It exhibited a molecular mass of 7.3 kDa in gel filtration and also in SDS-polyacrylamide gel electrophoresis, indicating that it is a single-chained protein. The N-terminal sequence of the peptide was DGVCFGGLANGDRT. The peptide exerted an antifungal action on Fusarium oxysporum with an IC50 of 3.8±0.4 μM (mean±SD, n=3). It also inhibited mycelial growth in Mycosphaerella arachidicola. It suppressed growth of lymphoma MBL2 cells and leukemia L1210 cells with an IC50 of 5.2±0.4 μM and 7.6±0.6 μM, respectively. HIV-1 reverse transcriptase was inhibited with an IC50 of 40±3.2 μM. However, no activity was demonstrated toward other viral enzymes.

Escherichia coli YajC is a small integral membrane protein with a single transmembrane helix. The gene yajC is part of the secD operon and the protein is identified in the SecDF-YajC complex. However, the exact function of YajC remains a mystery. While its function is usually discussed in the context of the SecDF-YajC complex, studies have shown that SecD/F, rather than YajC, are essential for those functions. Recently YajC is identified as the mysterious protein that co-crystallized with AcrB. To further investigate the structure of YajC, we expressed and purified the protein in a detergent solubilized state. The protein assumed a folded structure containing mixed α/β secondary structures, consistent with the structural prediction. Using signal Cys mutations and thiol-specific probes, we found the C-terminus of YajC was cytoplasmic, while the N-terminus of YajC was buried in the membrane. In addition, we expressed and purified a truncated fragment of YajC that corresponded to the C-terminal cytoplasmic domain (YajCCT). YajCCT formed a compact structure rich in β-strands and existed as a trimer.

A novel method is presented for predicting β-hairpin motifs in protein sequences. That is Random Forest algorithm on the basis of the multi-characteristic parameters, which include amino acids component of position, hydropathy component of position, predicted secondary structure information and value of auto-correlation function. Firstly, the method is trained and tested on a set of 8,291 β-hairpin motifs and 6,865 non-β-hairpin motifs. The overall accuracy and Matthew's correlation coefficient achieve 82.2% and 0.64 using 5-fold cross-validation, while they achieve 81.7% and 0.63 using the independent test. Secondly, the method is also tested on a set of 4,884 β-hairpin motifs and 4,310 non-β- hairpin motifs which is used in previous studies. The overall accuracy and Matthew's correlation coefficient achieve 80.9% and 0.61 for 5-fold cross-validation, while they achieve 80.6% and 0.60 for the independent test. Compared with the previous, the present result is better. Thirdly, 4,884 β-hairpin motifs and 4,310 non-β-hairpin motifs selected as the training set, and 8,291 β-hairpin motifs and 6,865 non-β-hairpin motifs selected as the independent testing set, the overall accuracy and Matthew's correlation coefficient achieve 81.5% and 0.63 with the independent test.

Two Calvin Cycle enzymes, NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a multiprotein complex with CP12, a small intrinsically-unstructured protein. Under oxidizing conditions, association with CP12 confers redox-sensitivity to the otherwise redox-insensitive A isoform of GAPDH (GapA) and provides an additional level of down-regulation to the redox-regulated PRK. To determine if CP12- mediated regulation is specific for GAPDH and PRK in vivo, a high molecular weight complex containing CP12 was isolated from tobacco chloroplasts and leaves and its protein composition was characterized. Gel electrophoresis and immunoblot analyses after separation of stromal proteins by size fractionation verified that the GAPDH (both isoforms) and PRK co-migrated with CP12 in dark- but not light-adapted chloroplasts. Nano-liquid-chromatography-mass-spectrometry of the isolated complex identified only CP12, GAPDH and PRK. Since nearly all of the CP12 from darkened chloroplasts migrates with GADPH and PRK as a high molecular mass species, these data indicate that the tight association of tobacco CP12 with GAPDH and PRK is specific and involves no other Calvin Cycle enzymes.

The study of rat proteins is an indispensable task in experimental medicine and drug development. The function of a rat protein is closely related to its subcellular location. Based on the above concept, we construct the benchmark rat proteins dataset and develop a combined approach for predicting the subcellular localization of rat proteins. From protein primary sequence, the multiple sequential features are obtained by using of discrete Fourier analysis, position conservation scoring function and increment of diversity, and these sequential features are selected as input parameters of the support vector machine. By the jackknife test, the overall success rate of prediction is 95.6% on the rat proteins dataset. Our method are performed on the apoptosis proteins dataset and the Gram-negative bacterial proteins dataset with the jackknife test, the overall success rates are 89.9% and 96.4%, respectively. The above results indicate that our proposed method is quite promising and may play a complementary role to the existing predictors in this area.

The virulent form of malaria is caused by Plasmodium falciparum that infects red blood cells. In order to survive inside the host, the parasite remodels the infected erythrocytes by exporting more than 300 effector proteins outside the parasitophorous vacuole membrane into the cytosol. The main feature of all the export proteins is the presence of a pentapeptide sequence motif; RxLxE/Q/D. This sequence motif is hydrolysed between L - x and the proteins with the acetylated new N-terminus xE/Q/D are exported. The enzyme responsible for this hydrolysis is plasmepsin V which is one of the ten aspartic proteases in P. falciparum. In order to understand the structural rationale for the specificity of this protease towards cleavage of the above motif, we generated three-dimensional models of seven plasmepsins (I, V to X) for which experimental structures are not available and compared these along with the crystal structures of three P. falciparum plasmepsins (II to IV). The structure comparisons revealed the importance of Tyr13, Glu77 and Ala117 specific to plasmepsin V that facilitates the accommodation of arginine at P3 in the RxLxE/Q/D motif. Our analysis correlates the structure- function relationship of plasmepsin V.

Recent studies have been supporting that the generation of Aβ42 oligomers is responsible for Alzheimer's disease. Therefore, those peptides which bind to Aβ42 are scientifically interesting and can be possible candidates for the diagnosis and therapy of Alzheimer's disease. A systemic in vitro evolution, developed recently and the designated progressive library method (PLM), was applied to obtain Aβ42-binding aptamers peptides. As a result, high affinity peptide aptamers made of 8 or 9 amino acids could be identified by this approach, endorsing the methodological effectiveness. Namely, the selection products from the secondary library of diversified peptides, which was constructed based on the information obtained from the primary library selection, were confirmed to be superior to those selected from the primary library as had been reported previously. The affinities of those peptides measured by SPR (surface plasmon resonance) were comparable to or higher than that of those peptides so far reported (Kd of 10-7). The other peptides selected were confirmed of their binding by a novel mode of gel shift assay (fluorescence enhancement caused by the binding). Thus, novel Aβ42-binding peptides with high affinity were provided for the future Alzheimer's disease study. The demonstration of the effectiveness of the systemic in vitro evolution of PLM is very encouraging for the study of identifying novel functional peptides.