Protein and Peptide Letters - Volume 18, Issue 5, 2011

The reassortment in proteins from influenza A viruses among human, swine, and Eurasian avian strains formed a new influenza A virus leading to the first pandemic in this century, which suggests that the barrier between species and between subtypes would not be strong enough to prevent the cross-species infection and cross-subtype reassortment from occurring. In this study, we intensively used the ANOVA including its model I and model II to analyze 2430 polymerase basic proteins 2 (PB2) of influenza A viruses in order to determine whether there is a barrier between species and between subtypes. The results show that (i) there is a barrier between HA subtypes, between NA subtypes and between hosting species in some cases, however, there is no barrier in most cases, which can lead to cross-species infection and crosssubtype reassortment, and (ii) the intra-subtype/species variation is larger than the inter-subtype/species variation in most cases, which can lead mutations/reassortments in PB2 to easily jump from species to species or from subtype to subtype. These results are in agreement with our previous studies along this research line in the hemagglutinin, neuraminidase, matrix protein 1 and 2 from influenza A virus, and provide further explanations for the possible reason for cross-subtype reassortment and cross-species infection.

In this manuscript, three-dimensional quantitative structure-activity relationship (3D-QSAR) studies using comparative molecule field analysis (CoMFA) techniques were applied to provide the structural information of Bcl-2 inhibitors. The CoMFA model obtained from the training set were all statistically significant with the cross-validated coefficients (q2) of 0.568 and conventional coefficients (r2) of 0.991. The contribution of steric field and the electrostatic field is 0.635 and 0.365, respectively. The 3D-QSAR model was furthermore validated by a test set of 6 molecules. The predicted correlation coefficient (r2 pred) on the test set is 0.582. Therefore, the 3D-QSAR models built may be used to exhibit the necessary ligand-based structural environment as well as to design novel Bcl-2 inhibitors with increasing activities.

B-factor from X-ray crystal structure can well measure protein structural flexibility, which plays an important role in different biological processes, such as catalysis, binding and molecular recognition. Understanding the essence of flexibility can be helpful for the further study of the protein function. In this study, we attempted to correlate the flexibility of a residue to its interactions with other residues by representing the protein structure as a residue contact network. Here, several well established network topological parameters were employed to feature such interactions. A prediction model was constructed for B-factor of a residue by using support vector regression (SVR). Pearson correlation coefficient (CC) was used as the performance measure. CC values were 0.63 and 0.62 for single amino acid and for the whole sequence, respectively. Our results revealed well correlations between B-factors and network topological parameters. This suggests that the protein structural flexibility could be well characterized by the inter-amino acid interactions in a protein.

Crystal structures of Sr2+, Ni2+ and Cu2+ of human insulin complexes have been determined. The structures of Sr2+ and Ni2+ complexes are similar to Zn2+ insulin and are in T6 conformation. (All the six monomers in the insulin hexamer are in Tensed conformation (T), which means the first eight residues of B-chain are in an extended conformation). Cu2+ complex, though it assumes T6 conformation, has more structural differences due to lowering of crystal symmetry and space group shift from H3 (Hexagonal crystal system) to P3 (Trigonal crystal system) and a doubling of the c axis. 2Ni2+ human insulin when compared to 4Ni2+ Arg insulin suggests that terminal modifications may be responsible for additional metal binding. All the three metals have been shown to have a role in diabetes and hence may be therapeutically useful.

The C-terminal β-hairpin of RNase A contains a turn with a cis Asn113-Pro114 peptide bond. Pioneering pulsed HX experiments have shown that the C-terminal β-hairpin forms early during refolding. This is puzzling since the Asn113-Pro114 bond is predominately trans at this stage and this conformation destabilizes the native monomer. RNase A, when refolded at high concentration, forms a series of 3D domain-swapped oligomers. In the oligomers formed by C terminal β-strand swapping, Asn113-Pro114 is trans and permits the formation of a new intersubunit β-sheet. We hypothesize that oligomeric species with trans Asn113-Pro114 may form during refolding. Such species could account for the HX results while comfortably accommodating Asn113-Pro114 in the trans conformation. Here, we test this hypothesis by employing chromatographic methods to detect oligomers forming in refolding conditions and find significant amounts of dimer. We propose that a 3D domain-swapped dimeric intermediate provides a minor alternative pathway for RNase A refolding.

Winged bean chymotrypsin trypsin inhibitor (WbCTI) is a Kunitz type serine protease inhibitor that inhibits both trypsin and chymotrypsin at 1:1 molar ratio. Site-directed mutagenesis study was employed to generate two mutants of WbCTI, with an aim to explore its dual inhibitory properties against the proteases. The mutants were expressed in Escherichia coli and, were purified to homogeneity using a single step immunoaffinity column. The two mutants, each containing a single mutation at the amino acid sequence positions of 63 and 64, were named as L63A and R64A, respectively. Purified L63A-WbCTI exhibited anti-trypsin activity with no anti-chymotrypsin activity whereas R64A-WbCTI could inhibit chymotrypsin but not trypsin. To investigate the binding interactions between the mutated forms of WbCTI with the putative proteases, binding studies were carried out using gel filtration chromatography which further confirmed the formation of enzyme-inhibitor complexes. Finally, 3D model structure of WbCTI was designed using computer simulations which further emphasize the roles of L63 and R64 residues for dual inhibitory characteristics of WbCTI.

Information of protein subcellular location plays an important role in molecular cell biology. Prediction of the subcellular location of proteins will help to understand their functions and interactions. In this paper, a different mode of pseudo amino acid composition was proposed to represent protein samples for predicting their subcellular localization via the following procedures: based on the optimal splice site of each protein sequence, we divided a sequence into sorting signal part and mature protein part, and extracted sequence features from each part separately. Then, the combined features were fed into the SVM classifier to perform the prediction. By the jackknife test on a benchmark dataset in which none of proteins included has more than 90% pairwise sequence identity to any other, the overall accuracies achieved by the method are 94.5% and 90.3% for prokaryotic and eukaryotic proteins, respectively. The results indicate that the prediction quality by our method is quite satisfactory. It is anticipated that the current method may serve as an alternative approach to the existing prediction methods.

A specific treatment of recurrent structural motifs that represent the local bias information has been proven to be an important ingredient in de novo protein structure predication. Significant majority of methods for local structure are based on building blocks, which still suffer from its inherent discrete nature. Instead of using building blocks, this work presents a new protocol framework for local structural motifs prediction based on the direct locating along protein sequence and probabilistic sampling in a continuous (φ,ψ) space. The protein sequence was first scanned by an algorithm of sliding window with variable length of 7 to 19 residues, to match local segments to one of 82 motifs patterns in the fragment library. Identified segments were then labeled and modeled as the correlations of backbone torsion angles with mixture of bivariate cosine distributions in continuous (φ,ψ) space. 3D conformations of corresponding segments were finally sampled by using a backtrack algorithm to the hidden Markov model with single output of (φ,ψ). For local motifs in 50 proteins of testing set, about 62% of eight-residue segments located with high confidence value were predicted within 1.5 Å of their native structures by the method. Majority of local structural motifs were identified and sampled, which indicates the proposed protocol may at least serve as the foundation to obtain better protein tertiary structure prediction.

Mass spectrometric profiling using ProteinChip and magnetic beads has rapidly grown over the past years, particularly to generate serum profiles for cancer diagnosis. The molecular weights of these distinguishing peaks are usually under 30 kDa. To identify those low molecular weight proteins and peptides is important for specific assays to be developed and increases biological insight. In this study, low molecular weight proteins and peptides from serum were purified by a combination of weak cation exchange magnetic beads and high performance liquid chromatography. The purified proteins and peptides were analyzed by 1D SDS PAGE, SELDI and LC-MS/MS. 246 proteins were identified from the HPLC fractions by LC-MS/MS. 95(38.62%) proteins were first identified in serum compare with Sys-BodyFluid database. 11(11/96) proteins were documented cancer associated proteins. We also observed about 109 proteins/peptides in SELDI mass spectrum, and 13 of the SELDI features were identified.

The distinct biochemical function of endoplasmic reticulum (ER) protein Calreticulin (CR) catalyzing the transfer of acyl group from acyloxycoumarin to a receptor protein was termed calreticulin transacylase (CRTAase). The present study, unlike the previous reports of others utilizing CR-deficient cells alone, dealt with the recombinant CR domains of Heamonchus contortus (rhCRTAase) in order to examine their CRTAase activity. P-domain of rhCR unlike N- and C-domains was found to be endowed with CRTAase function. We have also observed for the first time acetyl CoA, as a substrate for rhCRTAase/P-domain mediated acetylation of recombinant Schistosoma japonicum glutathione Stransferase (rGST). rhCRTAase/P-domain were also found to undergo autoacylation by acyloxycoumarins. Also, the isolated autoacylated rhCRTAase/P-domain in non-denatured form alone exhibited the ability to transfer acyl group to rGST indicating the stable intermediate nature of acylated CR. P-domain catalyzed acetylation of rGST by 7,8-Diacetoxy-4- methylcoumarin or acetyl CoA resulted in the modification of several lysine residues in common was evidenced by LCMS/ MS analysis. The putative site of the binding of acyloxycoumarins with CR was predicted by computational blind docking studies. The results showed the involvement of two lysine residues Lys-173 and Lys-174 present in P-domain for binding acyloxycoumarins and acetyl CoA thus highlighting that the active site for the CRTAase activity would reside in the P-domain of CR. Certain ER proteins are known to undergo acetylation under the physiological conditions involving acetyl CoA. These results demonstrating CRTAase mediated protein acetylation by acetyl CoA may hint at CR as the possible protein acetyltransferase of the ER lumen.

Human rhinovirus (HRV), cause of the common cold, is a leading cause of exacerbations of asthma and chronic obstruction pulmonary disease (COPD). Binding of HRV to ICAM (intercellular adhesion molecule)-1, its major receptor, induces a profound inflammatory response from airway epithelial cells. My laboratory has identified Syk tyrosine kinase to be an early regulator of HRV-ICAM-1 signalling: Syk mediates replication-independent p38 mitogen-activated protein (MAP) kinase and phosphatidyl-inositol 3 (PI3)-kinase activation, interleukin (IL)-8 expression, as well as HRV internalization via clathrin-mediated endocytosis. Syk activation is accompanied by formation of a protein complex consisting of ICAM-1, ezrin and Syk at the plasma membrane. However, the molecular mechanisms that regulate this process are not understood. In this report, we investigated the role of the Syk-SH2 domains and the ezrin-ITAM (immuno-tyrosine activation motif)-like motif in HRV-induced cell activation using the human BEAS-2B airway epithelial cells. Our observations suggest that the ezrin-ITAM plays a role in Syk recruitment and activation by binding to the Syk tandem SH2 domains, as originally described in the canonical ITAM-mediating signal transduction pathway in hematopoietic cells. This report is the first to demonstrate ITAM-mediated signaling in non-hematopoietic cells, suggesting that this signaling paradigm may be more ubiquitous than previously recognized.

Quantum mechanical fragment molecular orbital calculations have been performed for receptor binding of the hemagglutinin protein of the recently pandemic influenza 2009 H1N1 (2009/HIN1pdm), A/swine/Iowa/1930, and A/Puerto Rico/8/1934 viruses to α2-6 linked sialyloligosaccharides, as analogs of human receptors. The strongest receptor binding affinity was observed for the 2009/H1N1pdm. The inter-fragment interaction energy analysis revealed that the amino acid mutation of 2009/H1N1pdm, Ser145Lys, was a major cause of such strong binding affinity. Strong ionic pair interaction between the sialic acid and Lys145 was observed only in the 2009/H1N1pdm, in addition to the hydrogen bond between the sialic acid and Gln226 observed in all the HAs. Therefore, pandemic 2009/H1N1pdm has been found to recognize the α2-6 receptor much stronger than the 1930-swine and 1934-human.