Pex, analytical tools for PDB files. II. H-Pex: noncanonical H-bonds in alpha-helices

We use the H-Pex (Thomas et al., this issue) to analyze the main chain interactions in 131 proteins. In antiparallel beta-sheets, the geometry of the N...O bond is: median N...O distances, 2.9 SA, C==O...N angles at 154 degrees and the C alpha--C==O...H angles are dispersed around 3 degrees. In some instances, the other side of the C==O axis is occupied by a HC alpha. As recently supported by Vargas et al. (J Am Chem Soc 2000;122:4750-4755) C alpha H...O and NH...O could cooperate to sheet stability. In alpha-helices, the main chain C==O interact with the NH of their n + 4 neighbor on one side, and with a C beta H or C gamma H on the other side. The median O...N distance (3.0 A) and C==N angle (147 degrees) suggest a canonical H-bond, but the C alpha--C==O...H dihedral angle invalidates this option, since the hydrogen attacks the oxygen at 122 degrees, i.e., between the sp(2) and pi orbitals. This supports that the H-bond is noncanonical. In many instances, the C gamma H or the C beta H of the n + 4 residue stands opposite to the NH with respect to the oxygen. Therefore, we propose that, in alpha-helices, the C gamma H or C beta H and the NH of the n + 4 residue hold the oxygen like an electrostatic pincher. Proteins 2001;43:37-44.     

PDBToSDF: Create ligand structure files from PDB file

Protein Data Bank (PDB) file contains atomic data for protein and ligand in protein-ligand complexes. Structure data file (SDF) contains data for atoms, bonds, connectivity and coordinates of molecule for ligands. We describe PDBToSDF as a tool to separate the ligand data from pdb file for the calculation of ligand properties like molecular weight, number of hydrogen bond acceptors, hydrogen bond receptors easily.     

gmconvert: file conversion for genemapper output files

gmconvert is a platform‐independent program provided in GUI (for Apple OS X and Windows XP) and command‐line versions (for other platforms). gmconvert allows rapid reformatting of microsatellite data from output files produced by Applied Biosystems genemapper software (version 3.x). The program will re‐array data into three formats commonly used in downstream analysis: genepop , cervus , and gerud . gmconvert will greatly increase the speed of data preparation prior to analysis and aid in reducing transpositional errors associated with manual re‐arraying and reformatting steps. gmconvert is available from http://gallus.forestry.uga.edu/software/ .     

PDB@: An offline toolkit for exploration and analysis of PDB files

Protein Data Bank (PDB) is a freely accessible archive of the 3-D structural data of biological molecules. Structure based studies offers a unique vantage point in inferring the properties of a protein molecule from structural data. This is too big a task to be done manually. Moreover, there is no single tool, software or server that comprehensively analyses all structure-based properties. The objective of the present work is to develop an offline computational toolkit, PDB@ containing in-built algorithms that help categorizing the structural properties of a protein molecule. The user has the facility to view and edit the PDB file to his need. Some features of the present work are unique in itself and others are an improvement over existing tools. Also, the representation of protein properties in both graphical and textual formats helps in predicting all the necessary details of a protein molecule on a single platform.     

OMEGA: a three-dimensional databank for protein structures (a complement to PDB)

OMEGA is a compilation of recent structural information on proteinsderived from X-ray crystallography or NMR and published in journalsreferenced by Current Contents. To date, 401 entries have beenincluded (334 X-ray, 28 NMR, 5 NMR + X-ray, S electron microscopy,3 neutron scattering, 2 neutron diffraction, 1 electron microscopy+ X-ray, 12 model, 11 miscellaneous), with 5–10 new proteinsbeing added each week. OMEGA can be accessed on Macintosh andis interrog ated through 32 key words (space group, resolution,secondary structure, number of residues, etc). This pool ofproteins could be used for various purposes, including searchesfor proteins with a particular set of secondary structures.OMEGA will be continuously updated (every 6 months) and maylater include all proteins already reported in the PDB, as wellas structures reported in journals with smaller readerships.     

Multi-PK antibodies: Powerful analytical tools to explore the protein kinase world

Diverse biological events are regulated through protein phosphorylation mediated by protein kinases. Some of these protein kinases are known to be involved in the pathogenesis of various diseases. Although 518 protein kinase genes were identified in the human genome, it remains unclear how many and what kind of protein kinases are expressed and activated in cells and tissues under varying situations. To investigate cellular signaling by protein kinases, we developed monoclonal antibodies, designated as Multi-PK antibodies, that can recognize multiple protein kinases in various biological species. These Multi-PK antibodies can be used to profile the kinases expressed in cells and tissues, identify the kinases of special interest, and analyze protein kinase expression and phosphorylation state. Here we introduce some applications of Multi-PK antibodies to identify and characterize the protein kinases involved in epigenetics, glucotoxicity in type 2 diabetes, and pathogenesis of ulcerative colitis. In this review, we focus on the recently developed technologies for kinomics studies using the powerful analytical tools of Multi-PK antibodies.     

Pex14p is a member of the protein linkage map of Pex5p.

To identify members of the translocation machinery for peroxisomal proteins, we made use of the two-hybrid system to establish a protein linkage map centered around Pex5p from Saccharomyces cerevisiae, the receptor for the C-terminal peroxisomal targeting signal (PTS1). Among the five interaction partners identified, Pex14p was found to be induced under conditions allowing peroxisome proliferation. Deletion of the corresponding gene resulted in the inability of yeast cells to grow on oleate as well as the absence of peroxisomal structures. The PEX14 gene product of approximately 38 kDa was biochemically and ultrastructurally demonstrated to be a peroxisomal membrane protein, despite the lack of a membrane-spanning domain. This protein was shown to interact with itself, with Pex13p and with both PTS receptors, Pex5p and Pex7p, indicating a central function for the import of peroxisomal matrix proteins, either as a docking protein or as a releasing factor at the organellar membrane.     

Substrate specificity for myelin basic protein-specific protein methylase I.

The substrate specificity of bovine brain myelin basic protein (MBP)-specific protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), which methylates arginine residues of protein, has been studied using various MBPs, several synthetic peptides and heterogeneous nuclear ribonucleoprotein complex protein (hnRNP). (1) Among MBPs from different species of brain, the carp MBP was found to be the best substrate for MBP-specific protein methylase I. This high degree of methyl acceptability is most likely due to the fact that carp MBP is not in vivo methylated at the arginine residue (Deibler, G.E. and Martenson, R.E. (1973) J. Biol. Chem. 248, 2387-2391) and that the methylatable amino acid sequence is present in this protein. (2) In order to study the minimum chain length of MBP polypeptide which functions as the methyl acceptor, several synthetic polypeptides whose sequences are identical to the region surrounding the residue 107 of bovine MBP (the in vivo methylation site) were synthesized. It was found that the hexapeptide, Gly-Lys-Gly-Arg-Gly-Leu (corresponding to residues 104-109 of bovine MBP), was the shortest methyl accepting peptide, while the tetrapeptide, Gly-Arg-Gly-Leu (corresponding to residues 106-109) was inactive as a substrate. (3) hnRNP protein is known to contain methylarginine at residue 193 (Williams, K.R., Stone, K.L., LoPresti, M.B., Merrill, B.M. and Plank, S.R. (1985) Proc. Natl. Acad. Sci. USA 82, 5666-5670) which is post-translationally modified. Thus, the RNP protein overproduced in Escherichia coli and therefore did not contain methylarginine was examined for its methyl acceptability. It was found that neither MBP-specific nor histone-specific protein methylase I could methylate this methylarginine-less RNP protein. This suggests a possible existence of a distinct protein methylase I specific for this nuclear protein.     

Characterization of the mammalian peroxisomal import machinery: Pex2p, Pex5p, Pex12p, and Pex14p are subunits of the same protein assembly

Although many of the proteins involved in the biogenesis of the mammalian peroxisome have already been identified, our knowledge of the architecture of all this machinery is still very limited. In this work we used native gel electrophoresis and sucrose gradient sedimentation analysis in combination with immunoprecipitation experiments to address this issue. After solubilization of rat liver peroxisomes with the mild detergent digitonin, comigration of Pex5p, Pex14p, and a fraction of Pex12p was observed upon native electrophoresis and sucrose gradient sedimentation. The existence of a complex comprising Pex2p, Pex5p, Pex12p, and Pex14p was demonstrated by preparative coimmunoprecipitation experiments using an antibody directed to Pex14p. No stoichiometric amounts of Pex13p were detected in the Pex2p-Pex5p-Pex12p-Pex14p complex, although the presence of a small fraction of Pex13p in this complex could be demonstrated by Western blot analysis. Pex13p is also a component of a high molecular mass complex. Strikingly, partial purification of this Pex13p-containing complex revealed Pex13p as the major (if not the only) component. Taken together, our data indicate that Pex2p, Pex5p, Pex12p, and Pex14p, on one side, and Pex13p, on the other, are subunits of two stable protein complexes that probably interact with each other in the peroxisomal membrane.     

Improved process analytical technology for protein a chromatography using predictive principal component analysis tools

Protein A chromatography is widely employed for the capture and purification of antibodies and Fc‐fusion proteins. Due to the high cost of protein A resins, there is a significant economic driving force for using these chromatographic materials for a large number of cycles. The maintenance of column performance over the resin lifetime is also a significant concern in large‐scale manufacturing. In this work, several statistical methods are employed to develop a novel principal component analysis (PCA)‐based tool for predicting protein A chromatographic column performance over time. A method is developed to carry out detection of column integrity failures before their occurrence without the need for a separate integrity test. In addition, analysis of various transitions in the chromatograms was also employed to develop PCA‐based models to predict both subtle and general trends in real‐time protein A column yield decay. The developed approach has significant potential for facilitating timely and improved decisions in large‐scale chromatographic operations in line with the process analytical technology (PAT) guidance from the Food and Drug Administration (FDA). Biotechnol. Bioeng. 2011; 108:59–68. © 2010 Wiley Periodicals, Inc.     

Pex14p, more than just a docking protein

After binding newly synthesized peroxisomal matrix proteins in the cytosol, the second task of Pex5p, the peroxisomal cycling receptor, is to carry these proteins to the peroxisomal membrane. Defining the nature of the events that occur at this membrane system and which ultimately result in the translocation of the cargo proteins into the matrix of the organelle and in the recycling of Pex5p back to the cytosol, is one of the major goals of the research in this field. Presently, it is generally accepted that all these steps are promoted by a large protein complex embedded in the peroxisomal membrane. This docking/translocation machinery or importomer, as it is often called, comprises many different peroxins of which one of the best characterized is Pex14p. Here, we review data regarding this membrane peroxin with emphasis on the interactions that it establishes with Pex5p. The available evidence suggests that the key to understand how folded proteins are capable of passing an apparently impermeable membrane may largely reside in this pair of peroxins.     

The peroxisomal membrane protein import receptor Pex3p is directly transported to peroxisomes by a novel Pex19p- and Pex16p-dependent pathway

Two distinct pathways have recently been proposed for the import of peroxisomal membrane proteins (PMPs): a Pex19p- and Pex3p-dependent class I pathway and a Pex19p- and Pex3p-independent class II pathway. We show here that Pex19p plays an essential role as the chaperone for full-length Pex3p in the cytosol. Pex19p forms a soluble complex with newly synthesized Pex3p in the cytosol and directly translocates it to peroxisomes. Knockdown of Pex19p inhibits peroxisomal targeting of newly synthesized full-length Pex3p and results in failure of the peroxisomal localization of Pex3p. Moreover, we demonstrate that Pex16p functions as the Pex3p-docking site and serves as the peroxisomal membrane receptor that is specific to the Pex3p–Pex19p complexes. Based on these novel findings, we suggest a model for the import of PMPs that provides new insights into the molecular mechanisms underlying the biogenesis of peroxisomes and its regulation involving Pex3p, Pex19p, and Pex16p.     

biobambam: tools for read pair collation based algorithms on BAM files

Background

Sequence alignment data is often ordered by coordinate (id of the reference sequence plus position on the sequence where the fragment was mapped) when stored in BAM files, as this simplifies the extraction of variants between the mapped data and the reference or of variants within the mapped data. In this order paired reads are usually separated in the file, which complicates some other applications like duplicate marking or conversion to the FastQ format which require to access the full information of the pairs.

Results

In this paper we introduce biobambam, a set of tools based on the efficient collation of alignments in BAM files by read name. The employed collation algorithm avoids time and space consuming sorting of alignments by read name where this is possible without using more than a specified amount of main memory. Using this algorithm tasks like duplicate marking in BAM files and conversion of BAM files to the FastQ format can be performed very efficiently with limited resources. We also make the collation algorithm available in the form of an API for other projects. This API is part of the libmaus package.

Conclusions

In comparison with previous approaches to problems involving the collation of alignments by read name like the BAM to FastQ or duplication marking utilities our approach can often perform an equivalent task more efficiently in terms of the required main memory and run-time. Our BAM to FastQ conversion is faster than all widely known alternatives including Picard and bamUtil. Our duplicate marking is about as fast as the closest competitor bamUtil for small data sets and faster than all known alternatives on large and complex data sets.

     

Fraction I protein concentration in plants by analytical ultracentrifuge.

The fraction I protein content of plants was determined by centrifuging extracts in the analytical ultracentrifuge until the protein boundary was well separated. The refractive index increase across the boundary was determined with the interference optical system and converted to fraction I protein concentration with the factor 0.245 mg/ml/fringe.     

Characterization of peroxisomal Pex5p from rat liver. Pex5p in the Pex5p-Pex14p membrane complex is a transmembrane protein

Pex5p is the receptor for the vast majority of peroxisomal matrix proteins. Here, we show that about 15% of rat liver Pex5p is found in the peroxisomal fraction representing 0.06% of total peroxisomal protein. This population of Pex5p displays all the characteristics of an intrinsic membrane protein. Protease protection assays indicate that this pool of Pex5p has domains exposed on both sides of the peroxisomal membrane. The strong interaction of Pex5p with the membrane of the organelle is not affected by mild protease treatment of intact organelles, conditions that result in the partial degradation of Pex13p. Cytosolic Pex5p is a monomeric protein. In contrast, virtually all peroxisomal Pex5p was found to be part of a stable 250-kDa protein assembly. This complex was isolated and shown to comprise just two subunits, Pex5p and Pex14p.     

Proteomics: posttranslational modifications, immune responses and current analytical tools

The publication of the human genome sequence enables most of the still unknown protein sequences to be added to the current databases. A sequence alone does not, however, give information about the possible expression level of the corresponding protein, neither does it inform about the possible posttranslational modifications, like phosphorylation, glycosylation or changes in individual amino acids. Thus, the human proteome project, a large scale analysis of the functions of gene products, will have an enormous impact on our understanding of the biochemistry of proteins, processes and pathways they are involved in. The diversity in proteins is considerably expanded by various posttranslational modifications. These also pose problems to the investigators, but their careful analysis often pays back because they can reveal important properties in proteins or peptides--like an increased antigenicity leading to (auto)immune responses or an active form of a signaling protein. Immune tolerance usually exists towards self-proteins, but in specific cases it may be broken by posttranslational modifications in the proteins. Novel mass spectrometric, affinity and display techniques offer valuable tools for the large-scale analysis of proteomes. In the present paper we discuss their use for the detection of posttranslational modifications, functional interactions and possible disease-associated abnormalities in proteins.     

New tools for protein sequence analysis.

Analysis of protein sequence is an important tool in studies of both native and recombinant proteins. Novel techniques and instrumentation which facilitate determination of protein primary structure have recently been developed.     

T.I.M.S: TaqMan Information Management System,tools to organize data flow in a genotyping laboratory

Bacskground  

Single Nucleotide Polymorphism (SNP) genotyping is a major activity in biomedical research. The Taqman technology is one of the most commonly used approaches. It produces large amounts of data that are difficult to process by hand. Laboratories not equipped with a Laboratory Information Management System (LIMS) need tools to organize the data flow.     

ExSer: A standalone tool to mine protein data bank (PDB) for secondary structural elements

Detailed structural analysis of protein necessitates investigation at primary, secondary and tertiary levels, respectively. Insight into protein secondary structures pave way for understanding the type of secondary structural elements involved (α-helices, β-strands etc.), the amino acid sequence that encode the secondary structural elements, number of residues, length and, percentage composition of the respective elements in the protein. Here we present a standalone tool entitled "ExSer" which facilitate an automated extraction of the amino acid sequence that encode for the secondary structural regions of a protein from the protein data bank (PDB) file. AVAILABILITY: ExSer is freely downloadable from http://code.google.com/p/tool-exser/