Protein and Peptide Letters - Volume 20, Issue 2, 2013

Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) is a key enzyme in the synthesis of isoprenoids in the malaria parasite, using a pathway that is absent in the human host. This enzyme is receiving attention as it has been validated as a promising drug target. However, an impediment to the characterisation of this enzyme has been the inability to obtain sufficient quantities of the enzyme in a soluble and functional form. The expression of PfDXR from the codon harmonised coding region, under conditions of strongly controlled transcription and induction, resulted in a yield of 2 - 4 mg/L of enzyme, which is 8 to 10-fold higher than previously reported yields. The kinetic parameters Km, Vmax and kcat were determined for PfDXR using an NADPH-dependent assay. Residues K295 and K297, unique to species of Plasmodium and located in the catalytic hatch region; and residues V114 and N115, essential for NADPH binding, were mutated to resemble those found in E. coli DXR. Interestingly, these mutations decreased the substrate affinity of PfDXR to values resembling that of E. coli DXR. PfDXR-K295N, K297S and PfDXR-V114A, N115G demonstrated a decreased ability to turnover substrate by 4-fold and 2-fold respectively in comparison to PfDXR. This study indicates a difference in the role of the catalytic hatch in capturing substrate by species of Plasmodium. The results of this study could contribute to the development of inhibitors of PfDXR.

Structural information deduced from the new crystal form of xylanase from Bacillus sp (NCL 87-6-10) (ATBXYL- C) helped us to identify the active site and interpret the stability of the enzyme. The analysis of the tetragonal crystal structure of ATBXYL-C with a bound and cleaved xylotriose revealed the two glutamic acid residues in the structure that could act as nucleophile (Glu94) and base (Glu184) in the enzyme activity and also the tryptophan residues interacting with the substrate. The cleavage of xylotriose in the crystal showed xylobiose to be the major product. Intrinsic fluorescence of the enzyme showed the presence of tryptophans in partially exposed to the solvent at the active site and surface tryptophans in electropositive environment. The titration experiments with xylobiose and xylotriose revealed slightly enhanced preference for longer chain X3 compared with X2. The crystal structure also account for some of the factors, such as increased number of ionic interactions and additional interactions at the N-terminus, which contributed to increased alkalophilicity and thermostability of the enzyme.

Shigella flexneri is a Gram-negative bacillus that is responsible for a severe form of dysentery called Shigellosis, which mainly affects children and the elderly in both underdeveloped and developed countries. Pathogenic S. flexneri strains possess a large virulence plasmid that codes for effector proteins that are required for the entry and spread of the bacteria into colonocytes. Among these proteins is the translocator IpaC, which plays an important role in the invasion process; IpaC is implicated in pore formation in the host cell membrane and induces cytoskeletal rearrangements in macrophages and epithelial cells, thereby promoting bacterial entry. The ability of IpaC to insert into the plasma membrane is due to a large nonpolar region of the protein structure. This characteristic also renders difficulties in recovery and purification when the protein is expressed in E. coli. Several works have considered different methodologies for the improved production and purification of IpaC. Herein, we propose an alternative method that is based on changes in the induction temperature and extraction buffer to facilitate the accumulation of high yields of soluble proteins for their further processing and ultimate use in biotechnological approaches.

The stability of Merrifield linker in Fmoc deprotection process was quantitatively investigated by establishing working curve of two major decomposition components from two resin bound dipeptide models. By sampling reaction solution and analyzing with RP-HPLC, decomposition rate was determined. The results indicated that either α-amino acid or β-amino acid anchored Merrifield linker was endurable for Fmoc strategy peptide synthesis in common de-Fmoc conditions such as 20% piperidine/DMF and 2% DBU/2% piperidine/DMF under room temperature treatments. However, Fmoc-deprotection with microwave assistance of α-amino acid anchored peptide resin with 20% piperidine/DMF more than 20 times or β-amino acid anchored peptide resin with 2% DBU/2% piperidine/DMF more than 30 times is not recommended. Feasibility of the proposed compatibility was verified by design and synthesis of a thymic humoral factor derived peptide via Fmoc strategy on Merrifield resin. Thus by choosing moderate de-Fmoc protocol, Merrifield resin is feasible for Fmoc strategy oligopeptide synthesis.

A series of shorter peptide analogues of Bactenecin7 (RP, PRP, GPRP and RPRP) were synthesized and conjugated to 3-(4-oxo-3,4-dihydroquinazolin-2-yl)propanoic acid to study the effect of conjugation. All the peptides and their conjugates were characterized by analytical and spectroscopic techniques. The synthesized compounds viz., peptides, heterocyclic conjugates and the hydrogenolyzed products were evaluated for antimicrobial activity against a panel of pathogens. The results revealed that all the conjugates have shown enhanced activity than their counterparts. Further, hydrogenolyzed tetrapeptide conjugates (10 and 13) have exerted highly potent activity nearly 3-4 times than the standard drugs used.

Protein structure prediction is often assisted by predicting one-dimensional structural properties including relative solvent accessibility (RSA) surface and backbone torsion angles (BTA) of residues, and these two properties are continuously varying variables because proteins can move freely in a three-dimensional space. Instead of subdividing them into a few arbitrarily defined states that many popular approaches used, this paper proposes an integrated system for realvalue prediction of protein structural motifs' two local properties, based on the modified Hidden Markov Model that we previously presented. The model was used to capture the relevance of RSA and the dependency of BTA between adjacent residues along the local protein chain in motifs with definite probabilities. These two properties were predicted according to their own probability distribution. The method was applied to a protein fragment library. For nine different classes of motifs, real values of RSA were predicted with mean absolute error (MAE) of 0.122-0.175 and Pearson's correlation coefficient (PCC) of 0.623-0.714 between predicted and actual RSA. Meanwhile, real values of BTA were obtained with MAE of 8.50-29.40 for φ angles, 11.20-38.50 for ψ angles and PCC of 0.601-0.716 for φ, 0.597-0.713 for ψ. The results were compared with well-known Real-SPINE Server, and indicate the proposed method may at least serve as the foundation to obtain better local properties from structural motifs for protein structure prediction.

Combining stem cell with phage display to discover novel biomarkers is a new field in stem cell studies. Novel peptides obtained through phage display panning have been functionally identified and involved into initial applications as cell culture support or cell label. In the present study, we designed a glutathione S-transferase (GST)-tagged peptide complex to construct an easy and efficient prokaryotic expression and purification platform for peptides obtained from phage display panning. The purified GST-peptide protein could specifically bind to neural stem (NS) cells and could be flexibly used for GST pull down assay or NS cell labeling in future studies. The results of the present study would be useful for cell labeling and future investigations of special receptors on stem cell surface.

An extracellular antifungal protein of 28 kDa (exAFP-C28) was identified from an endophytic fungus Colletotrichum sp. DM-06. After purification, the MIC value of exAFP-C28 against Candida albicans, a well-known human pathogenic fungus was found to be 32 μg/mL that unaffected the human red blood cells. The antifungal activity associated with exAFP-C28 was manifested by the increased membrane permeability of C. albicans cells followed by disruption. Proteomics and bioinformatics analyses revealed that several peptide fragments of exAFP-C28 have identity with the bacterial 50S ribosomal protein L10, and a stretch of 55 amino acids of two peptide fragments corresponding to the Nterminus of L10 protein is capable of forming amphipathic helix required for membrane penetration. Taken together, our results suggest that the exAFP-C28 protein from Colletotrichum sp. DM-06 is a promising therapeutic agent in controlling candidiasis disease in animals including humans.

Microbial resistance to antibiotics is a rising concern among health care professionals, driving them to search for alternative therapies. In the past few years, antimicrobial peptides (AMPs) have attracted a lot of attention as a substitute for conventional antibiotics. Antimicrobial peptides have a broad spectrum of activity and can act as antibacterial, antifungal, antiviral and sometimes even as anticancer drugs. The antibacterial peptides have little sequence homology, despite common properties. Since there is a need to develop a computational method for predicting the antibacterial peptides, in the present study, we have applied the concept of Chou's pseudo-amino acid composition (PseAAC) and machine learning methods for their classification. Our results demonstrate that using the concept of PseAAC and applying Support Vector Machine (SVM) can provide useful information to predict antibacterial peptides.

In this communication we describe an approach in which guanidine hydrochloride-induced dissociation of a protein inhibitor-serine protease complex is used to explore the molecular basis of protein denaturation. The rationale behind this approach is that the inhibitor-protease complex is stabilized by the same types of non-covalent interactions that stabilize the native state of a protein. The dissociation of inhibitor-protease complex can be performed at concentrations of guanidine hydrochloride at which the inhibitor and the protease retain their native conformations. Here, we present our results on the effect of 0.1M to 0.4M guanidine hydrochloride concentrations on the association equilibrium constants (reciprocal of dissociation constant) of P1G, P1A, P1V, P1N, and P1S variants of turkey ovomucoid third domain with bovine α-chymotrypsin. We use these results to calculate the free energy change in the dissociation of inhibitor-protease complexes (the m value) per mol of guanidine hydrochloride concentration. Our results agree with the general consensus that the denaturing effect of guanidine hydrochloride is due to its favorable interaction with the polar parts of proteins and that the non-polar side chains have no or little favorable interaction with guanidine hydrochloride.

Muscular necrosis is a serious consequence of Bothrops snake bites that may lead to permanent loss of tissue or function. Myonecrosis may be due to injury to blood vessels, destabilization and/or rupture of plasma membrane, and inflammatory mechanisms triggered by different proteins from the snake venom. In this work we describe the isolation and partial functional characterization of a myotoxin from B. alternatus snake venom. The myotoxin was isolated by a combination of ion exchange and gel filtration chromatography and displayed a molecular weight of approximately 15,000, as estimated by SDS-PAGE under reducing conditions. In non-reducing conditions a protein band of approximately 25,000 was also observed, suggesting that its native form is a homodimer. The myotoxin induced myonecrosis, but had no proteolytic and phospholipase A2 activities. The myotoxic activity was assessed on the basis of the histological and ultrastructural alterations induced by the toxin in the gastrocnemius skeletal muscle of Swiss mice. The toxin led to a series of drastic degenerative events characterized by extensive cellular destruction, loss of the arrangements of skeletal fibers, intense infiltration of inflammatory cells, fatty degeneration and hemorrhage. Electron microscopy analyses revealed that the myotoxin caused cell swelling, mitochondrial alterations and dilation of the sarcoplasmic reticulum, but did not affect the integrity of the muscle cell membranes. The myonecrosis caused by this toxin was related to the perturbation in the membrane permeability, intracellular alterations and inflammatory reaction.

Muramyl dipeptide (MDP) and tuftsin are known biologically active compound displaying a significant influence on various cell populations of innate immune response. MDP, as a fragment of bacterial cell wall, stimulates not only macrophages and monocytes, but also dendritic cells. In contrast, little is known about tuftsin influence on these cells. Therefore it seemed vital to access whether tuftsin or its derivatives conjugated with MDP could influence the activity of this subpopulation of antigen presenting cells (APC). Immature dendritic cells (iDCs) were derived from human monocytes through eight-day tissue culture supplemented with hrIL-4 and hrGM-CSF. On the day 9 DCs were stimulated with newly synthesized conjugates of tuftsin and muramyl dipeptide. The influence of the examined compounds on the activity and maturity of monocyte-derived DCs was estimated by flow cytometry analysis. The flow cytometry analysis revealed that tuftsin and some of its analogues do stimulate maturation and activity of DCs but to a lesser extend in comparison to MDP. The obtained results suggest further development of the experiments concerning the influence of MDP and tuftsin analogues on the activity of dendritic cells.

Lethal toxin of Clostridium sordellii (MLD 150 ng/kg) is one of the most potent Clostridial toxins and is responsible for most of the diseases including sudden death syndrome in cattle, sheep and toxic shock syndrome, necrotizing faciitis, neonatal omphalitis and gangrene in humans. Lethal toxin (TcsL) is a single chain protein of about 270 kDa. In the present study, 1.6 kb DNA fragment encoding for the catalytic domain of TcsL was PCR amplified, cloned in pQE30 UA vector and expressed in E. coli SG 13009. The expression of recombinant lethal toxin protein (rTcsL) was optimized and it was purified under native conditions using a single step Ni-NTA affinity chromatography. The purified recombinant protein was used for the production of polyclonal antibodies in mice and rabbit. The raised antibodies reacted specifically with the purified rTcsL and intact native lethal toxin on Western blot. The biological activity of the recombinant protein was tested in HeLa cells where it showed the cytotoxicity. Further, the polyclonal antibodies were used for in-vitro neutralization of purified rTcsL, acid precipitated C. sordellii and C. difficile native toxins in HeLa cells. Mice and rabbit anti-rTcsL sera effectively neutralized the cytotoxicity of rTcsL and C. sordellii native toxin but it did not neutralize the cytotoxicity of C. difficile toxin in HeLa cells.

A highly cost-effective and easy-to-assemble cultivation system suitable for middle scale-size culturing of bacterial cells is described. In the culture, from a flat-shaped air-stone with large surface area, fine bubbles are generated with a low-cost air pump available in an aquarium fish shop, and cell-agitation and oxygen supply are efficiently conducted by fine bubbles simultaneously. Growth properties of the cells and their saturation density are comparable to those in a conventional culture system. The expression of recombinant protein was revealed to be similar to conventional methods. The system does not require any expensive machines or equipments. In addition, all equipments except plastic flat-shaped airstone are reusable after sterilization. Due to the low cost, the ease to use and multiple cultivations at once, our system may enable to find better culture conditions, to scale-up with ease and to perform timesaving efficient protein production.

The identification of interfaces in protein complexes is effective for the elucidation of protein function and helps us to understand their roles in biological processes. With the exponentially growing amount of protein sequence data, an exploration of new methods that predict protein interaction sites based solely on sequence information is becoming increasingly urgent. Because a combination of different methods could produce better results than a single method, interaction site prediction can be improved through the utilization of different methods. This paper describes a new method that predicts interaction sites based on protein sequences by integrating five different algorithms employing meta-method, Majority Vote and SVMhmm Regression techniques. The ‘metaPIS’ web-server was implemented for meta-prediction. An evaluation of the meta-methods using independent datasets revealed that Majority Vote achieved the highest average Matthews correlation coefficient (0.181) among all the methods assessed. SVMhmm Regression achieved a lower score but provided a more stable result. The metaPIS server allows experimental biologists to speculate regarding protein function by identifying potential interaction sites based on protein sequence. As a web server, metaPIS is freely accessible to the public at http://202.116.74.5:84/metapis.

Haemostasis depends on the balanced participation of von Willebrand factor (vWF), a large multimeric and multidomain glycoprotein with essential role during the initial steps of blood clotting. Mature vWF circulates in plasma with the form of multimers comprised of several domains with diverse functions. More specifically, the A1 domain of vWF plays crucial role in haemostasis, regulating the mechanism of platelet adhesion in sites of vascular injury while A2 domain regulates the normal turnover of vWF. Recent studies have implied that an intramolecular interaction between A1 and A2 domains exists, which prevents platelets adhesion and subsequently inhibits the initial step of the blood coagulation mechanism. In an effort to elucidate the essential nature of the interaction between these two domains, we produced and purified the corresponding recombinant unmodified polypeptides. The secondary structure of the two domains was studied individually and as a mixture using circular dichroism spectroscopy. The observed interaction was verified by ELISA competition assays using antibodies and their ability to form productive interactions was further characterized kinetically. In silico analysis (docking and molecular dynamics simulations) of the A1-A2 binding indicated three possible structural models highlighting the crucial, for this interaction, region.