Prediction of the β-Hairpins in Proteins Using Support Vector Machine

By using of the composite vector with increment of diversity and scoring function to express the information of sequence, a support vector machine (SVM) algorithm for predicting β-hairpin motifs is proposed. The prediction is done on a dataset of 3,088 non homologous proteins containing 6,027 β-hairpins. The overall accuracy of prediction and Matthew’s correlation coefficient are 79.9% and 0.59 for the independent testing dataset. In addition, a higher accuracy of 83.3% and Matthew’s correlation coefficient of 0.67 in the independent testing dataset are obtained on a dataset previously used by Kumar et al. (Nuclic Acid Res 33:154–159). The performance of the method is also evaluated by predicting the β-hairpins of in the CASP6 proteins, and the better results are obtained. Moreover, this method is used to predict four kinds of supersecondary structures. The overall accuracy of prediction is 64.5% for the independent testing dataset.     

A ′Fragment Fitting Approach′ to Model Disulfide Loops by Utilizing Homologous Peptide Fragments from Unrelated Proteins of Known Structures: Application to the V3 Loop of the HIV-1 Envelope Glycoprotein gp120

The V3 loop of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has gained considerable attention for developing subunit vaccines against HIV-1 and also as a target to develop anti-HIV-1 drugs. These endeavors would be significantly enhanced by understanding the structural aspects of this loop. The structure of the full-length V3 loop has not been defined yet. Therefore, a novel modeling technique, termed `Fragment Fitting Approach′ (FFA), was developed to model the V3 loop. This technique utilizes fragments (³ 6 residue long) with local sequence and secondary structure similarity from unrelated proteins with known x-ray crystallographic structure and concatenating the fragments to build the model. A systematic search method was devised to identify the fragments using the combined criteria of sequence and secondary structure identity and/or similarity, predicted by a combination of methods. FFA requires partial three-dimensional coordinates of the target sequence to be modelled to get the overall coordinate path correct. The method was validated with nine disulfide-bonded loops from the Protein data bank. The modelled structures conform well with the corresponding x-ray crystallographic structures. As the models were built using the x-ray coordinates with reasonable resolution (£ 3 Å), they are expected to have stereochemically correct structures. The modelled V3 loop structure might assist in structure-based drug design of anti-HIV-1 agents targeted to this loop.     

A ′Fragment Fitting Approach′ to Model Disulfide Loops by Utilizing Homologous Peptide Fragments from Unrelated Proteins of Known Structures: Application to the V3 Loop of the HIV-1 Envelope Glycoprotein gp120

The V3 loop of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has gained considerable attention for developing subunit vaccines against HIV-1 and also as a target to develop anti-HIV-1 drugs. These endeavors would be significantly enhanced by understanding the structural aspects of this loop. The structure of the full-length V3 loop has not been defined yet. Therefore, a novel modeling technique, termed `Fragment Fitting Approach′ (FFA), was developed to model the V3 loop. This technique utilizes fragments (³ 6 residue long) with local sequence and secondary structure similarity from unrelated proteins with known x-ray crystallographic structure and concatenating the fragments to build the model. A systematic search method was devised to identify the fragments using the combined criteria of sequence and secondary structure identity and/or similarity, predicted by a combination of methods. FFA requires partial three-dimensional coordinates of the target sequence to be modelled to get the overall coordinate path correct. The method was validated with nine disulfide-bonded loops from the Protein data bank. The modelled structures conform well with the corresponding x-ray crystallographic structures. As the models were built using the x-ray coordinates with reasonable resolution (£ 3 Å), they are expected to have stereochemically correct structures. The modelled V3 loop structure might assist in structure-based drug design of anti-HIV-1 agents targeted to this loop.     

Comparison of the Predicted Structures of Loops in the ras–SOS Protein Bound to a Single ras-p21 Protein with the Crystallographically Determined Structures in SOS Bound to Two ras-p21 Proteins

We have previously computed the structures of three loops, residues 591–596, 654–675 and 742–751, in the ras-p21 protein-binding domain (residues 568–1044) of the guanine nucleotide-exchange-promoting SOS protein that were crystallographically undefined when one molecule of ras-p21 (unbound to nucleotide) binds to SOS. Based on our computational results, we synthesized three peptides corresponding to sequences of each of these three loops and found that all three peptides strongly inhibit ras-p21 signaling. More recently, a new crystal structure of SOS has been determined in which this protein binds to two molecules of ras-p21, one unbound to GTP and one bound to GTP. In this structure, the 654–675 loop and residues 742–743 and 750–751 are now crystallographically defined. We have superimposed our energy-minimized structure of the ras-binding domain of SOS bound to one molecule of ras-p21 on the X-ray structure for SOS bound to two molecules of ras-p21. We find that, while the two structures are superimposable, there are large deviations of the residues 673 and 676 and 741 and 752, flanking the two loop segments. This suggests that the binding of the extra ras-p21 molecule, which is far from each of the three loops, induces conformational changes in these domains and further supports their role in signal transduction. In spite of these differences, we have superimposed our computed structures for the loop residues on those from the more recent X-ray structure. Our structure for the 654–675 segment is an anti-parallel beta-sheet with a reverse turn at residues 663–665; in the X-ray structure residues 655–662 adopt an alpha-helical conformation; on the other hand, our computed structure for residues 663–675 superimpose on the X-ray structure for these residues. We further find that our computed structures for residues 742–743 and 750–751 are superimposable on the X-ray structure for these residues.     

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gα_i. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.     

Flexibility of A · Pairs in Left-Handed DNA Helices

Abstract

We have analysed by various approaches the structure of cloned synthetic sequences in supercoiled plasmids. Individual inserts were formed by d(C-G)_n blocks interrupted by the presence of A · T pairs positioned either in phase or out of phase of pur-pyr alternation. Based on the thermodynamic analysis we obtained results confirming that A · T pairs are easily incorporated into left-handed helices without significant energetic penalty. Sequences GTAC which are known to form cruciform structures in multiple repetition underwent a B-Z transition. In the case of plasmids containing AA/TT code words and substantial discontinuities in purine-pyrimidine alternation our analysis indicates that Z-Z junctions formed by A · T pairs contributed little to the overall energetic demands of the B-Z transition probably thanks to their high conformational flexibility.     

Residual Structures in the Acid-Unfolded States of Vλ6 Proteins Affect Amyloid Fibrillation

Many proteins form amyloid-like fibrils in vitro under partially or highly unfolding conditions. Recently, we showed that the residual structure in highly unfolded state is closely related to amyloid fibril formation in hen lysozyme. Thus, to better understand the role of the residual structure on amyloid fibril formation, we focused on AL amyloidosis, which results from the extracellular deposition of monoclonal immunoglobulin light-chain variable domains (V_Ls) as insoluble fibrils. We examined the relationship between the residual structure and amyloid fibril formation on three λ6 recombinant V_L (rVλ6) proteins, wild type, Jto, and Wil. Although rVλ6 proteins are highly unfolded in pH 2, ¹⁵N NMR transverse relaxation experiments revealed nonrandom structures in regions, which include some hydrophobic residues and a single disulfide bond, indicating the existence of residual structure in rVλ6 proteins. However, the residual structure of Wil was markedly disrupted compared with those of the other proteins, despite there being no significant differences in amino acid sequences. Fibrillation experiments revealed that Wil had a longer lag time for fibril formation than the others. When the single disulfide bond was reduced and alkylated, the residual structure was largely disrupted and fibril formation was delayed in all three rVλ6 proteins. It was suggested that the residual structure in highly unfolded state has a crucial role in amyloid fibril formation in many proteins, even pathogenic ones.     

Selection and Characterization of Artificial Proteins Targeting the Tubulin α Subunit

1. Download high-res image (241KB)
2. Download full-size image

     

A conserved motif in the C-terminal tail of DNA polymerase α tethers primase to the eukaryotic replisome

The DNA polymerase α-primase complex forms an essential part of the eukaryotic replisome. The catalytic subunits of primase and pol α synthesize composite RNA-DNA primers that initiate the leading and lagging DNA strands at replication forks. The physical basis and physiological significance of tethering primase to the eukaryotic replisome via pol α remain poorly characterized. We have identified a short conserved motif at the extreme C terminus of pol α that is critical for interaction of the yeast ortholog pol1 with primase. We show that truncation of the C-terminal residues 1452-1468 of Pol1 abrogates the interaction with the primase, as does mutation to alanine of the invariant amino acid Phe(1463). Conversely, a pol1 peptide spanning the last 16 residues binds primase with high affinity, and the equivalent peptide from human Pol α binds primase in an analogous fashion. These in vitro data are mirrored by experiments in yeast cells, as primase does not interact in cell extracts with pol1 that either terminates at residue 1452 or has the F1463A mutation. The ability to disrupt the association between primase and pol α allowed us to assess the physiological significance of primase being tethered to the eukaryotic replisome in this way. We find that the F1463A mutation in Pol1 renders yeast cells dependent on the S phase checkpoint, whereas truncation of Pol1 at amino acid 1452 blocks yeast cell proliferation. These findings indicate that tethering of primase to the replisome by pol α is critical for the normal action of DNA replication forks in eukaryotic cells.     

Prediction of Protein–Protein Interaction Sites in Sequences and 3D Structures by Random Forests

Identifying interaction sites in proteins provides important clues to the function of a protein and is becoming increasingly relevant in topics such as systems biology and drug discovery. Although there are numerous papers on the prediction of interaction sites using information derived from structure, there are only a few case reports on the prediction of interaction residues based solely on protein sequence. Here, a sliding window approach is combined with the Random Forests method to predict protein interaction sites using (i) a combination of sequence- and structure-derived parameters and (ii) sequence information alone. For sequence-based prediction we achieved a precision of 84% with a 26% recall and an F-measure of 40%. When combined with structural information, the prediction performance increases to a precision of 76% and a recall of 38% with an F-measure of 51%. We also present an attempt to rationalize the sliding window size and demonstrate that a nine-residue window is the most suitable for predictor construction. Finally, we demonstrate the applicability of our prediction methods by modeling the Ras–Raf complex using predicted interaction sites as target binding interfaces. Our results suggest that it is possible to predict protein interaction sites with quite a high accuracy using only sequence information.     

A Comparison of the Whole Genome Approach of MeDIP-Seq to the Targeted Approach of the Infinium HumanMethylation450 BeadChip? for Methylome Profiling

DNA methylation is one of the most studied epigenetic marks in the human genome, with the result that the desire to map the human methylome has driven the development of several methods to map DNA methylation on a genomic scale. Our study presents the first comparison of two of these techniques - the targeted approach of the Infinium HumanMethylation450 BeadChip® with the immunoprecipitation and sequencing-based method, MeDIP-seq. Both methods were initially validated with respect to bisulfite sequencing as the gold standard and then assessed in terms of coverage, resolution and accuracy. The regions of the methylome that can be assayed by both methods and those that can only be assayed by one method were determined and the discovery of differentially methylated regions (DMRs) by both techniques was examined. Our results show that the Infinium HumanMethylation450 BeadChip® and MeDIP-seq show a good positive correlation (Spearman correlation of 0.68) on a genome-wide scale and can both be used successfully to determine differentially methylated loci in RefSeq genes, CpG islands, shores and shelves. MeDIP-seq however, allows a wider interrogation of methylated regions of the human genome, including thousands of non-RefSeq genes and repetitive elements, all of which may be of importance in disease. In our study MeDIP-seq allowed the detection of 15,709 differentially methylated regions, nearly twice as many as the array-based method (8070), which may result in a more comprehensive study of the methylome.     

A Novel Approach for Secretion of Heterologous Proteins with Correct N-Terminal Processing by Using α-Factor Pre Sequence in Pichia pastoris

Numerous proteins have been secreted in P. pastoris by fusing the target gene with α-factor pre-pro sequence at Kex2 endopeptidase cleavage site. However, in some instances the product cannot be correctly processed due to aberrant cleavage by Kex2 endopeptidase such as aprotinin. In this study, an aprotinin gene was cloned into pPIC9K at the signal peptidase cleavage site through a single NheI restriction site designed at the 3'end of the α-factor signal sequence preregion, and transformed into GS115 host cell. By G418 resistance and ELISA assay, a high-yield recombinant was selected. After fed-batch cultivation in a 7-L bioreactor, the product was efficiently secreted into culture medium and accumulated up to ～ 4.7 mg L-1. MALDI-TOF/MS and N-terminal analyses confirmed its authenticity. Thus, a novel cloning strategy for secretion of aprotinin with correct N-terminal processing in P. pastoris has been developed which can be potentially applied to other proteins.     

A Quantitative Approach to the Prioritization of Zoonotic Diseases in North America: A Health Professionals’ Perspective

Background

Currently, zoonoses account for 58% to 61% of all communicable diseases causing illness in humans globally and up to 75% of emerging human pathogens. Although the impact of zoonoses on animal health and public health in North America is significant, there has been no published research involving health professionals on the prioritization of zoonoses in this region.

Methodology/Principal Findings

We used conjoint analysis (CA), a well-established quantitative method in market research, to identify the relative importance of 21 key characteristics of zoonotic diseases for their prioritization in Canada and the US. Relative importance weights from the CA were used to develop a point-scoring system to derive a recommended list of zoonoses for prioritization in Canada and the US. Study participants with a background in epidemiology, public health, medical sciences, veterinary sciences and infectious disease research were recruited to complete the online survey (707 from Canada and 764 from the US). Hierarchical Bayes models were fitted to the survey data to derive CA-weighted scores for disease criteria. Scores were applied to 62 zoonotic diseases to rank diseases in order of priority.

Conclusions/Significance

We present the first zoonoses prioritization exercise involving health professionals in North America. Our previous study indicated individuals with no prior knowledge in infectious diseases were capable of producing meaningful results with acceptable model fits (79.4%). This study suggests health professionals with some knowledge in infectious diseases were capable of producing meaningful results with better-fitted models than the general public (83.7% and 84.2%). Despite more similarities in demographics and model fit between the combined public and combined professional groups, there was more uniformity across priority lists between the Canadian public and Canadian professionals and between the US public and US professionals. Our study suggests that CA can be used as a potential tool for the prioritization of zoonoses.