Protein and Peptide Letters - Volume 19, Issue 8, 2012

Mechanical single molecule techniques offer exciting possibilities for investigating protein folding and stability in native environments at sub-nanometer resolutions. Compatible solutes show osmotic activity which even at molar concentrations do not interfere with cell metabolism. They are known to protect proteins against external stress like temperature, high salt concentrations and dehydrating conditions. We studied the impact of the compatible solute ectoine (1M) on membrane proteins by analyzing the mechanical properties of Bacteriorhodopsin (BR) in its presence and absence by single molecule force spectroscopy. The unfolding experiments on BR revealed that ectoine decreases the persistence length of its polypeptide chain thereby increasing its tendency to coil up. In addition, we found higher unfolding forces indicating strengthening of those intra molecular interactions which are crucial for stability. This shows that force spectroscopy is well suited to study the effect of compatible solutes to stabilize membrane proteins against unfolding. In addition, it may lead to a better understanding of their detailed mechanism of action.

Heparin is naturally occurring polysaccharides which interacts with seminal plasma proteins and regulate multiple steps in fertilization process. Qualitative and quantitative information regarding the affinity for heparin-seminal plasma proteins interactions is not generally well documented and there are no reports of a comprehensive analysis of these interactions in human seminal plasma. Such information should improve our understanding of how GAGs especially heparin present in the reproductive tract regulate fertilization. In this study, we use SPR to study interactions of heparin with various seminal plasma heparin-binding proteins (HBPs). HBPs like lactoferrin (LF), fibronectin fragment (FNIII), semenogelinI (SGI) and prostate specific antigen (PSA) all bind heparin with different binding kinetics and affinities. Kinetic data suggests that FNIII binds heparin with a high affinity (KD=3.2 nM), while PSA binds heparin with a micromolar affinity (KD=11.1 μM). Preincubation of SGI with heparin inhibits the binding of SGI to immobilized PSA in a dosedependent manner, while FNIII incubated with heparin binds with an increased affinity to PSA. Solution-competition studies show that the minimum size of a heparin oligosaccharide capable of binding with PSA is greater than a tetrasaccharide, with LF and SGI is larger than a hexasaccharide and for FNIII is larger than an octasaccharide.

The in vitro permeation and absorption of calcium ions across the small intestine were measured at different concentrations of calcium gluconate solutions (1.0, 10.0 and 20.0 mM) with or without prolactin. The calcium ions permeated through the small intestine from a donor environment to an acceptor environment that mimicked the conditions in the stomach to ileum segment of the digestive tract. The permeation and absorption of calcium were directly dependent on the calcium concentration of the solutions. At 10 and 20 mM permeation was significantly higher than that at 1.0 mM (p<0.05). In the presence of prolactin both permeation and absorption increase considerably. At the lowest concentration (1.0 mM) simulating calcium deficiency, there was compensation by the small intestine, suggesting that such deficiency stimulates its mobilization from intestinal tissue. Prolactin enhances the calcium mobilization process even at sufficient calcium intakes. It is suggested that prolactin takes part in regulation of calcium homeostasis in the organism.

The plant peptide hormone ENOD40B was produced in a protein production strain of Escherichia coli harboring an induction controller plasmid (Rosetta(DE3)pLysS) as a His6-tagged ubiquitin fusion protein. The fusion protein product was denatured and refolded as part of the isolation procedure and purified by immobilized metal ion chromatography. The peptide hormone was released from its fusion partner by adding yeast ubiquitin hydrolase (YUH) and subsequently purified by reversed phase chromatography. The purity of the resulting peptide fragment was assayed by MALDITOF mass spectrometry and NMR spectroscopy. The final yields of the target peptide were 7.0 mg per liter of LB medium and 3.4 mg per liter of minimal medium.

Adhesion of calcium oxalate (CaOx) crystals to kidney cells is a key event in kidney stones associated with marked hyperoxaluria. As the propensity of stone recurrence and persistent side effects are not altered by surgical techniques available, phytotherapeutic agents could be useful as an adjuvant therapy. The present study is aimed at examining the antilithiatic potency of the protein biomolecules of Tribulus terrestris, a plant which is a common constituent of herbal marketed preparations to treat urolithiasis. Various biochemical methods with mass spectrometry were used to purify and characterize the purified protein. The protective potency of the protein was tested on the oxalate induced injury on renal epithelial cell lines (NRK 52E). An antilithiatic protein having molecular weight of ~ 60kDa was purified. This purified protein showed similarities with Carotenoid cleavage dioxygenase 7 (CCD7) of Arabidopsis thaliana after matching peptide mass fingerprints in MASCOT search engine. An EF hand domain was identified in CCD7 by SCAN PROSITE. Presence of an EF hand domain, a characteristic feature of calcium binding proteins and a role in the synthesis of retinol which is transported by retinol binding protein, a protein found in kidney stone matrix; of CCD7 support the role of TTP as an antilithiatic protein. The protective potency of TTP on NRK 52E was quite comparable to the aqueous extract of cystone. Our findings suggest that this purified protein biomolecule from Tribulus terrestris could open new vista in medical management of urolithiasis.

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant proteins bound to the plant cell wall containing leucine-rich repeats (LRR). They play an important role in plant defence being able to inhibit fungal endopolygalacturonases (EPGs), the first enzymes secreted by phytopathogenic fungi during plant infection. In the present work, a novel PGIP (LsPGIP) has been isolated from Lathyrus sativus seeds. LsPGIP exhibited an inhibitory activity towards EPGs from Aspergillus niger and Rhizopus spp. A pI value of 8.3 and a molecular mass of 40 kDa were determined for the purified inhibitor. Furthermore, N-terminal sequence up to residue 20 revealed that LsPGIP exhibit a high percentage of identity with PGIP from Actinidia deliciosa. A secondary structure similar to those of other polygalacturonase inhibitors was also inferred form circular dichroism data.

Amyloid plaque is associated with several neuronal and non-neuronal degenerative diseases. More than twenty human proteins can fold abnormally to form pathological deposits like amyloid plaque. Strategies for treating such diseases include therapies designed to decrease protein plaque formation or its complete clearance, but monitoring/clinical trials of these treatments are limited by the lack of effective methods to monitor amyloid deposits in the organs/tissues of living patients. The current study shows binding and staining ability of quinacrine to protein amyloid deposits, using Hen Egg White Lysozyme (HEWL) as model system and characterization of its binding interaction with HEWL, employing several biophysical techniques. Since quinacrine can pass the blood brain barrier, the current report suggests potential application of quinacrine for antemortem diagnostic of amyloid.

We report a refinement in implicit water of the previously published solution structure of the Grb7-SH2 domain bound to the erbB2 receptor peptide pY1139. Structure quality measures indicate substantial improvement, with residues in the most favored regions of the Ramachandran plot increasing by 14 % and with WHAT IF statistics (Vriend, G. J. Mol. Graph., 1990, 8(1), 52-56) falling closer to expected values for well-refined structures.

A novel anti-proliferative lectin was purified from Morus alba L. (Mulberry) leaves by a two step chromatographic procedure namely, immobilized metal ion affinity chromatography (IMAC) and convective interaction media (CIM) based anion exchange chromatography. The purified mulberry leaf lectin (MLL) was specific to galactose, galactosamine and N-acetyl galactosamine (GalNAc). MLL was homogenous with a molecular weight of ~56kDa in silver stained SDS-PAGE. The lectin showed RBC agglutination activity up to 40°C and was independent of pH above pH 6. Haemagglutination activity of purified MLL was not dependent on any metal ions. However, with high concentration of trivalent metal ions, Fe3+ and Al3+ and the divalent metal ion Fe2+, a three fold increase in agglutination activity was observed. The purified MLL showed an anti-proliferative activity towards human breast cancer cells (MCF-7) and colon cancer cells (HCT-15) with a higher potency towards MCF-7 cells. This is the first report on the anti-proliferative activity of a GalNAc specific lectin from M. alba.

Understanding the molecular basis of neurodegenerative diseases has enormous implications for the development of effective therapeutic strategies. One of the most puzzling features of these pathologies is the occurrence of distinct strains, which are believed to be generated by alternative conformational transitions of the same protein/peptide. Very recently, it has been discovered that small model peptides are able to form alternative tightly packed assemblies (polymorphs) in the crystalline state. Intriguingly, it has been postulated that the different polymorphs of the same polypeptide sequence may be representative of distinct strains. As the organization of crystalline aggregates of small peptides may be heavily biased by crystal packing, we have here performed MD simulations on steric zipper polymorphs formed by of the IAPP-derived fragment SSTNVG. Our analyses show that these aggregates are rather stable also in a non-crystalline environment. This finding corroborates the hypothesis that steric zipper assemblies are good candidates to account for the phenomenon of strain in neurodegenerative diseases. Present investigations also provide clues on the factors that favour the formation of polymorphs. Indeed, the intrinsic stability of individual β-sheets formed by SSTNVG strands is very poor. Therefore, the formation of these aggregates is essentially dictated by inter-sheet interactions established within the steric zipper assembly.

The kinetics of thermal inactivation of bovine carbonic anhydrase (BCA) was studied in a 50 mM Tris-HCl buffer, pH 7.8 using p-nitrophenyl acetate as substrate in absorbance of 400 nm by UV-VIS spectrophotometry. The number of conformational locks and inter-subunit amino acid residues of BCA were obtained by thermal inactivation analysis. The cleavage bonds between dimers of BCA during thermal dissociation and type of interactions between specific amino acid residues were also detected. The thermal inactivation curves were plotted in temperatures ranging between 40-70°C. It was shown several phases for inactivation of BCA at 65°C. Analyses of the curves were done by the conformational lock theory. The subunits are dissociated and several intermediates appear during inactivation through increasing the temperature in comparison with native state. Dynamic light scattering measurements was done to study the changes in hydrodynamic radius during thermal inactivation. Three distinct zones were shown in DLS data. Biochemical computation using ligplot is performed to find the inter-subunit amino acid residues for BCA.

Polymyositis is an inflammatory myopathy characterized by muscle invasion of T-cells penetrating the basal lamina and displacing the plasma membrane of normal muscle fibers. In order to understand the different adhesive mechanisms at the T-cell surface, Schubert randomly selected 19 proteins expressed at the T-cell surface and studied them using MELK technique [4], among which 15 proteins are picked up for further study by us. Two types of functional similarity networks are constructed for these proteins. The first type is MELK similarity network, which is constructed based on their MELK data by using the McNemar’s test [24]. The second type is GO similarity network, which is constructed based on their GO annotation data by using the RSS method to measuring functional similarity. Then the subset surprisology theory is employed to measure the degree of similarity between two networks. Our computing results show that these two types of networks are high related. This conclusion added new values on MELK technique and expanded its applications greatly.

The extracellular domains of death ligands and those of death receptors are closely related to many serious human diseases through the initiation of apoptosis. Recombinant production of the extracellular domains has been investigated due to demand for a large amount of purified samples, which are a prerequisite for their biochemical characterization and constitute the fundamentals of medical applications. This review focuses on the recombinant production of extracellular domains of the major members of death ligand and death receptor families using non-mammalian expression systems with an emphasis on Fas ligand and Fas receptor. In contrast to the efficient production of the functional extracellular domains of TRAIL, TNFα and LTα by intracellular expression systems using Escherichia coli or Pichia pastoris, that of Fas ligand requires the secretory expression systems using P. pastoris or Dictyostelium discoideum, and the productivity in P. pastoris was largely dependent on tag sequence, potential N-glycosylation site and expressed protein region. On the other hand, the exploitation of insect cell systems is generally useful for the preparation of functional extracellular domains of death receptors containing many disulfide bridges in the absence of extended secondary structure, and a Bombyx mori larvae secretion system presented a superior productivity for human Fas receptor extracellular domain. Based on the results obtained so far, further efforts should be devoted to the artificial control of death ligand – death receptor interactions in order to make a contribution to medicine, represented by the development of novel biopharmaceuticals.

Proteomics techniques were used to identify the underlying mechanism of the early stage of symbiosis between the common bean (Phaseolus vulgaris L.) and bacteria. Proteins from roots of common beans inoculated with bacteria were separated using two-dimensional polyacrylamide gel electrophoresis and identified using mass spectrometry. From 483 protein spots, 29 plant and 3 bacterial proteins involved in the early stage of symbiosis were identified. Of the 29 plant proteins, the expression of 19 was upregulated and the expression of 10 was downregulated. Upregulated proteins included those involved in protein destination/storage, energy production, and protein synthesis; whereas the downregulated proteins included those involved in metabolism. Many upregulated proteins involved in protein destination/storage were chaperonins and proteasome subunits. These results suggest that defense mechanisms associated with induction of chaperonins and protein degradation regulated by proteasomes occur during the early stage of symbiosis between the common bean and bacteria.

G-protein coupled receptor (GPCR) is a membrane protein family, which serves as an interface between cell and the outside world. They are involved in various physiological processes and are the targets of more than 50% of the marketed drugs. The function of GPCRs can be known by conducting Biological experiments. However, the rapid increase of GPCR sequences entering into databanks, it is very time consuming and expensive to determine their function based only on experimental techniques. Hence, the computational prediction of GPCRs is very much demanding for both pharmaceutical and educational research. Feature extraction of GPCRs in the proposed research is performed using three techniques i.e. Pseudo amino acid composition, Wavelet based multi-scale energy and Evolutionary information based feature extraction by utilizing the position specific scoring matrices. For classification purpose, a majority voting based ensemble method is used; whose weights are optimized using genetic algorithm. Four classifiers are used in the ensemble i.e. Nearest Neighbor, Probabilistic Neural Network, Support Vector Machine and Grey Incidence Degree. The performance of the proposed method is assessed using Jackknife test for a number of datasets. First, the individual performances of classifiers are assessed for each dataset using Jackknife test. After that, the performance for each dataset is improved by using weighted ensemble classification. The weights of ensemble are optimized using various runs of Genetic Algorithm. We have compared our method with various other methods. The significance in performance of the proposed method depicts it to be useful for GPCRs classification.