Protein and Peptide Letters - Volume 15, Issue 10, 2008

It is important but challenging to determine the binding specificity of MHC-peptide interactions accurately and to predict their binding affinity quantitatively. In this paper, we discuss the application of an effective amino acid descriptor to model and predict the binding affinities between the MHC protein and its peptide ligands. This amino acid descriptor was derived from 23 electronic properties, 37 steric properties, 54 hydrophobic properties and 5 hydrogen bond properties of coded amino acids using principal component analysis (PCA), called the divided physicochemical property scores (DPPS). The DPPS descriptor was used to characterize a set of mouse MHC (H-2KK) binding peptides, and genetic algorithm-partial least square (GA-PLS) models were then constructed. In analyses, these models were statistically consistent with previous reports and molecular graphics exhibition. Hydrophobic interactions and hydrogen bonds were important to antigen recognition and presentation, especially exerting effects on anchor residues of peptides.

The activity of caspase-2 was examined under varying biochemical conditions with the synthetic and protein substrates, Bid and procaspase-7. The results indicate that it was largely influenced by pH which might be one reason behind the inconsistency for the cleavage of its established substrates during caspase-2-induced apoptosis.

Bacillus amyloliquefaciens protease-catalyzed peptide bond formation was investigated using the carbamoylmethyl (Cam) ester as the acyl donor. A series of N-protected L-amino acid Cam esters were coupled with an Lamino acid amide in acetonitrile in good to excellent yields. The superiority of the Cam ester for segment condensations was demonstrated in several 2+1 couplings. Furthermore, the couplings of racemic amino acid Cam esters as carboxyl components afforded the L-L-peptides in a highly diastereoselective manner.

Prefoldin is a hetero-hexameric ATP-independent chaperone, shared by eukaryotes and archaea, which binds non-native proteins preventing them from aggregation. We report the identification and characterization in vivo and in vitro of the first prefoldin from a crenarchaeon, the hyperthermophile Sulfolobus solfataricus. A functional complex was obtained either co-expressing the α- and β-prefoldin subunits in Escherichia coli, or incubating at high temperature the separately expressed subunits. In S. solfataricus, prefoldin expression and apparent molecular weight were not affected by either heat or cold shock.

Insulin nitration mediated by peroxynitrite (ONOO(-)) has been implicated in diabetes and diabetic cardiovascular complications. In this study, we identified the nitration sites of porcine insulin by infusion of ONOO(-) and quantified its secondary structural change. Insulin was cleaved with V8 protease to six peptides (four of them contained each tyrosine residue), then analyzed by HPLC-MS and further confirmed the nitration sites by HPLC-MS/MS. At low accumulated doses of peroxynitrite, the main products were two different mono-nitrated insulin species at Tyr-A19 and Tyr-B26 with Tyr-A19 being predominant as shown by peptide mapping. Also, the content of α-helix structure of insulin reduced to 22.9 % and random-coil structure increased to 30.2 % (compare with native insulin of 41.7 % and 13.7 %, respectively) as determined by FTIR spectra.

A new, effective and easily accessible carboxyl activating group , 3-mercapto-5, 6 - diphenyl-1, 2, 4 - triazine was developed and the effectiveness was investigated using acyl derivatives, chemically by the preparation of respective amides, in addition to monitoring spectrophotometrically exploiting the UV- visible spectra. The carboxyl activating nature of the above mercapto heterocycle was further confirmed by solid phase peptide synthesis. Here CLPSER resin as polymeric support was applied. We synthesized Acyl Carrier Protein and smaller peptides by replacing this thiol for other conventional carboxyl activating reagents. These peptides synthesized were purified by HPLC and characterized by molecular weight method.

Expression of plant proteins in E. coli is frequently unsuccessful, but soluble and functional rice catalase-B can be produced in E. coli when it is co-expressed with the chaperone GroEL/ES. The rice catalase exhibited properties typical for a catalase including the decomposition of H2O2 and inhibition by aminotriazole, a specific inhibitor for plant and animal catalases. This achievement records for first time the successful expression of a both native and variant rice plant catalase in bacterial cytosol suggesting that it may be an option to be considered for the expression of other plant proteins in E. coli.

Certain metals lead to increased risk of Parkinson's disease (PD) and the aggregation of α-synuclein is implicated in the PD pathology. Although α-synuclein fibrillation has been extensively studied in dilute solutions in vitro, the intracellular environment is highly crowded. We are showing here that certain metals cause a significant acceleration of α-synuclein fibrillation in the presence of high concentrations of various macromolecules mostly through decreasing the fibrillation lagtime. The faster fibrillation in crowded environments in the presence of heavy metals suggests a simple molecular basis for the observed elevated risk of PD due to exposure to metals.

The studies presented here explore antimicrobial peptide preferential binding behavior for a target pathogen, Escherichia coli O157:H7. A modified immunoassay and surface plasmon resonance were employed to evaluate immobilized peptide binding of whole bacterial cells. The knowledge gained may guide the rational design of peptides with enhanced species binding selectivity.

A novel peptide complex was synthesized by a solid-solid reaction of glycyl-L-glutamine monohydrate and bismuth trichloride powders. The formula of the complex is (BiCl3)2[NH2CH2CONHCH(COOH)CH2CH2COOH·H2O]3. The crystal structure of the complex belongs to monoclinic system with the lattice parameters: a = 0.6914 nm, b = 0.7378 nm, c = 1.0005 nm, β = 194.1° . The characterization results of IR, Raman spectra and thermal analysis show that complex is dimer and unidentate coordination. Each bismuth atom and three chlorine atoms are coplanar in the complex.

Botulinum (BoNT) and tetanus (TeNT) neurotoxins are bacterial zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. There are seven serotypes of BoNT, while TeNT is found in one serotype. In order to characterize their enzymatic activities and to propose serotype-differentiation an enzymatic assay based on their metalloprotease activity was developed. The assays were conducted with FRET peptides derived from SNAP-25, synaptobrevin and syntaxin. The substrates were cleaved by 2 ng/mL of toxin at different rates (Kcat /KM from 0.028 to 75.9 μM.s-) at a single bond, as confirmed by Q-TOF mass spectrometry. Inhibition of the hydrolysis was obtained with EDTA or with specific antibodies directed to each neurotoxin. Different substrate selectivities, especially by BoNT- A and E, suggest that these substrates can be used as a putative method for clostridial toxin quantification and serotype differentiation and could be easily adapted to a high-throughput protocols.

Function prediction by sequence-similarity based methods identifies only ∼50% of the proteins deduced from newly sequenced genomes. We have developed an approach to annotate the ‘leftover proteins’ i.e., those which cannot be assigned function using sequence similarity. Our method (MOPS) is pan-taxonomic, predicting fine-grained molecular function (rather than a broad functional category) with high performance. In addition, we developed a validation scheme that assesses predictions using domain-specific knowledge.

The spatial orientation of domains of the heat shock protein 70 from Plasmodium falciparum (PfHsp70) were mapped based on a three-dimensional model of the protein. Purified PfHsp70 displayed chaperone activity in vitro. Amino acid substitutions introduced in the chaperone's substrate binding cavity compromised the protein's chaperone function.

To elucidate whether eukaryotic elongation factor 1A (eEF-1A) in a human hepidermoid cancer cell line (H1355) belonged to the family of the Ni-interacting protein, we analyzed the sequence of peptides obtained by on-Ni- NTA-agarose tryptic digestion of proteins from H1355 cell extract. LC/MS analysis showed the presence of several peptides mainly from abundant cellular proteins corresponding to eEF-1A, tubulin and actin. The results indicated that F-actin strongly binds to Ni-NTA-agarose whereas the other proteins are indirectly bound to the resin because of the formation of a protein-protein complex with actin.

By proposing a improved Chou's pseudo amino acid composition approach to extract the features of the sequences, a powerful predictor based on k-nearest neighbor was introduced to identify the types of lipases according to their sequences. To avoid redundancy and bias, demonstrations were performed on a dataset where none of the proteins has ≥ 25%sequence identity to any other. The overall success rate thus obtained by the 10-fold cross-validation test was over 90%, indicating that the improved Chou's pseudo amino acid composition might be a useful tool for extracting the features of protein sequences, or at lease can play a complementary role to many of the other existing approaches.

Erythromycin A is produced by Saccharopolyspora erythraea via a secondary metabolic pathway using several steps including glycosylations and hydroxylations of the first macrolide intermediate 6-deoxyerythronolide B. Erythromycin C-12 hydroxylase (CYP113A1), the P450 cytochrome active in the final stages of erythromycin biosynthesis, was cloned and expressed in E. coli. Different crystal forms were harvested from distinct crystallization conditions: two ligandfree forms, one substrate bound and two inhibitors-bound. All crystals belong either to the monoclinc P21 or to the orthorhombic P212121 space groups, and exhibit diffraction limits ranging from 2.3 to 1.6 Å. The structures will be determined by molecular replacement.

Thermophilic endo-1,3(4)-β-glucanase (laminarinase) from Rhodothermus marinus was crystallized by the hanging-drop vapor diffusion method. The needle-like crystals belong to space group P21 and contain two protein molecules in the asymmetric unit with a solvent content of 51.75 %. Diffraction data were collected to a resolution of 1.95Å and resulted in a dataset with an overall Rmerge of 10.4% and a completeness of 97.8%. Analysis of the structure factors revealed pseudomerohedral twinning of the crystals with a twin fraction of approximately 42%.