CiteULike: neils's Wang

The Structure of an Archaeal Pilus.

Ying A Wang — 2008-07-09T11:51:07-00:00

Journal of molecular biology (12 June 2008)

Bacterial pili are involved in a host of activities, including motility, adhesion, transformation, and immune escape. Structural studies of these pili have shown that several distinctly different classes exist, with no common origin. Remarkably, it is now known that the archaeal flagellar filament appears to have a common origin with the bacterial type IV pilus, and assembly in both systems involves hydrophobic N-terminal alpha-helices that form three-stranded coils in the center of these filaments. Recent work has identified further genes in archaea as being similar to bacterial type IV pilins, but the function or structures formed by such gene products was unknown. Using electron cryo-microscopy, we show that an archaeal pilus from Methanococcus maripaludis has a structure entirely different from that of any of the known bacterial pili. Two subunit packing arrangements were identified: one has rings of four subunits spaced by approximately 44 A and the other has a one-start helical symmetry with approximately 2.6 subunits per turn of a approximately 30 A pitch helix. Remarkably, these schemes appear to coexist within the same filaments. For the segments composed of rings, the twist between adjacent rings is quite variable, while for the segments having a one-start helix there is a large variability in both the axial rise and the twist per subunit. Since this pilus appears to be assembled from a type IV pilin-like protein with a hydrophobic N-terminal helix, it provides yet another example of how different quaternary structures can be formed from similar building blocks. This result has many implications for understanding the evolutionary divergence of bacteria and archaea.

Life Sciences and the web: a new era for collaboration

Jonathan Sagotsky — 2008-07-03T09:46:41-00:00

Mol Syst Biol, Vol. 4 (1 July 2008)

A General System for Studying Protein-Protein Interactions in Gram-Negative Bacteria

Dale Pelletier — 2008-07-01T12:23:36-00:00

J. Proteome Res. (1 July 2008)

Abstract: One of the most promising methods for large-scale studies of protein interactions is isolation of an affinity-tagged protein with its in vivo interaction partners, followed by mass spectrometric identification of the copurified proteins. Previous studies have generated affinity-tagged proteins using genetic tools or cloning systems that are specific to a particular organism. To enable proteinprotein interaction studies across a wider range of Gram-negative bacteria, we have developed a methodology based on expression of affinity-tagged bait proteins from a medium copy-number plasmid. This construct is based on a broad-host-range vector backbone (pBBR1MCS5). The vector has been modified to incorporate the Gateway DEST vector recombination region, to facilitate cloning and expression of fusion proteins bearing a variety of affinity, fluorescent, or other tags. We demonstrate this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram-negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results compared favorably with those for both plasmid and chromosomally encoded affinity-tagged fusion proteins expressed in a model organism, Escherichia coli.

Comparative proteogenomics: Combining mass spectrometry and comparative genomics to analyze multiple genomes

Nitin Gupta — 2008-06-03T02:17:56-00:00

Genome Res. (2008)

Recent proliferation of low-cost DNA sequencing techniques will soon lead to an explosive growth in the number of sequenced genomes and will turn manual annotations into a luxury. Mass spectrometry recently emerged as a valuable technique for proteogenomic annotations that improves on the state-of-the-art in predicting genes and other features. However, previous proteogenomic approaches were limited to a single genome and did not take advantage of analyzing mass spectrometry data from multiple genomes at once. We show that such a comparative proteogenomics approach (like comparative genomics) allows one to address the problems that remained beyond the reach of the traditional "single proteome" approach in mass spectrometry. In particular, we show how comparative proteogenomics addresses the notoriously difficult problem of "one-hit-wonders" in proteomics, improves on the existing gene prediction tools in genomics, and allows identification of rare post-translational modifications. We therefore argue that complementing DNA sequencing projects by comparative proteogenomics projects can be a viable approach to improve both genomic and proteomic annotations.

Genomics of an extreme psychrophile, Psychromonas ingrahamii

Monica Riley — 2008-05-06T13:58:51-00:00

BMC Genomics, Vol. 9, No. 1. (2008)

BACKGROUND:The genome sequence of the sea-ice bacterium Psychromonas ingrahamii 37, which grows exponentially at -12C, may reveal features that help to explain how this extreme psychrophile is able to grow at such low temperatures. Determination of the whole genome sequence allows comparison with genes of other psychrophiles and mesophiles.RESULTS:Correspondence analysis of the composition of all P. ingrahamii proteins showed that (1) there are 6 classes of proteins wheras most other bacteria have three to four, (2) integral inner membrane proteins are not sharply separated from bulk proteins suggesting that, overall, they may have a lower hydrophobic character, and (3) there is strong opposition between asparagine and the oxygen-sensitive amino acids methionine, arginine, cysteine and histidine and (4) one of the previously unseen clusters of proteins has a high proportion of aorphana hypothetical proteins, raising the possibility these are cold-specific proteins. Based on annotation of proteins by sequence similarity, (1) P. ingrahamii has a large number (61) of regulators of cyclic GDP, suggesting that this bacterium produces an extracellular polysaccharide that may help sequester water or lower the freezing point in the vicinity of the cell. (2) P. ingrahamii has genes for production of the osmolyte, betaine choline, which may balance the osmotic pressure as sea ice freezes. (3) P. ingrahamii has a large number (11) of three-subunit TRAP systems that may play an important role in the transport of nutrients into the cell at low temperatures. (4) Chaperones and stress proteins may play a critical role in transforming nascent polypeptides into 3-dimensional configurations that permit low temperature growth. (5) Metabolic properties of P. ingrahamii were deduced. Finally, a few small sets of proteins of unknown function which may play a role in psychrophily have been singled out as worthy of future study. CONCLUSIONS:The results of this genomic analysis provide a springboard for further investigations into mechanisms of psychrophily. Potentially unique operons have been identified. Focus on the role of asparagine excess in proteins, targeted phenotypic characterizations and gene expression investigations are needed to ascertain if and how the organism regulates various proteins in response to growth at lower temperatures.

The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus)

Ray Ming — 2008-04-23T19:37:29-00:00

Nature, Vol. 452, No. 7190. (24 April 2008), pp. 991-996.

Comparative proteogenomics: Combining mass spectrometry and comparative genomics to analyze multiple genomes

Nitin Gupta — 2008-04-22T01:55:58-00:00

Genome Res. (April 2008)

Recent proliferation of low-cost DNA sequencing techniques will soon lead to an explosive growth in the number of sequenced genomes and will turn manual annotations into a luxury. Mass spectrometry recently emerged as a valuable technique for proteogenomic annotations that improves on the state-of-the-art in predicting genes and other features. However, previous proteogenomic approaches were limited to a single genome and did not take advantage of analyzing mass spectrometry data from multiple genomes at once. We show that such comparative proteogenomics approach (like comparative genomics) allows one to address the problems that remained beyond the reach of the traditional "single proteome" approach in mass-spectrometry. In particular, we show how comparative proteogenomics addresses the notoriously difficult problem of "one-hit-wonders" in proteomics, improves on the existing gene prediction tools in genomics and allows identification of rare post-translational modifications. We therefore argue that complementing DNA sequencing projects by comparative proteogenomics projects can be a viable approach to improve both genomic and proteomic annotations.

A Systematic Assessment of MHC Class II Peptide Binding Predictions and Evaluation of a Consensus Approach

Peng Wang — 2008-04-15T02:01:11-00:00

PLoS Comput Biol, Vol. 4, No. 4. (Apr 2008)

The identification of MHC class II restricted peptide epitopes is an important goal in immunological research. A number of computational tools have been developed for this purpose, but there is a lack of large-scale systematic evaluation of their performance. Herein, we used a comprehensive dataset consisting of more than 10,000 previously unpublished MHC-peptide binding affinities, 29 peptide/MHC crystal structures, and 664 peptides experimentally tested for CD4+ T cell responses to systematically evaluate the performances of publicly available MHC class II binding prediction tools. While in selected instances the best tools were associated with AUC values up to 0.86, in general, class II predictions did not perform as well as historically noted for class I predictions. It appears that the ability of MHC class II molecules to bind variable length peptides, which requires the correct assignment of peptide binding cores, is a critical factor limiting the performance of existing prediction tools. To improve performance, we implemented a consensus prediction approach that combines methods with top performances. We show that this consensus approach achieved best overall performance. Finally, we make the large datasets used publicly available as a benchmark to facilitate further development of MHC class II binding peptide prediction methods.

Scanning the human genome at kilobase resolution

Jun Chen — 2008-03-28T02:51:51-00:00

Genome Res. (2008)

Normal genome variation and pathogenic genome alteration frequently affect small regions in the genome. Identifying those genomic changes remains a technical challenge. We report here the development of the DGS (Ditag Genome Scanning) technique for high-resolution analysis of genome structure. The basic features of DGS include (1) use of high-frequent restriction enzymes to fractionate the genome into small fragments; (2) collection of two tags from two ends of a given DNA fragment to form a ditag to represent the fragment; (3) application of the 454 sequencing system to reach a comprehensive ditag sequence collection; (4) determination of the genome origin of ditags by mapping to reference ditags from known genome sequences; (5) use of ditag sequences directly as the sense and antisense PCR primers to amplify the original DNA fragment. To study the relationship between ditags and genome structure, we performed a computational study by using the human genome reference sequences as a model, and analyzed the ditags experimentally collected from the well-characterized normal human DNA GM15510 and the leukemic human DNA of Kasumi-1 cells. Our studies show that DGS provides a kilobase resolution for studying genome structure with high specificity and high genome coverage. DGS can be applied to validate genome assembly, to compare genome similarity and variation in normal populations, and to identify genomic abnormality including insertion, inversion, deletion, translocation, and amplification in pathological genomes such as cancer genomes.

Protein production and purification.

S Gräslund — 2008-02-01T14:36:35-00:00

Nat Methods, Vol. 5, No. 2. (February 2008), pp. 135-146.

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

Computational analysis and prediction of the binding motif and protein interacting partners of the Abl SH3 domain.

T Hou — 2006-04-16T13:55:55-00:00

PLoS Comput Biol, Vol. 2, No. 1. (January 2006)

Protein-protein interactions, particularly weak and transient ones, are often mediated by peptide recognition domains, such as Src Homology 2 and 3 (SH2 and SH3) domains, which bind to specific sequence and structural motifs. It is important but challenging to determine the binding specificity of these domains accurately and to predict their physiological interacting partners. In this study, the interactions between 35 peptide ligands (15 binders and 20 non-binders) and the Abl SH3 domain were analyzed using molecular dynamics simulation and the Molecular Mechanics/Poisson-Boltzmann Solvent Area method. The calculated binding free energies correlated well with the rank order of the binding peptides and clearly distinguished binders from non-binders. Free energy component analysis revealed that the van der Waals interactions dictate the binding strength of peptides, whereas the binding specificity is determined by the electrostatic interaction and the polar contribution of desolvation. The binding motif of the Abl SH3 domain was then determined by a virtual mutagenesis method, which mutates the residue at each position of the template peptide relative to all other 19 amino acids and calculates the binding free energy difference between the template and the mutated peptides using the Molecular Mechanics/Poisson-Boltzmann Solvent Area method. A single position mutation free energy profile was thus established and used as a scoring matrix to search peptides recognized by the Abl SH3 domain in the human genome. Our approach successfully picked ten out of 13 experimentally determined binding partners of the Abl SH3 domain among the top 600 candidates from the 218,540 decapeptides with the PXXP motif in the SWISS-PROT database. We expect that this physical-principle based method can be applied to other protein domains as well.

Predicting gene ontology functions from protein's regional surface structures

Zhi Liu — 2007-12-11T06:25:02-00:00

BMC Bioinformatics, Vol. 8, No. 1. (2007)

BACKGROUND:Annotation of protein functions is an important task in the post-genomic era. Most early approaches for this task exploit only the sequence or global structure information. However, protein surfaces are believed to be crucial to protein functions because they are the main interfaces to facilitate biological interactions. Recently, several databases related to structural surfaces, such as pockets and cavities, have been constructed with a comprehensive library of identified surface structures. For example, CASTp provides identification and measurements of surface accessible pockets as well as interior inaccessible cavities.RESULTS:A novel method was proposed to predict the Gene Ontology (GO) functions of proteins from the pocket similarity network, which is constructed according to the structure similarities of pockets. The statistics of the networks were presented to explore the relationship between the similar pockets and GO functions of proteins. Cross-validation experiments were conducted to evaluate the performance of the proposed method. Results and codes are available at: http://zhangroup.aporc.org/bioinfo/PSN/.CONCLUSIONS:The computational results demonstrate that the proposed method based on the pocket similarity network is effective and efficient for predicting GO functions of proteins in terms of both computational complexity and prediction accuracy. The proposed method revealed strong relationship between small surface patterns (or pockets) and GO functions, which can be further used to identify active sites or functional motifs. The high quality performance of the prediction method together with the statistics also indicates that pockets play essential roles in biological interactions or the GO functions. Moreover, in addition to pockets, the proposed network framework can also be used for adopting other protein spatial surface patterns to predict the protein functions.

The maximal size of protein to diffuse through the nuclear pore is larger than 60kDa.

R Wang — 2007-12-05T03:26:56-00:00

FEBS Lett, Vol. 581, No. 17. (10 July 2007), pp. 3164-3170.

It has generally been believed that the diffusion limit set by the nuclear pore for protein is 60kDa. We here studied the cellular localization of several artificial proteins and found that the diffusion limit set by the nuclear pore is not as small as previously thought. The results indicate that the maximal size of protein to diffuse through the nuclear pore complex could be quite larger than 60kDa, thus greatly extending the diffusion limit that the nuclear pore can accommodate.

PPSP: prediction of PK-specific phosphorylation site with Bayesian decision theory.

Yu Xue — 2007-12-04T03:22:11-00:00

BMC Bioinformatics, Vol. 7 (2006)

BACKGROUND: As a reversible and dynamic post-translational modification (PTM) of proteins, phosphorylation plays essential regulatory roles in a broad spectrum of the biological processes. Although many studies have been contributed on the molecular mechanism of phosphorylation dynamics, the intrinsic feature of substrates specificity is still elusive and remains to be delineated. RESULTS: In this work, we present a novel, versatile and comprehensive program, PPSP (Prediction of PK-specific Phosphorylation site), deployed with approach of Bayesian decision theory (BDT). PPSP could predict the potential phosphorylation sites accurately for approximately 70 PK (Protein Kinase) groups. Compared with four existing tools Scansite, NetPhosK, KinasePhos and GPS, PPSP is more accurate and powerful than these tools. Moreover, PPSP also provides the prediction for many novel PKs, say, TRK, mTOR, SyK and MET/RON, etc. The accuracy of these novel PKs are also satisfying. CONCLUSION: Taken together, we propose that PPSP could be a potentially powerful tool for the experimentalists who are focusing on phosphorylation substrates with their PK-specific sites identification. Moreover, the BDT strategy could also be a ubiquitous approach for PTMs, such as sumoylation and ubiquitination, etc.

BRSK2 is activated by cyclic AMP-dependent protein kinase A through phosphorylation at Thr260.

Zekun Guo — 2007-12-04T03:22:09-00:00

Biochem Biophys Res Commun, Vol. 347, No. 4. (Sep 2006), pp. 867-871.

Brain selective kinase 2 (BRSK2) has been identified as a member of AMPK related kinases. LKB1 can phosphorylate the Thr174 of BRSK2, increasing its activity >50-fold. In this study, we identified cAMP-dependent protein kinase A (PKA) as another upstream kinase of BRSK2, which can phosphorylate BRSK2 at Thr260. The association between these two proteins was confirmed by GST pull-down. Furthermore, our study indicated that the kinase activity of BRSK2 can be increased through phosphorylation by PKA.

AgBase: a unified resource for functional analysis in agriculture.

Fiona Mccarthy — 2007-12-04T01:53:36-00:00

Nucleic Acids Res, Vol. 35, No. Database issue. (Jan 2007), pp. D599-D603.

Analysis of functional genomics (transcriptomics and proteomics) datasets is hindered in agricultural species because agricultural genome sequences have relatively poor structural and functional annotation. To facilitate systems biology in these species we have established the curated, web-accessible, public resource 'AgBase' (www.agbase.msstate.edu). We have improved the structural annotation of agriculturally important genomes by experimentally confirming the in vivo expression of electronically predicted proteins and by proteogenomic mapping. Proteogenomic data are available from the AgBase proteogenomics link. We contribute Gene Ontology (GO) annotations and we provide a two tier system of GO annotations for users. The 'GO Consortium' gene association file contains the most rigorous GO annotations based solely on experimental data. The 'Community' gene association file contains GO annotations based on expert community knowledge (annotations based directly from author statements and submitted annotations from the community) and annotations for predicted proteins. We have developed two tools for proteomics analysis and these are freely available on request. A suite of tools for analyzing functional genomics datasets using the GO is available online at the AgBase site. We encourage and publicly acknowledge GO annotations from researchers and provide an online mechanism for agricultural researchers to submit requests for GO annotations.

Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase Ibeta.

J Schlossmann — 2007-11-22T14:54:37-00:00

Nature, Vol. 404, No. 6774. (9 March 2000), pp. 197-201.

Calcium release from the endoplasmic reticulum controls a number of cellular processes, including proliferation and contraction of smooth muscle and other cells. Calcium release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores is negatively regulated by binding of calmodulin to the IP3 receptor (IP3R) and the NO/cGMP/cGMP kinase I (cGKI) signalling pathway. Activation of cGKI decreases IP3-stimulated elevations in intracellular calcium, induces smooth muscle relaxation and contributes to the antiproliferative and pro-apoptotic effects of NO/cGMP. Here we show that, in microsomal smooth muscle membranes, cGKIbeta phosphorylated the IP3R and cGKIbeta, and a protein of relative molecular mass 125,000 which we now identify as the IP3R-associated cGMP kinase substrate (IRAG). These proteins were co-immunoprecipitated by antibodies directed against cGKI, IP3R or IRAG. IRAG was found in many tissues including aorta, trachea and uterus, and was localized perinuclearly after heterologous expression in COS-7 cells. Bradykinin-stimulated calcium release was not affected by the expression of either IRAG or cGKIbeta, which we tested in the absence and presence of cGMP. However, calcium release was inhibited after co-expression of IRAG and cGKIbeta in the presence of cGMP. These results identify IRAG as an essential NO/cGKI-dependent regulator of IP3-induced calcium release.

Phosphate groups as substrate determinants for casein kinase I action.

H Flotow — 2007-11-22T13:08:32-00:00

J Biol Chem, Vol. 265, No. 24. (25 August 1990), pp. 14264-14269.

Phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase has been shown to enhance subsequent phosphorylation by casein kinase I (Flotow, H., and Roach, P. J. (1989) J. Biol. Chem. 264, 9126-9128). In the present study, synthetic peptides based on the sequences of the four phosphorylated regions in muscle glycogen synthase were used to probe the role of substrate phosphorylation in casein kinase I action. With all four peptides, prior phosphorylation significantly stimulated phosphorylation by casein kinase I. A series of peptides was synthesized based on the NH2-terminal glycogen synthase sequence PLSRTLS7VSS10LPGL, in which phosphorylation at Ser7 is required for modification of Ser10 by casein kinase I. The spacing between the P-Ser and the acceptor Ser was varied to have 1, 2, or 3 intervening residues. The peptide with a 2-residue spacing (-S(P)-X-X-S-) was by far the best casein kinase I substrate. When the P-Ser residue at Ser7 was replaced with P-Thr, the resulting peptide was still a casein kinase I substrate. However, substitution of Asp or Glu residues at Ser7 led to peptides that were not phosphorylated by casein kinase I. Phosphorylation of one of the other peptides showed that Thr could also be the phosphate acceptor. From these results, we propose that there are substrates for casein kinase I for which prior phosphorylation is a critical determinant of protein kinase action. In these instances, an important recognition motif for casein kinase I appears to be -S(P)/T(P)-Xn-S/T- with n = 2 much more effective than n = 1 or n = 3. Thus, casein kinase I may be involved in hierarchal substrate phosphorylation schemes in which its activity is controlled by the phosphorylation state of its substrates.

Cyclic GMP-dependent protein kinase substrates in rat brain.

X Wang — 2007-11-21T06:39:43-00:00

J Neurochem, Vol. 65, No. 2. (August 1995), pp. 595-604.

Cyclic GMP (cGMP)-dependent protein kinase (PKG) has a limited substrate specificity, and only cerebellar G-substrate has been demonstrated in brain. In view of the physiological importance of cGMP and PKG in the nervous system, it is important to identify endogenous PKG substrates in rat brain. We devised a combination of ion-exchange and hydrophobic chromatographies to identify potential PKG substrates. Extracts from cytosol, peripheral membrane proteins, or a fraction enriched in Ca(2+)-sensitive lipid-binding proteins were partly purified and phosphorylated with purified PKG. Using whole extracts only a single specific PKG substrate-P34-was found. However, after chromatography we detected > 40 distinct proteins that were phosphorylated by PKG to a much greater extent than by cyclic AMP-dependent protein kinase or protein kinase C. Four PKG substrates--P140, P65, P32, and P18--were detected in the cytosol. Six PKG substrates--P130, P85 (doublet), P58, P54, and P38--were enriched from the Ca(2+)-sensitive lipid-binding protein fraction. In peripheral membrane fractions > 30 relatively specific PKG substrates were enriched after chromatography, especially P130, P94, P58, P52, P45, P40, P36, P34, P28, P26, P24, and P20. These results indicate that brain is not lacking in PKG substrates and show that many are apparently quite specific substrates for this enzyme. The identification of some of these novel PKG substrates will facilitate understanding the role of cGMP signaling in the brain.

Cyclic GMP-dependent protein kinase and cellular signaling in the nervous system.

X Wang — 2007-11-21T06:37:46-00:00

J Neurochem, Vol. 68, No. 2. (February 1997), pp. 443-456.

Nitric oxide (NO) and natriuretic peptide hormones play key roles in a surprising number of neuronal functions, including learning and memory. Most data suggest that they exert converging actions by elevation of intracellular cyclic GMP (cGMP) levels through activation of soluble and particulate guanylyl cyclases. However, cGMP is only the starting point for multiple signaling cascades, which are now beginning to be defined. A primary action of elevated cGMP levels is the stimulation of cGMP-dependent protein kinase (PKG), the major intracellular receptor protein for cGMP, which phosphorylates substrate proteins to exert its actions. It has become increasingly clear that PKG mediates some of the neuronal effects of cGMP, but how is not yet clear. One clear illustration of this pathway has been reported in striatonigral nerve terminals, where NO mediates phosphorylation of the protein phosphatase regulator dopamine- and cyclic AMP-regulated phosphoprotein having a molecular mass of 32,000 (DARPP-32) by PKG. There are remarkably few PKG substrates in brain whose identities are known. A survey of these proteins and those known from other tissues that might also be found in the nervous system reveals the key molecular sites where cGMP and PKG signaling is likely to be regulating neural function. These potential substrates are critically placed to have profound effects on the protein phosphorylation network through regulation of protein phosphatases, intracellular calcium levels, and the function of many ion channels and neurotransmitter receptors. The brain also contains a rich diversity of specific PKG substrates whose identities are not yet known. Their future identification will provide exciting new leads that will permit better understanding of the role of PKG signaling in both basic and higher orders of brain function.