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1.
Proteomics ; 11(7): 1277-86, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21319301

ABSTRACT

A wide range of chemical reagents are available to study the protein-protein interactions or protein structures. After reaction with such chemicals, covalently modified proteins are digested, resulting in shorter peptides that are analyzed by mass spectrometry (MS). Used especially when NMR of X-ray data are lacking, this methodology requires the identification of modified species carrying relevant information, among the unmodified peptides. To overcome the drawbacks of existing methods, we propose a more direct strategy relying on the synthesis of solid-supported cleavable monofunctional reagents and cross-linkers that react with proteins and that selectively release, after protein digestion and washings, the modified peptide fragments ready for MS analysis. Using this Solid-Phase Cross-Linking (SPCL) strategy, only modified sequences are analyzed and consistent data can be easily obtained since the signals of interest are not masked or suppressed by over-represented unmodified materials.


Subject(s)
Cross-Linking Reagents/chemistry , Peptide Fragments/analysis , Proteins/analysis , Animals , Binding Sites , Chromatography, High Pressure Liquid , Horses , Mass Spectrometry/methods , Models, Molecular , Peptide Fragments/chemistry , Protein Binding , Protein Interaction Domains and Motifs , Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Trypsin/metabolism
2.
Proteomics ; 9(23): 5384-8, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19902427

ABSTRACT

We designed a new cross-linker bearing a CHCA moiety. The use of the CHCA-tagged cross-linker JMV 3378 in conjunction with a neutral MALDI matrix alpha-cyano-4-hydroxycinnamic methyl ester enabled specific signal enhancement in MALDI-TOF MS of cross-link containing peptides. Discrimination between modified and non-modified peptides can be achieved by comparison of two spectra, one using CHCA and the other using the alpha-cyano-4-hydroxycinnamic methyl ester matrix. The methodology was validated using cytochrome c and apo-myoglobine as model proteins.


Subject(s)
Cross-Linking Reagents/chemistry , Proteins/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Amino Acid Sequence , Animals , Apoproteins/analysis , Cross-Linking Reagents/chemical synthesis , Cytochromes c/analysis , Horses , Molecular Sequence Data , Molecular Structure , Myoglobin/analysis , Peptides/analysis , Protein Conformation , Sensitivity and Specificity
3.
Bioinformatics ; 24(23): 2782-3, 2008 Dec 01.
Article in English | MEDLINE | ID: mdl-18826958

ABSTRACT

MOTIVATION: The technique of chemical cross-linking followed by mass spectrometry has proven to bring valuable information about the protein structure and interactions between proteic subunits. It is an effective and efficient way to experimentally investigate some aspects of a protein structure when NMR and X-ray crystallography data are lacking. RESULTS: We introduce MSX-3D, a tool specifically geared to validate protein models using mass spectrometry. In addition to classical peptides identifications, it allows an interactive 3D visualization of the distance constraints derived from a cross-linking experiment. AVAILABILITY: Freely available at http://proteomics-pbil.ibcp.fr


Subject(s)
Mass Spectrometry/methods , Protein Conformation , Software , Computer Simulation , Databases, Protein , Models, Molecular , Proteins/chemistry , Proteomics/methods
4.
FEMS Microbiol Lett ; 274(2): 252-9, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17627778

ABSTRACT

The catalytic mechanism of bacterial tyrosine-kinases (PTK) is poorly understood. These enzymes possess Walker A and B ATP-binding motifs, which are effectively required for their autophosphorylation whereas these motifs are usually found in ATP-binding proteins but not in eukaryotic protein-kinases. It was previously shown that the PTK Wzc in Escherichia coli undergoes intra- and interphosphorylation. In this work, it is shown that, in addition to its kinase activity, Wzc produces free inorganic phosphate. It is demonstrated that this ATPase activity is increased significantly by intraphosphorylation of Wzc. The fact that intraphosphorylation of Wzc does not affect Wzc affinity for ATP was also demonstrated and it was suggested that it could rather modify the local environment of the ATP molecule in the catalytic site so as to render Wzc more liable to catalyze ATP hydrolysis and interphosphorylation. These results should contribute to better understanding of the catalytic mechanism of this particular class of tyrosine-kinases, which seems, so far, restricted to bacteria.


Subject(s)
Adenosine Triphosphatases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Membrane Proteins/metabolism , Protein-Tyrosine Kinases/metabolism , Catalytic Domain , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Gene Expression Regulation, Bacterial , Membrane Proteins/chemistry , Phosphorylation , Protein-Tyrosine Kinases/chemistry
6.
Nucleic Acids Res ; 35(Database issue): D363-6, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17142229

ABSTRACT

The hepatitis C virus (HCV) genome shows remarkable sequence variability, leading to the classification of at least six major genotypes, numerous subtypes and a myriad of quasispecies within a given host. A database allowing researchers to investigate the genetic and structural variability of all available HCV sequences is an essential tool for studies on the molecular virology and pathogenesis of hepatitis C as well as drug design and vaccine development. We describe here the European Hepatitis C Virus Database (euHCVdb, http://euhcvdb.ibcp.fr), a collection of computer-annotated sequences based on reference genomes. The annotations include genome mapping of sequences, use of recommended nomenclature, subtyping as well as three-dimensional (3D) molecular models of proteins. A WWW interface has been developed to facilitate database searches and the export of data for sequence and structure analyses. As part of an international collaborative effort with the US and Japanese databases, the European HCV Database (euHCVdb) is mainly dedicated to HCV protein sequences, 3D structures and functional analyses.


Subject(s)
Databases, Protein , Hepacivirus/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Genome, Viral , Genomics , Internet , Models, Molecular , Protein Conformation , Sequence Analysis, Protein , User-Computer Interface
7.
Mol Biol Evol ; 23(12): 2288-302, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16945979

ABSTRACT

Collagens are thought to represent one of the most important molecular innovations in the metazoan line. Basement membrane type IV collagen is present in all Eumetazoa and was found in Homoscleromorpha, a sponge group with a well-organized epithelium, which may represent the first stage of tissue differentiation during animal evolution. In contrast, spongin seems to be a demosponge-specific collagenous protein, which can totally substitute an inorganic skeleton, such as in the well-known bath sponge. In the freshwater sponge Ephydatia mülleri, we previously characterized a family of short-chain collagens that are likely to be main components of spongins. Using a combination of sequence- and structure-based methods, we present evidence of remote homology between the carboxyl-terminal noncollagenous NC1 domain of spongin short-chain collagens and type IV collagen. Unexpectedly, spongin short-chain collagen-related proteins were retrieved in nonsponge animals, suggesting that a family related to spongin constitutes an evolutionary sister to the type IV collagen family. Formation of the ancestral NC1 domain and divergence of the spongin short-chain collagen-related and type IV collagen families may have occurred before the parazoan-eumetazoan split, the earliest divergence among extant animal phyla. Molecular phylogenetics based on NC1 domain sequences suggest distinct evolutionary histories for spongin short-chain collagen-related and type IV collagen families that include spongin short-chain collagen-related gene loss in the ancestors of Ecdyzosoa and of vertebrates. The fact that a majority of invertebrates encodes spongin short-chain collagen-related proteins raises the important question to the possible function of its members. Considering the importance of collagens for animal structure and substratum attachment, both families may have played crucial roles in animal diversification.


Subject(s)
Collagen Type IV/genetics , Evolution, Molecular , Extracellular Matrix/genetics , Non-Fibrillar Collagens/genetics , Porifera/genetics , Amino Acid Sequence , Animals , Collagen Type IV/chemistry , Invertebrates/genetics , Membrane Proteins/genetics , Models, Biological , Models, Molecular , Molecular Sequence Data , Multigene Family/genetics , Non-Fibrillar Collagens/chemistry , Phylogeny , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid
8.
Hepatology ; 43(5): 1157-65, 2006 May.
Article in English | MEDLINE | ID: mdl-16628639

ABSTRACT

Part of the effort to develop hepatitis C-specific drugs a nd vaccines is the study of genetic variability of allpublicly available HCV sequences. Three HCV databases are currently available to aid this effort and to provide additional insight into the basic biology, immunology, and evolution of the virus. The Japanese HCV database (http://s2as02.genes.nig.ac.jp) gives access to a genomic mapping of sequences as well as their phylogenetic relationships. The European HCV database (http://euhcvdb.ibcp.fr) offers access to a computer-annotated set of sequences and molecular models of HCV proteins and focuses on protein sequence, structure and function analysis. The HCV database at the Los Alamos National Laboratory in the United States (http://hcv.lanl.gov) provides access to a manually annotated sequence database and a database of immunological epitopes which contains concise descriptions of experimental results. In this paper, we briefly describe each of these databases and their associated websites and tools, and give some examples of their use in furthering HCV research.


Subject(s)
Biomedical Research , Databases, Factual , Hepacivirus , Genomics , Hepacivirus/genetics , Hepacivirus/immunology , Models, Molecular , Phylogeny
9.
J Biol Chem ; 281(20): 14048-56, 2006 May 19.
Article in English | MEDLINE | ID: mdl-16565080

ABSTRACT

Protein phosphorylation on tyrosine has been originally characterized in animal systems and has been shown to be involved in several fundamental processes including signal transduction, growth control, and malignancy. It has been later demonstrated to occur also in a number of bacteria, and recent data suggest that it may participate in the control of bacterial pathogenicity. In this work, we provide evidence that the gram-positive human pathogen Staphylococcus aureus harbors a protein-tyrosine kinase activity. This activity is borne by a protein, termed Cap5B2, whose phosphorylating capacity is expressed only in the presence of a stimulatory protein, either Cap5A1 or Cap5A2, that enhances its affinity for the phosphoryl donor ATP. In fact, the last 27/29 amino acids of the C-terminal domain of either polypeptide are sufficient for stimulating Cap5B2 activity. The stimulation of Cap5B2 by Cap5A1 involves essentially three amino acid residues in a helix of Cap5A1 (Asp202, Glu203, and Asp205) and three residues in a helix (helix 7) of Cap5B2 (Glu190, Lys192, and Lys193), thus suggesting helix-helix interaction between these two proteins. This type of helix-helix interaction resembles the interaction required for the activation of MinD ATPase by MinE protein in the process of septum-site determination, MinD sharing sequence similarity with Cap5B2. Such activation mechanism is described here in a gram-positive bacterial tyrosine kinase, and differs from the activation mechanism previously proposed for gram-negative bacteria. Therefore, it appears that S. aureus, and possibly other gram-positive bacteria, utilizes a specific molecular mechanism for triggering protein-tyrosine kinase activity.


Subject(s)
Protein-Tyrosine Kinases/metabolism , Staphylococcus aureus/physiology , Amino Acid Sequence , Bacterial Proteins/metabolism , Dose-Response Relationship, Drug , Escherichia coli/metabolism , Humans , Models, Biological , Molecular Sequence Data , Phosphorylation , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Staphylococcus aureus/metabolism
10.
Bioinformatics ; 21(20): 3929-30, 2005 Oct 15.
Article in English | MEDLINE | ID: mdl-16141250

ABSTRACT

UNLABELLED: We provide the scientific community with a web server which gives access to SuMo, a bioinformatic system for finding similarities in arbitrary 3D structures or substructures of proteins. SuMo is based on a unique representation of macromolecules using selected triplets of chemical groups having their own geometry and symmetry, regardless of the restrictive notions of main chain and lateral chains of amino acids. The heuristic for extracting similar sites was used to drive two major large-scale approaches. First, searching for ligand binding sites onto a query structure has been made possible by comparing the structure against each of the ligand binding sites found in the Protein Data Bank (PDB). Second, the reciprocal process, i.e. searching for a given 3D site of interest among the structures of the PDB is also possible and helps detect cross-reacting targets in drug design projects. AVAILABILITY: The web server is freely accessible to academia through http://sumo-pbil.ibcp.fr and full support is available from MEDIT (http://www.medit.fr). CONTACT: mjambon@burnham.org.


Subject(s)
Imaging, Three-Dimensional/methods , Internet , Protein Interaction Mapping/methods , Proteins/chemistry , Sequence Analysis, Protein/methods , Software , User-Computer Interface , Amino Acid Sequence , Binding Sites , Molecular Sequence Data , Protein Binding , Protein Conformation , Proteins/analysis , Structure-Activity Relationship
11.
J Biol Chem ; 280(44): 36857-64, 2005 Nov 04.
Article in English | MEDLINE | ID: mdl-16107340

ABSTRACT

The ATP-binding cassette is the most abundant family of transporters including many medically relevant members and gathers both importers and exporters involved in the transport of a wide variety of substrates. Although three high resolution three-dimensional structures have been obtained for a prototypic exporter, MsbA, two have been subjected to much criticism. Here, conformational changes of BmrA, a multidrug bacterial transporter structurally related to MsbA, have been studied. A three-dimensional model of BmrA, based on the "open" conformation of Escherichia coli MsbA, was probed by simultaneously introducing two cysteine residues, one in the first intracellular loop of the transmembrane domain and the other in the Q-loop of the nucleotide-binding domain (NBD). Intramolecular disulfide bonds could be created in the absence of any effectors, which prevented both drug transport and ATPase activity. Interestingly, addition of ATP/Mg plus vanadate strongly prevented this bond formation in a cysteine double mutant, whereas ATP/Mg alone was sufficient when the ATPase-inactive E504Q mutation was also introduced, in agreement with additional BmrA models where the ATP-binding sites are positioned at the NBD/NBD interface. Furthermore, cross-linking between the two cysteine residues could still be achieved in the presence of ATP/Mg plus vanadate when homobifunctional cross-linkers separated by more than 13 Angstrom were added. Altogether, these results give support to the existence, in the resting state, of a monomeric conformation of BmrA similar to that found within the open MsbA dimer and show that a large motion is required between intracellular loop 1 and the nucleotide-binding domain for the proper functioning of a multidrug ATP-binding cassette transporter.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Benzimidazoles/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Protein Folding , ATP-Binding Cassette Transporters/chemistry , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Binding Sites , Biological Transport , Catalysis , Cross-Linking Reagents/metabolism , Dimerization , Disulfides , Escherichia coli/chemistry , Escherichia coli/metabolism , Membrane Transport Proteins/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Protein Conformation , Protein Structure, Tertiary , Sequence Homology, Amino Acid
12.
J Mol Recognit ; 18(3): 225-35, 2005.
Article in English | MEDLINE | ID: mdl-15593306

ABSTRACT

Prostate-specific antigen (PSA) is widely used as a serum marker for the diagnosis of prostate cancer. To evaluate two anti-free PSA monoclonal antibodies (mAbs) as potential tools in new generations of more relevant PSA assays, we report here their properties towards the recognition of specific forms of free PSA in seminal fluids, LNCaP supernatants, 'non-binding' PSA and sera from cancer patients. PSA from these different origins was immunopurified by the two anti-free PSA mAbs (5D3D11 and 6C8D8) as well as by an anti-total PSA mAb. The composition of the different immunopurified PSA fractions was analysed and their respective enzymatic activities were determined. In seminal fluid, enzymatically active PSA was equally purified with the three mAbs. In LNCaP supernatants and human sera, 5D3D11 immunopurified active PSA mainly, whereas 6C8D8 immunopurified PSA with residual activity. In sera of prostate cancer patients, we identified the presence of a mature inactive PSA form which can be activated into active PSA by use of high saline concentration or capture by an anti-total PSA mAb capable of enhancing PSA activity. According to PSA models built by comparative modelling with the crystal structure of horse prostate kallikrein described previously, we assume that active and activable PSA could correspond to mature intact PSA with open and closed conformations of the kallikrein loop. The specificity of 5D3D11 was restricted to both active and activable PSA, whereas 6C8D8 recognized all free PSA including intact PSA, proforms and internally cleaved PSA.


Subject(s)
Antibodies, Monoclonal , Prostate-Specific Antigen/immunology , Prostate-Specific Antigen/metabolism , Prostatic Neoplasms/blood , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/pharmacology , Antibody Specificity , Horses , Humans , Kallikreins/chemistry , Male , Models, Molecular , Molecular Sequence Data , Prostate-Specific Antigen/antagonists & inhibitors , Prostatic Neoplasms/pathology , Semen/chemistry , Sequence Homology, Amino Acid , Tumor Cells, Cultured
13.
Nucleic Acids Res ; 33(Database issue): D641-6, 2005 Jan 01.
Article in English | MEDLINE | ID: mdl-15608279

ABSTRACT

Genomic projects heavily depend on genome annotations and are limited by the current deficiencies in the published predictions of gene structure and function. It follows that, improved annotation will allow better data mining of genomes, and more secure planning and design of experiments. The purpose of the GeneFarm project is to obtain homogeneous, reliable, documented and traceable annotations for Arabidopsis nuclear genes and gene products, and to enter them into an added-value database. This re-annotation project is being performed exhaustively on every member of each gene family. Performing a family-wide annotation makes the task easier and more efficient than a gene-by-gene approach since many features obtained for one gene can be extrapolated to some or all the other genes of a family. A complete annotation procedure based on the most efficient prediction tools available is being used by 16 partner laboratories, each contributing annotated families from its field of expertise. A database, named GeneFarm, and an associated user-friendly interface to query the annotations have been developed. More than 3000 genes distributed over 300 families have been annotated and are available at http://genoplante-info.infobiogen.fr/Genefarm/. Furthermore, collaboration with the Swiss Institute of Bioinformatics is underway to integrate the GeneFarm data into the protein knowledgebase Swiss-Prot.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Databases, Genetic , Genes, Plant , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/physiology , Philosophy , Systems Integration , User-Computer Interface
14.
Appl Bioinformatics ; 3(4): 237-40, 2004.
Article in English | MEDLINE | ID: mdl-15702954

ABSTRACT

UNLABELLED: To date, more than 30 000 hepatitis C virus (HCV) sequences have been deposited in the generalist databases DNA Data Bank of Japan (DDBJ), EMBL Nucleotide Sequence Database (EMBL) and GenBank. The main difficulties with HCV sequences in these databases are their retrieval, annotation and analyses. To help HCV researchers face the increasing needs of HCV sequence analyses, we developed a specialised database of computer-annotated HCV sequences, called HCVDB. HCVDB is re-built every month from an up-to-date EMBL database by an automated process. HCVDB provides key data about the HCV sequences (e.g. genotype, genomic region, protein names and functions, known 3-dimensional structures) and ensures consistency of the annotations, which enables reliable keyword queries. The database is highly integrated with sequence and structure analysis tools and the SRS (LION bioscience) keywords query system. Thus, any user can extract subsets of sequences matching particular criteria or enter their own sequences and analyse them with various bioinformatics programs available on the same server. AVAILABILITY: HCVDB is available from http://hepatitis.ibcp.fr.


Subject(s)
Databases, Genetic , Hepacivirus/chemistry , Hepacivirus/metabolism , Sequence Analysis/methods , User-Computer Interface , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Sequence , Base Sequence , Chromosome Mapping/methods , Database Management Systems , Documentation/methods , Genome, Viral , Hepacivirus/genetics , Information Storage and Retrieval/methods , Molecular Sequence Data , Viral Proteins/genetics
15.
Proteins ; 52(2): 137-45, 2003 Aug 01.
Article in English | MEDLINE | ID: mdl-12833538

ABSTRACT

An innovative bioinformatic method has been designed and implemented to detect similar three-dimensional (3D) sites in proteins. This approach allows the comparison of protein structures or substructures and detects local spatial similarities: this method is completely independent from the amino acid sequence and from the backbone structure. In contrast to already existing tools, the basis for this method is a representation of the protein structure by a set of stereochemical groups that are defined independently from the notion of amino acid. An efficient heuristic for finding similarities that uses graphs of triangles of chemical groups to represent the protein structures has been developed. The implementation of this heuristic constitutes a software named SuMo (Surfing the Molecules), which allows the dynamic definition of chemical groups, the selection of sites in the proteins, and the management and screening of databases. To show the relevance of this approach, we focused on two extreme examples illustrating convergent and divergent evolution. In two unrelated serine proteases, SuMo detects one common site, which corresponds to the catalytic triad. In the legume lectins family composed of >100 structures that share similar sequences and folds but may have lost their ability to bind a carbohydrate molecule, SuMo discriminates between functional and non-functional lectins with a selectivity of 96%. The time needed for searching a given site in a protein structure is typically 0.1 s on a PIII 800MHz/Linux computer; thus, in further studies, SuMo will be used to screen the PDB.


Subject(s)
Computational Biology/methods , Models, Molecular , Proteins/chemistry , Algorithms , Catalytic Domain , Chymotrypsin/chemistry , Chymotrypsin/genetics , Evolution, Molecular , Fabaceae/chemistry , Plant Lectins/chemistry , Plant Lectins/genetics , Protein Conformation , Reproducibility of Results , Subtilisin/chemistry , Subtilisin/genetics
16.
Nucleic Acids Res ; 31(13): 3393-9, 2003 Jul 01.
Article in English | MEDLINE | ID: mdl-12824334

ABSTRACT

The World Wide Web server of the PBIL (Pôle Bioinformatique Lyonnais) provides on-line access to sequence databanks and to many tools of nucleic acid and protein sequence analyses. This server allows to query nucleotide sequence banks in the EMBL and GenBank formats and protein sequence banks in the SWISS-PROT and PIR formats. The query engine on which our data bank access is based is the ACNUC system. It allows the possibility to build complex queries to access functional zones of biological interest and to retrieve large sequence sets. Of special interest are the unique features provided by this system to query the data banks of gene families developed at the PBIL. The server also provides access to a wide range of sequence analysis methods: similarity search programs, multiple alignments, protein structure prediction and multivariate statistics. An originality of this server is the integration of these two aspects: sequence retrieval and sequence analysis. Indeed, thanks to the introduction of re-usable lists, it is possible to perform treatments on large sets of data. The PBIL server can be reached at: http://pbil.univ-lyon1.fr.


Subject(s)
Databases, Genetic , Protein Conformation , Sequence Analysis, DNA , Sequence Analysis, Protein , Sequence Analysis, RNA , Internet , Models, Molecular , Nucleic Acids/chemistry , Protein Structure, Secondary , Proteins/chemistry , Sequence Alignment , Sequence Homology , Software , Systems Integration , User-Computer Interface
17.
Bioinformatics ; 19(4): 506-12, 2003 Mar 01.
Article in English | MEDLINE | ID: mdl-12611806

ABSTRACT

MOTIVATION: Multiple sequence alignments are essential tools for establishing the homology relations between proteins. Essential amino acids for the function and/or the structure are generally conserved, thus providing key arguments to help in protein characterization. However for distant proteins, it is more difficult to establish, in a reliable way, the homology relations that may exist between them. In this article, we show that secondary structure prediction is a valuable way to validate protein families at low identity rate. RESULTS: We show that the analysis of the secondary structures compatibility is a reliable way to discard non-related proteins in low identity multiple alignment. AVAILABILITY: This validation is possible through our NPS@ server (http://npsa-pbil.ibcp.fr)


Subject(s)
Databases, Protein , Proteins/analysis , Proteins/chemistry , Sequence Alignment/methods , Sequence Analysis, Protein/methods , Algorithms , Amino Acid Sequence , Base Sequence , Protein Conformation , Protein Structure, Secondary , Proteins/classification , Sequence Homology, Amino Acid , Serine Proteinase Inhibitors/analysis , Serine Proteinase Inhibitors/chemistry
18.
J Bioinform Comput Biol ; 1(3): 505-520, 2003 Oct.
Article in English | MEDLINE | ID: mdl-15307241

ABSTRACT

We present an original strategy, that involves a bioinformatic software structure, in order to perform an exhaustive and objective statistical analysis of three-dimensional structures of proteins. We establish the relationship between multiple sequences alignments and various structural features of proteins. We show that amino acids implied in disulfide bonds, salt bridges and hydrophobic interactions have been studied. Furthermore, we point out that the more variable the sequences within a multiple alignment, the more informative the multiple alignment. The results support multiple alignments usefulness for predictions of structural features.


Subject(s)
Proteins/chemistry , Proteins/genetics , Sequence Alignment/statistics & numerical data , Amino Acid Sequence , Computational Biology , Conserved Sequence , Disulfides/chemistry , Hydrophobic and Hydrophilic Interactions , Protein Conformation , Protein Structure, Secondary , Salts/chemistry , Software
19.
J Biol Chem ; 278(9): 7199-205, 2003 Feb 28.
Article in English | MEDLINE | ID: mdl-12486138

ABSTRACT

Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that can stimulate the action of tolloid metalloproteinases, such as bone morphogenetic protein-1, on a procollagen substrate, by up to 20-fold. The PCPE molecule consists of two CUB domains followed by a C-terminal NTR (netrin-like) domain. In order to obtain structural insights into the function of PCPE, the recombinant protein was characterized by a range of biophysical techniques, including analytical ultracentrifugation, transmission electron microscopy, and small angle x-ray scattering. All three approaches showed PCPE to be a rod-like molecule, with a length of approximately 150 A. Homology modeling of both CUB domains and the NTR domain was consistent with the low-resolution structure of PCPE deduced from the small angle x-ray scattering data. Comparison with the low-resolution structure of the procollagen C-terminal region supports a recently proposed model (Ricard-Blum, S., Bernocco, S., Font, B., Moali, C., Eichenberger, D., Farjanel, J., Burchardt, E. R., van der Rest, M., Kessler, E., and Hulmes, D. J. S. (2002) J. Biol. Chem. 277, 33864-33869) for the mechanism of action of PCPE.


Subject(s)
Glycoproteins/chemistry , Amino Acid Sequence , Cell Line , Extracellular Matrix Proteins , Glycoproteins/ultrastructure , Humans , Mass Spectrometry , Microscopy, Electron , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Scattering, Radiation , Sequence Homology, Amino Acid , Ultracentrifugation , X-Rays
20.
Biochem J ; 368(Pt 1): 213-21, 2002 Nov 15.
Article in English | MEDLINE | ID: mdl-12133006

ABSTRACT

Nr-13 is an anti-apoptotic member of the Bcl-2 family previously shown to interact with Bax. The biological significance of this interaction was explored both in yeast and vertebrate cells and revealed that Nr-13 is able to counteract the pro-apoptotic activity of Bax. The Bax-interacting domain has been identified and corresponds to alpha-helices 5 and 6 in Nr-13. Site-directed mutagenesis has revealed that the N-terminal region of Nr-13 is essential for activity and corresponds to a genuine Bcl-2 homology domain (BH4). The modelling of Nr-13, based on its similarity with other Bcl-2 family proteins and energy minimization, suggests the possibility of electrostatic interactions between the two N-terminal-conserved domains BH4 and BH3. Disruption of these interactions severely affects Nr-13 anti-apoptotic activity. Together our results suggest that electrostatic interactions between BH4 and BH3 domains play a role in the control of activity of Nr-13 and a subset of Bcl-2 family members.


Subject(s)
Apoptosis/physiology , Avian Proteins , Membrane Proteins/chemistry , Proto-Oncogene Proteins c-bcl-2/chemistry , Amino Acid Sequence , Animals , COS Cells , Cells, Cultured , Chickens , Membrane Proteins/genetics , Mice , Models, Molecular , Mutation , Protein Conformation , Protein Structure, Tertiary , Rats , Sequence Homology, Amino Acid , Static Electricity , Subcellular Fractions
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